ELSEVIER
MICROBIOLOGY
REVIEWS
FEMS MicrobiologyReviews 18 (1996) 5-63
Tungsten in biological systems
Arnulf Kletzin
1 Michael W.W. Adams
*
Centerfor Metalloenzyme Studies, Departmentof BiochemistO' and Molecular Biology, UniL'ersi~'of Georgia, Athens,
GA 30602-7229, USA
Received 2 September 1995; accepted 29 September 1995
Abstract
Tungsten (atomic number 74) and the chemically analogous and very similar metal molybdenum (atomic number 42) are
minor yet equally abundant elements on this planet. The essential role of molybdenum in biology has been known for
decades and molybdoenzymes are ubiquitous. Yet, it is only recently that a biological role for tungsten has been established
in prokaryotes, although not as yet in eukaryotes. The best characterized organisms with regard to their metabolism of
tungsten are certain species of hyperthermophilic archaea (Pyrococcus furiosus and Thermococcus litoralis), methanogens
(Methanobacterium thermoautotrophicum and Mb. wolfei), Gram-positive bacteria (Clostridium thermoaceticum, C.
formicoaceticum and Eubacterium acidaminophilum), Gram-negative anaerobes (Desulfovibrio gigas and Pelobacter
acetylenicus) and Gram-negative aerobes (Methylobacterium sp. RXM). Of these, only the hyperthermophilic archaea
appear to be obligately tungsten-dependent. Four different types of tungstoenzyme have been purified: formate dehydrogenase, formyl methanofuran dehydrogenase, acetylene hydratase, and a class of phylogenetically related oxidoreductases that
catalyze the reversible oxidation of aldehydes. These are carboxylic reductase, and three ferredoxin-dependent oxidoreductases which oxidize various aldehydes, formaldehyde and glyceraldehyde 3-phosphate. All tungstoenzymes catalyze redox
reactions of very low potential ( < - 4 2 0 mV) except one (acetylene hydratase) which catalyzes a hydration reaction. The
tungsten in these enzymes is bound by a pterin moiety similar to that found in molybdoenzymes. The first crystal structure of
a tungsten- or pterin-containing enzyme, that of aldehyde ferredoxin oxidoreductase from P. furiosus, has revealed a
catalytic site with one W atom coordinated to two pterin molecules which are themselves bridged by a magnesium ion. The
geochemical, ecological, biochemical and phylogenetic basis for W- vs. Mo-dependent organisms is discussed.
Keywords: Tungsten;Molybdenum;Oxidoreductases; hyperthermophiles;Methanogenesis;Acetogenesis
Abbreviations:FDH, formate dehydrogenase;FMDH, formyl methanofurandehydrogenase;AH, acetylenehydratase; CAR, carboxylic
reductase; AOR, aldehyde ferredoxinoxidoreductase;FOR, formaldehydeferredoxinoxidoreductase;GAPOR, glyceraldehydei3-phosphate
ferredoxin oxidoreductase; ADH, aldehyde dehydrogenase; AOX, aldehyde oxidase; SOX, sulfite oxidase; DCPIP, 2,6-dichlorophenolindophenol; EPR, electron paramagnetic resonance; EXAFS, extended X-ray absorption fine structure; MCD, magnetic circular dichroism;
RR, resonanceRaman; Fd, ferredoxin;MFR, methanofuran;CHO-MFR, N-formylmethanofuran;GAP, glyceraldehyde-3-phosphate;3PG,
3-phosphoglycerate; MGD, MAD and MHD, the guanine, adenine and hypoxanthineforms of molybdopterindinucleotide,respectively
* Corresponding author, Tel.: + 1 (706) 542 2060; Fax: + 1 (706) 542 0229; E-mail: [email protected],
t Present address: Institut f'tirMikrobiologie,TechnischeHochschule Darmstadt, Schnittspahnstr. 10, 64287 Darmstadt, Germany.
0168-6445/96/$32.00 © 1996 Federationof European MicrobiologicalSocieties. All rights reserved
SSDI 0168-6445(95)00025-9
6
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reviews 18 (1996) 5-63
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
2. Physical and chemical properties of tungsten and molybdenum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
3. Abundance and chemical forms of tungsten and molybdenum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
4. Tungsten and microbial growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Methanogenic archaea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Hyperthermophiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Gram-positive bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4. Sulfate-reducing bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5. Nitrogen-fixing bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.6. Pelobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.7. Aerobic and facultatively aerobic bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10
10
13
19
21
22
23
24
5. Tungsten and eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1. Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2. Mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
26
26
27
6. Tungstoenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1. Formate dehydrogenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2. Formylmethanofuran dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3. Aldehyde-oxidizing enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4. Acetylene hydratase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5. The classification of tungstoenzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.6. The tungsten-pterin catalytic site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.7. Tungsten-substitutedmolybdoenzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
28
29
31
35
45
46
47
48
7. Conclusions: is tungsten really a phylogenetically ancient redox cofactor? . . . . . . . . . . . . . . . . . . . . . . . . . .
49
8. Note added in proof . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51
1. Introduction
The notion that tungsten (chemical symbol: W)
has a functional role in biological systems was substantiated only very recently. The first indications
were reported in the early 1970s when the growth of
certain acetogenic clostridia was shown to be stimulated by the addition of tungstate to their growth
media [1-7]. It was a decade later, however, before
the first naturally occurring W-containing protein
was purified to homogeneity [8]. At the present time,
more than ten tungstoenzymes have been isolated,
and there is substantial evidence that this number
will increase rapidly in the near future. With an
atomic number of 74, W is a most unlikely choice
for a metal with a biological function; indeed, there
are few examples of biologically relevant elements
with atomic numbers above 35. The most notable
exceptions are molybdenum (Mo, atomic number 42)
and iodine (I, 53), both of which are widely used in
biology, and there are a few examples of organisms
that require strontium (Sr, 38), niobium (Nb, 41),
A. Kletzin, M.W.W. Adams / FEMS Microbiology Retriews 18 (1996) 5-63
barium (Ba, 56) and tantalum (Ta, 73), (for reviews,
see [9,10]). In contrast to W, the essential role of the
chemically analogous element molybdenum (Mo) in
a variety of fundamental biological processes has
been known for over 60 years ([11-14]; for recent
reviews, see [15-22]). Moreover, Mo-containing enzymes are ubiquitous in nature and have been found
in the vast majority of different forms of life [19].
Yet, W and Mo have similar atomic and ionic radii
and exhibit comparable coordination chemistry. Prior
to the discovery of functional W-containing proteins,
it was suggested that replacing the Mo in a molybdoenzyme with W (as well as Nb and Ta, [23]) might
provide insight into the role of the metal in the
particular catalytic process [24-26]. This was attempted by growing, in the presence of tungstate,
various organisms, ranging from bacteria to rats
[5,25,26]. Such studies resulted, however, in the
production of either inactive Mo-enzymes lacking
any metal or W-substituted molybdoenzymes with
little or no catalytic activity. Thus, in spite of the
high degree of chemical similarity between these two
elements, organisms are highly selective. Even if W
does become incorporated into a molybdoenzyme, its
properties are different enough from Mo, with only a
few exceptions, to preclude significant catalytic activity.
So, does W have a very limited biological role?
On the contrary, research in the just the last year or
two suggests that 'natural' tungstoenzymes are present in a much wider range of microorganisms than
was previously thought, although so far they have
not been found in eukaryotes. The fact that some of
them are produced in well studied microorganisms
but only under unusual growth conditions [27-30],
while some others are found in 'exotic' species such
as hyperthermophilic archaea [31-34], has severely
hindered progress in this area. Similarly, many of the
known tungstoenzymes are very oxygen-sensitive
[1-3,8,27-29,32-35], which can make even their
detection problematic. One of the objectives o f this
review is to demonstrate that the full spectrum of
tungstoenzymes has yet to be fully recognized or
appreciated. We will focus mainly on the effect of W
on microbial growth and a description of known
tungstoenzymes. These topics were reviewed briefly
in [6,22,36,37]. Also included is a short discussion of
the physical and chemical properties of W and Mo,
and the formation of W-containing minerals, which
is, in part, a result of the same hydrothermal processes that ultimately provide the habitat fOr some
W-dependent hyperthermophiles. Since most, although not all, of known tungstoenzymes have Mocontaining counterparts which catalyze the same or a
Table I
Physical and chemical properties of the tungsten and molybdenum a
Element
Tungsten (W)
Atomic number
Average atomic weight
Stable isotopes (above 9% abundance)
Unstable isotopes used in labelling
Electronic configurationof outer shell
Atomic radii (,~)
Ionic radii for IV oxidation state (~,)
Electronegativity
pKa of oxo acid (MO4-/HMO4 ) c
Solubility (g/l) ofNa2MO 4 (25°C) ca
M = O bond length (A)
Melting point (°C)
Density (g/cm 3)
7
74
183.85
182, 183 b, 184, 186
185 (/3-)
4f ]4 5d4 6s2
Molybdenum (Mo)
42
95.94
92, 94, 95, 96, 97, 98
99(/3-)
4d5 5s I
1.40
1.40
0.68
0.68
1.4
1.3
4.60
0.86
3.87
1.33
1.76
1.76
3410
19
2610
10
Compiled from Refs. [38-44].
rS3W has an unstable nuclear isomer.
c M represents W or Mo.
a There is great variability in the reports on the solubility of tungstate salts [38,43].
8
A. Kletzin, M. W. W, Adams/FEMS Microbiology Reviews 18 (1996) 5-63
similar reaction, a comparison of the properties W
and Mo-containing isoenzymes will also be presented. The fundamental question to be addressed is:
why is W utilized by some microorganisms rather
than Mo?
2. Physical and chemical properties of tungsten
and molybdenum
Although the chemistry of both W and Mo is
varied and complex because of the range of possible
oxidation states ( - 2 to + 6) and of their ability to
form polynuclear complexes [38,39], only the +4,
+5, and + 6 oxidation states lie in the biological
range and only mononuclear systems have been identified in biology so far [16-19]. The great similarity
in their chemical properties is well established (see
Table 1), and these are quite different from other
group Via transition metals such as chromium (Cr)
[40]. The atomic and ionic radii of W and Mo, as
well as their electron affinity, are virtually identical
(Table 1), and both occur naturally as a mixture of
several stable isotopes together with several unstable
ones [40-44]. Radioactive isotopes suitable for biological research are available for both elements (~85W
and 99Mo), as are stable nuclear spin isotopes (t83W
and 95Mo) for the study of hyperfine interactions by
various spectroscopies.
W is valuable because it has the highest melting
point of all metals (Table l), great strength and good
conductivity. Tungsten alloys, with a hardness almost comparable to diamond, are major industrial
products, and the metal is also widely used in light
bulb filaments, as catalysts in the petroleum industry
(WS 2) and as a lubricating agent (WSe 2) [45]. The
annual production of W is about 35 000 metric tons
per year but it undergoes considerable fluctuation
largely depending on the demands of the military
and space industries.
3. Abundance and chemical forms of tungsten and
molybdenum
Both W and Mo are relatively scarce in nature•
Overall, they rank 54th and 53rd, respectively, in the
abundance of the elements on earth [40,43,44]. W is
usually found as oxo-rich tungstate minerals [W(VI)],
either as scheelite (CaWO4) or wolframite
([Fe/Mn]WO4), whereas the more reduced tungstenite [WS 2, W(IV)] is very rare [46,47]. In contrast,
Mo is mostly present as Mo(IV) in jordesite and
molybdenite (both MoS 2 [46,48]), minerals that are
often associated with wolframite. Reviews on the
geology, geochemistry and mineralogy of W can be
found in [44,46,47,49,50]. W deposits are usually
formed by hydrothermal mineralization and metamorphic processes, typically in subterranean environments, and W can be deposited at the discharge site
of thermal waters (see below, [46,51]). Sulfur-containing deposits are rare in part because WS 2 is
readily solubilized according to Eq. (1) [46]:
WS2+4H20~WO
~- + 2 H 2 S + 4 H + + 2 e
-
(1)
The formation of tungstates rather than molybdates
and of molybdenites rather than tungstenites in hydrothermal processes is determined by the redox
potential of the mineral-forming solution• This is
typically much higher than that required to reduce
W(VI) but below that necessary to oxidize Mo(IV).
In natural environments, the high stability of the oxo
compounds of W(VI) compared with W(IV), and of
sulfur derivatives of Mo(IV), such as MoS 2, compared with Mo(VI), such as molybdates, is due
mainly to the reaction shown in Eq. (2) (where
AH = - 31 kcal/mol; [46])•
H2MoO 4 + WS 2 --->MoS 2 + H2WO 4
(2)
Most natural environments also contain Ca, Fe and
Mn, and the reaction shown in Eq. (2) is reinforced
by the stability of the following associations: CaWO 4,
Ca(W,Mo)O 4 • MoS 2, FeWO 4 • MoS 2, and MnWO 4
• MoS 2 [46,52].
The W content of most rock formations is similar
to that of Mo, but in surface waters the solubility and
mobility of W is low compared to Mo due to intensive sorptional precipitation [43,46]. In freshwater
environments, the W concentration rarely exceeds 20
nM and is usually much less than 0.5 nM (outside of
areas rich in W minerals [43,46]), whereas Mo concentrations are typically two or more orders of magnitude higher. Similarly, the concentration of W in
sea water is extremely low, and about 500000 fold
less than that of Mo (see Table 2; [43,44,53]), although some marine sediments contain quite high W
A. Kletzin, M.W. W, Adams / FEMS Microbiology Ret'iews 18 (1996) 5- 63
Table 2
Distribution of W and Mo in natural environments a
Element
W
Mo
Earth's crust ( m g / k g )
Rank
Soils ( m g / k g )
Seawater ( / z g / k g ) h
Freshwater ( / x g / k g )
Terrestrial hot springs ( / ~ g / k g )
Keough hot spring (p~g/kg) d
Searles lake brines ( / x g / k g ) d
Juan de Fuca, vent fluids
(p~g/kg) ~
Juan de Fuca, vent fluids
(/xg/kg) f
Juan de Fuca, chimney walls
(mg/kg) g
Juan de Fuca, vent flanges
(mg/kg) e
TAG, mid-Atlantic relict zones
(mg/kg) h
Most abundant valance state
(complex)
1.55
54th
0.1-3.0
0.0002
< 0.1
15-300
300
70,000
0.37
1.55
53th
0.3-39
8.9-13.5
0. 1-5.0
3-60
n.r. c
n.r.
< 0.002
n.r.
< 0.1-3. l
n.r.
6-120
180-585
47
n.r.
40-240
VI (WO42- )
IV (MoS 2)
Compiled from Refs. [40,42,43,46,53,56],
b Some sources give much a higher WO~- concentration in sea
water (0.12 /zg/kg, [79,80]), in which case the M o / W ratio is
near 200 rather than 5 × 105. Although others regard such data as
unreliable [53,81], they appear in some reviews and textbooks
[82,83]. We cannot validate the reliability of individual sources,
but since the lower values given in the Table were obtained by
independent sources and are cited in most reviews on W [40,43,56],
they are assumed to be correct.
n.r,, not reported.
d Taken from Ref. [57].
Baross, J., Mukund, S. and Adams, M.W.W., unpublished data.
f Taken from Ref. [73].
g Taken from Ref. [74].
h Taken l¥om Ref. [75].
concentrations (10-60 /xg/kg; [54-56]). W is present in significant amounts ( > 50 riM) in four types
of natural waters: in groundwaters directly associated
with W-containing ore deposits; in alkaline, nitrogenous fissure-vein thermal waters of crystalline rocks;
in alkaline waters of lakes in arid zones; and in
hot-spring waters and hydrothermal vents (see below). The highest values reported are more than 0.27
mM in the highly concentrated brines of Searles
Lake, California, which drain an area of scheelite
depositions in an ultra-dry climate [57]. One of the
springs feeding into Searles lake contains 1.5 /xM
[57]. Values approaching I l /.tM have been found in
9
alkaline lakes of the Pamirs [46]. There are few data
on the W contents of soils, and values are typically
near 1 m g / k g . This is slightly below the overall
abundance of W in the lithosphere [58], but is usually an order of magnitude below that of Mo [518-61].
Deep sea hydrothermal vents represent unique
environments in which to consider W accumulation
and availability. These originate from the convective
circulation of sea water through newly formed
oceanic crust at seafloor spreading centers (for review, see [62]). The so-called white smoker vents
release clear or milky fluids with temperatures ranging from 200 to 330°C, whereas the black smokers
discharge jets of water blackened by sulfide precipitates at temperatures up to >_ 400°C. The black
smoker chimneys are transition metal precipitates
surrounded by a wall consisting mainly of precipitated CaSO 4 derived from seawater. White smokers
tend to form porous structures high in SiO 2 through
which the fluids percolate in multiple channels. Vents
of both types can form active mounds more than 200
m in diameter and 50 m height (e.g. [63] and references therein). The vents support complex ,ecosystems, including tube worms, clams, crabs, etc, which
are entirely dependent on cbemolitboautotrophic microorganisms which in turn use the vent fluids as
sources of minerals, sulfur and CO 2 [64-70].
The chemistry of the vent fluids results from the
prior passage of seawater through basalt at ~emperatures in most cases above 350°C. This leads to
enrichment, due to leaching, in Na, K and CI to
concentrations above 0.5 M, as well as in various
transition and rare earth metals (reviewed in [71])
and sulfide concentrations can reach 7 mM [71,72].
In contrast, Mo is removed from seawater during
hydrothermal cycling [73]. Vent fluids investigated
so far contain Mo at concentrations at least one order
of magnitude below that of seawater, and some of
them were virtually Mo-free (see Table 2). On the
other hand, the chimney walls of smokers ,are more
enriched in Mo with values ranging from 6 to 120
m g / k g [74] and 40 to 240 m g / k g [75]. So ;far, there
has been only one study on the abundance of W in
deep sea sites (see Table 2). It was found that W, in
contrast to Mo, was present in samples of vent fluids
at about one thousand times that found in sea water.
Similarly, in chimney walls, or more specifically, in
samples of protruding flanges [76], the concentration
10
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reuiews 18 (1996) 5-63
of W reached 580 m g / k g , more than ten times that
of Mo (47 m g / k g ; Baross, J.A., Mukund, S. and
Adams, M.W.W., unpublished data). These flanges
are porous metal sulfide-silica structures which result
from the deposition of material from the flow of
superheated white smoker fluid under a horizontal
overhang [76]. Hence, the W and Mo in the flanges
appear to have different origins. The W must co-precipitate with Fe, Mn and Ca minerals from the hot
vent fluids [46], while the deposition of Mo is more
likely the result of sea water intrusion. After reduction with sulfide, Mo would be deposited as MoS 2.
Interestingly, the chimney walls and flanges contain
high concentrations of microorganisms within all
temperature ranges up to 120°C [66,77,78].
4. Tungsten and microbial growth
The influence of W on the growth of a wide
variety of microorganisms has been investigated, and
in many cases the effects on various enzyme activities have also been reported. In addition, various
species have been grown in the presence of radioactive 185W and the cellular distribution of labelled
proteins has been examined. These studies have
shown that W stimulates the growth of many types
of prokaryote, but as yet, a positive influence of W
on the growth of a eukaryote has not been demonstrated. In the following, the effects of W on microbial growth are discussed with reference to systematic taxa or groups of organisms with common physiological capabilities. Thus, we begin with the
methanogens, all of which are classified as archaea
(formerly archaebacteria [84]), and then consider organisms which grow at 90°C, the so-called hyperthermophiles, most of which are also archaea. Representatives of the methanogens and the hyperthermophiles are among the most studied of all prokaryotes with regard to the metabolism of W, and several
types of tungstoenzyme have been purified from
them. The best characterized of the bacteria (formerly
eubacteria [84]) with respect to the effects of W are
the acetogens, and these are included within the
Gram-positive group. Other types of bacteria that
metabolize W and contain tungstoenzymes include
both anaerobes, such as those that reduce sulfate or
utilize acetylene, and aerobes, such as those that
oxidize methane. For the sake of completion, we also
briefly discuss some other organisms not known to
contain tungstoenzymes but whose growth is affected by W.
4.1. Methanogenic archaea
Methanogens are a unique group that possess
many physiological and biochemical characteristics
that are not shared by any other type of microorganism. The production of methane using H 2 and CO 2
as growth substrates is a defining property [85],
although many species can also use simple carbon
compounds, such as formate or acetate, as an energy
source. All methane-producing organisms are classified within the archaea (for a recent review on the
taxonomy of methanogens, see [86]). A stimulatory
effect of W on the growth of methanogens was first
reported in the late 1970s with Methanococcus uannielii [87-89]. Its growth was dramatically enhanced
by the addition of W, but not by Mo, to the growth
medium when formate was used as the carbon and
energy source, but not when the organism was grown
on H : and CO 2. This suggested the involvement of a
W-containing formate dehydrogenase (FDH). Partial
purification showed that the formate-oxidizing activity of Mc. vannielii resided with two types of FDH
[87,88]. One existed as a very high molecular mass
complex which contained W and also selenium (Se),
while the other had a M r of approximately 110 kDa
and contained Mo, Fe, and S but not Se. Cells grown
without added W but in a Se-supplemented medium
contained both types of FDH, whereas the FDH
containing W and Se was the predominant form in
cells grown in the presence of W [89].
The early work with FDH in Mc. uannielii stimulated significant interest in this area, and from the
early 1980s onwards, several newly isolated
methanogens were shown to require W for growth
[90-96]. With some species, the stimulatory effect of
W on cell growth was very concentration dependent.
For example, an obligately autotrophic ( H 2 / C O 2utilizing) methanogen, Methanobacterium wolfei,
was isolated from sewage sludge in 1984 and its
growth was dependent upon the addition of yeast
extract, ash of yeast extract (showing that an organic
cofactor was not involved), or W (the responsible
moiety: [91]). A linear correlation of growth rate and
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reviews 18 (1996) 5-63
1I
Table 3
Incorporation of tungsten into cell material of methanogenic archaea and of some bacteria ~
Genus
Species
Methanogenic Arehaea
Methanobrevibacter
arboriphilus DSM1125
smithii
Methanobacterium
formicicum ZZI
bryantii M.o.H. DSM 863
thermoautotrophicum
zS'SW uptake ~
T ~
(°C)
Growth
substrate
ZS~W supply
(Bq/ml)
37
37
37
37
60
H2/CO
H2/CO 2
H2/CO 2
1050
1175
1000
1160
1085
21
48
20
21
29
50
81
52
48
466
2.0
4.1
2.0
1.8
2,7
60
H 2/CO2
1050
162
247
15.4
60
60
80
37
37
60
H:/CO 2
H2/CO:
H2/CO 2
Formate
H2/CO 2
1240
1150
610
735
850
730
129
130
141
42
19
361
188
252
398
73
100
831
10.4
11.3
23.1
5,7
2.2
49.4
37
30
30
37
37
60
FI2/CO 2
H2/CO 2
Formate
H2/CO 2
Formate
H2/CO 2
730
900
725
700
685
840
177
702
300
541
33
840
532
1991
2586
I1)50
n.r.
24.2
78.0
41.4
77.2
4.8
42./)
60
H2/CO 2
1580
563
1440
35.7
60
40
37
37
37
37
30
37
37
37
37
37
Formate
n.r.
H2/CO 2
Formate
HJCO 2
Formate
CH3OH
H2/CO 2
H2/CO 2
Formate
H 2/C02
Formate
1060
770
720
890
1120
850
1050
500
1785
510
1360
35(1
3.1
86.0
87.2
11.3
37.8
1.8
0.5
6.8
77.6
71.8
79.3
31.4
37
37
74
74
Lactate/SO 4
Glucose
Yeast extract
Yeast extract
835
1090
1840
275
2
H2/CO:
H:/CO 2
(Bq/ml)
( B q / m g ¢)
Total ~1
(~)
DH DSM 1053
thermoautotrophicum
Marburg DSM 2113
Methanothermus
Methanococcus
thermoautotrophicum S / W
wolfeii DSM 2970
fervidus DSM 2088
rannielii DSM 1224
thermolithotrophicus
H2/CO:
DSM 2095
Methanogenium
marisnigri DSM 1498
cariaci DSM 1497
tatii DSM 2702
thermophilicum
n,r.
DSM 2373
thermophilicum
Ratisbona DSM 2640
Methanospiri[lum
liminatans DSM 4140 f
hungatei DSM 864
Methanomicrobium
paynteri DSM 2545
Methanolobus
Methanosarcina
Methanoplanus
tindarius DSM 2278
barkeri DSM 1538
limicola DSM 2279
Methanocorpusculum
parcum DSM 3823
Bacteria
Desulfot,ibrio
Eubacterium
Thermotoga
sp. MP47
limosum
sp. ZB
sp. ZB (with 63Ni)
H2/CO 2
33
n.r.
628
101
423
15
5
34
1386
366
1079
110
53
22
2
2
1441
2304
450
759
n.r.
n.r.
177
3433
1896
3309
606
197
180
n.r.
n.r.
Modified from Ref. [90].
6 Growth temperature of the organism.
c This represents the difference between the lSSw supply and the 18SW-content of the medium after cell growth.
a The percentage of the radiolabel incorporated was determined empirically and depends on the 1~5W supply.
Dry weight.
f Taken from Ref. [98].
6.3
2,0
0,1
0.7
12
A. Kletzin, M. W. W~ Adams / FEMS Microbiology' Retqews 18 (1996) 5-63
W-content was found up to a concentration of 7.6
# M W in the medium, but growth was inhibited at
W concentrations above 11.6 p~M. In this case, Mo
could not replace W. On the other hand, in some
methanogens, W has an antagonistic effect on Mo.
For example, with Methanobacterium formicicum
growing on H 2 / C O z in a Mo-supplemented medium,
FDH was found to comprise approximately 3% of
the total soluble protein [97]. Depletion of Mo led to
dramatic decreases in the intracellular concentration
of Mo, of total FDH activity, and of the amount of
immunologically detectable FDH apoprotein. When
cells were grown in the presence of both W and Mo,
intracellular W accumulated only when the W concentration in the medium exceeded that of Mo by at
least a 1000-fold. Under such conditions, W was
incorporated into FDH but this yielded virtually inactive enzyme. However, even in the presence of a
huge excess of W over Mo, the cell growth yields on
H 2 / C O 2 were not affected [97]. These results suggest that W prevents the accumulation of Mo and
that W is unable to replace the Mo requirement of
FDH.
Most investigations on the effects of W on the
growth and metabolism of methanogens have utilized H 2 / C O 2 or formate as growth substrates (see
Table 3), but this is not always the case. For example, methanogenic strains have been isolated from
both freshwater and seawater sources using 2-propanol and acetate as the growth substrates and these
produce acetone from 2-propanol [94]. One of the
marine strains investigated, which appeared to be a
species of Methanogenium, required W for growth,
as well as biotin and vitamin B~2. The optimal W
concentration was 0.1 /zM but Mo (or Se) was not
required. [94]. The related species, Methanogenium
tatii (Topt = 45°C), which was isolated from a solfataric field, grows on H 2 / C O 2 or formate but also
requires acetate. In this case, no growth was observed if W was not present in the growth medium,
although the role of W was not investigated [92]. In
contrast, the growth on H 2 / C O 2 of the related organism, Mg. liminatans, which was isolated from the
effluent of a waste water reactor, was stimulated by
W ( 1 - 2 /zM) but it was not shown that the element
was absolutely required for growth [98].
There have also been some unusual reports on the
effect of W on the growth of methanogens. For
example, three different and independently isolated
strains of Mg. thermophilicum (Toot = 55-60°C) have
been reported [93] and these grow on formate or
H J C O 2 and require the presence of acetate, yeast
extract and peptone for optimal growth. When yeast
extract and peptone were replaced by casamino acids,
the doubling time did not change but the final density decreased by 80%. In all three strains, the
essential growth factor present in the complex
medium was shown to be nickel (Ni), but in one of
them, Ni could be replaced by W for at least eight
successive transfers [93]. No comment was made on
this rather curious result, and the effects of combinations of different metal ions on cell growth were not
reported. Ni is an essential component of several
methanogenic enzymes including hydrogenase and
methyl CoM reductase [85] and its replacement by
W, and least in known Ni-containing enzymes, is not
possible. Contaminating Ni in the tungstate salt that
was used in these studies is the most likely explanation for the observed results. A second and most
intriguing report concerned the sapropelic, anaerobic,
marine ciliate Metopus contortus Quennerstedt. This
organism inhabits sulfide-containing environments
and lacks mitochondria and cytochrome c oxidase
[95]. However, it contains numerous methanogenic
endosymbionts (approx. 4500 per cell), the most
abundant of which is Mb. formicicum. From crushed
cilicate ceils, a second, non-related and W-dependent
methanogen, Methanoplanus endosymbiosus, was
isolated. The new organism required 0.1 mM W for
optimal growth on H 2 / C O 2 or formate [95]. Unfortunately, details on its incorporation of W into proteins or of W-transport by the host ciliate cells were
not reported.
Up until recently, the only known enzyme to
show a response to W in at least some methanogens
was FDH. Hence, all enzymatic aspects of W-dependent growth and W accumulation properties of
methanogenic species focused on this enzyme. However, in 1992, it was shown that the stimulatory
effect of W on the growth of Mb. wolfei and Mb.
thermoautotrophicum was associated with another
enzyme, f o r m y l m e t h a n o f u r a n dehydrogenase
(FMDH), a key enzyme in the methanogenic pathway [27,99]. FMDH catalyzes the first step in autotrophic methanogenesis, the reduction of CO 2 to
formaldehyde, wherein the formaldehyde is bound to
A. Kletzin, M. W. W. Adams/FEMS Microbiology Rez'iews 18 (1996) 5-63
methanofuran, a cofactor unique to methanogens
[100,101]. In fact, it is now known that both Mb.
wolfei [27,99,102] and Mb. thermoautotrophicum
strain Marburg [28,29] contain two FMDHs, one
dependent upon W and one dependent on Mo. However, this is not a general phenomenon in
methanogens since, for example, W does not support
the synthesis of active FMDH in Methanosarcina
barkeri [103]. Nevertheless, the finding of a second
W-containing enzyme in at least some of these organisms necessitates a reevaluation of the likely
effects of W on cell growth and metabolism. For
example, Methanocorpusculum parvum [96,104],
which was isolated in 1987, grows on H2/CO2,
formate and 2-propanol/CO 2, The addition of both
Ni and W to the medium stimulated growth, and
radiolabelling studies showed that 63Ni was incorporated mainly into cofactor F430 of methyl reductase
but ~85W was also found in the protein fraction. The
optimal concentrations of W for growth on H 2/CO2
and formate were 1 p~M and 0.5 /zM, respectively.
W at a concentration of 1 /~M inhibited growth on
formate, but this effect could be alleviated by the
simultaneous addition of Mo and Se (both at final
concentrations of 0,1 p~M). A possible explanation
for these results is the antagonistic effect of W on
FDH, in which case Mp. paruurn probably contains
a W-dependent FMDH and a Mo and Se-dependent
FDH [90,96]. The ~SW label in cell extracts of Mp.
par~,um chromatographed as a single protein traction, consistent with this interpretation. Two other
species of Methanocorpusculum have been isolated,
Mp. sinense and Mp. bal,aricum [104], and these
have W-dependent growth properties similar to those
of Mp. paruum.
The results of an extensive and systematic survey
of tSSW uptake by various methanogens reported in
1987 [90] also have to be reevaluated in terms of
FMDH. In this study, cells of various species were
grown in the presence of the radiolabel, the dry
weight of the culture was determined and the W
uptake was calculated from the difference in W
content of the cell pellet and the culture supernatant
[90]. From the results, which are summarized in
Table 3, several interesting patterns can be seen.
First, almost 50% of the species investigated (13 out
of 22 if different strains are counted separately)
incorporate significant amounts of W. Second, in
13
general, growth on H J C O 2 required W to a greater
extent than did growth on formate. In several cases,
no W is incorporated during formate-dependent
growth, e.g. Mg. thermophilicum Ratisbona, while in
others the fraction of W that is incorporated is much
lower than during Hz-dependent growth, e.g. Mp.
parvum, and only Methanoplanus limicola incorporates equal amounts of W under both growth regimes.
In all strains shown in Table 3 that grow on
H J C O 2, we assume that W is required for the
expression of functional FMDH. At this point it is
not clear if an additional Mo-dependent FMDH is
also present in the organisms studied, which is the
case with both Mb. therrnoautotrophicum and Mb.
wolfei [28,29,99,102]. The decreased demand for W
during growth on formate suggests that the synthesis
of W-dependent FMDH is repressed, and presumably
W is incorporated into FDH. Another important point
is that the methanogens studied obviously do not
incorporate W indiscriminately, in contrast to what
has been found in some bacteria [105-107].
Methanogens must possess an uptake mechanism
which is selective for W or Mo. FMDH activity has
been found in all methanogens when grown on
H 2 / C O 2, while its presence during growth on organic C~-compounds will obviously depend on
whether (i) the growth substrate is disproportionated
to CO, and CH~ [108]; (ii) only the reduction reaction is used [109]; or (iii) FDH is present [89]. Thus,
at this point, the relationship between FMDH and
FDH under different growth conditions is far from
clear. The situation is complicated by the fact that, at
least in some methanogens, both W- and Mo-forms
of both enzymes appear to exist, and this is compounded by the presence of both elements in the
media that are typically used to grow these organisms. Although it can be concluded that W does play
a key role in the metabolism of some, although not
all, methanogens, further studies with additional
species (see Table 3) are obviously required before
the precise roles of W in methanogenesis can be
defined.
4.2. Hyperthermophiles
Hyperthermophilic microorganisms have the remarkable property of growing at temperatures approaching, and in some cases, in excess of, IO0°C.
14
A. Kletzin, M. W.W. Adams/ FEMSMicrobiologyReviews 18 (1996) 5-63
Although the temperature extremes that define hyperthermophiles have changed over the years, we
will refer here to hyperthermophiles as organisms
with an optimal growth temperature of at least 80°C
or a maximal growth temperature of at least 90°C. So
far about 20 genera of hyperthermophiles are known,
virtually all of which were isolated in the last decade
(reviewed in [66,110-120]). All but two of them are
classified as archaea (formerly archaebacteria) rather
than bacteria (formerly eubacteria, see [84,121 ]). The
h y p e r t h e r m o p h i l i c a r c h a e a include three
methanogenic genera and a unique sulfate-reducing
genus termed Archaeoglobus. The majority of these
organisms, however, are obligate heterotrophs whose
growth is dependent upon elemental sulfur (S°),
which they reduce to H2S via a respiratory-type
metabolism. Most of them use proteinaceous growth
substrates, although some can also utilize carbohydrates and a few autotrophic species are known. Of
the two hyperthermophilic bacterial genera, Thermotoga sp. are anaerobic heterotrophs with a fermentative-type metabolism, while Aquifex sp. are microaerophilic autotrophs (see below).
For the isolation and culture of hyperthermophiles, Mo is usually included in trace element
solutions (as are many other metals), although there
has been no systematic study on its effectiveness
[122-126]. On the other hand, in 1989, it was reported that W stimulated the growth of the hyperthermophilic archaeon, Pyrococcus furiosus [126].
This organism grows optimally at 100°C using sugars as a carbon and energy source in a complex
medium which also contains peptides. The fermentation products are H 2, CO 2 and acetate. It was isolated from a shallow, marine hydrothermal discharge
site [127] and related species have been found near
deep sea hydrothermal vents [66,77,128]. In an attempt to enhance the growth yields of P. furiosus,
the addition of salts of elements such as V, Se, Cs, F,
Pb, Rb and additional Ni to the mineral salts solution
were unsuccessful, whereas the addition of W (10
/xM) and additional Fe (final concentration, 25 /zM)
to the growth medium gave a 5-10 fold increase in
cell yields (wet weight) from large scale fermenter
runs. Although the effect of W on growth kinetics
and final cell densities were not reported, this preliminary study clearly indicated a role for W in the
metabolism of P. furiosus, and perhaps in other
heterotrophic archaea ([126], reviewed in [22,3638,129-133]). Moreover, it led to the purification of
the first W-containing iron-sulfur protein from this
organism [31 ]. This was shown to catalyze the oxidation of various aldehydes to the corresponding acid
using P. furiosus ferredoxin as the electron carder,
and was termed aldehyde ferredoxin oxidoreductase
(AOR; [32]).
The effect of W on the growth of P. furiosus and
on the fermentation of maltose and peptides was
subsequently extended to chemostat experiments under energy-limiting conditions [134,135]. These
showed that the addition of maltose to a peptidelimited culture increased the cell yields significantly
(as measured by dry weight and protein content), but
the greatest effect was observed when W was also
added. This was accompanied by a 40-fold increase
in the specific activity of AOR, which was over
twice that observed in peptide-grown cells (grown
with added W but in the absence of maltose). These
results were consistent with the postulated role of
AOR as part of an unusual Entner-Doudoroff pathway for glucose degradation ([32,136]; reviewed in
[36,37,129-133]) a modified version of which was
recently proposed [137]. However, other studies suggest a different interpretation. First, a ~3C-labelling
investigation reported in 1994 [138] showed that the
predominant route for glucose oxidation in P. furiosus is an unusual Embden-Meyerhof pathway, and
this does not involve a non-phosphorylated aldehyde
oxidation step, the proposed role of AOR. Second, in
1995, another W-containing iron-sulfur enzyme
termed glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR) was purified from P. furiosus
[34]. As shown in Fig. 1, GAPOR is thought to
replace glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) in this Embden-Meyerhof pathway. The
production of 3-phosphoglycerate by GAPOR, rather
than 1,3-bisphosphoglycerate by GAPDH, was consistent with the reported growth yield data from P.
furiosus [ 139].
Hence, the stimulation of cell growth by the
addition of maltose and W to P. furiosus is presumably due to the activation of GAPOR rather than
AOR. At this point it is not clear why the activity of
AOR increases upon maltose addition since, as discussed below, this enzyme is now thought to play a
role in amino acid oxidation. In addition to GAPOR,
A. Kletzin, M. W. W. Adams/FEMS Microbiology Reviews 18 (1996) 5-63
Glucose
Hcxokinase
(ADP)
V
ADP
~",~AMP
Glucosc-6-phosphate
1
Fmctosc-6-phosphate
Phospho-
V
ADP
fructokinasc
(ADP)
~"~ AMP
Fructose-1,6~bisphosphate
Glycera|dehyde-3-phosphate--q-----Dihydmxyacetonephosphate
!
GAPOR
~ ' ~ Fd - - -~- FNOR - - ~ NADP - - -~-
3-Phosphoglyceratc
I
/'~
2H +
H2
2-Phosphoglyceratc
Enolas¢
~ . ~ HaO
Phosphoenolpyruvate
ADP
~
AlP
Pymvate
Pvruvate
~"-CoASH
Ferr~lom
K2c2c
Oxidoreductas¢ ~""~ F~ - - "~ FNOR - - - ~ NADP - - .~- H ~
v
Acetyl CoA + CO2
Acetyl CoA [/,.-ADP + Pi
Synthetase
/f~
2H+
H2
~X.~ATP+ CoASH
Acetate
Fig. 1. Proposed role of GAPOR in the carbohydrate-utilizing
hyperthermophilic archaea. Enzymes that have been purified from
P. furiosus are shown in bold and underlined, unusual enzymes
are indicated, and dotted lines indicate electron transfer reactions.
The abbreviations are: Fd, ferredoxin; FNOR, ferredoxin NADP
oxidoreductase: H~_-ase, hydrogenase. Modified from [34].
the pathway for the metabolism of sugars to acetate
in P. furiosus (Fig. 1) contains other unusual enzymes such as ADP-dependent hexose(-phosphate)
kinases [138] and acetyl-CoA synthetase [140]. A
conventional GAPDH is also present in P. furiosus;
in fact, the enzyme has been purified and its gene
has been cloned from the closely related organism,
P. woesei [141]. The specific activity of GAPDH is
very low in maltose-grown P. furiosus and the enzyme is thought to function in gluconeogenesis
[34,142].
The role of W in the metabolism of the hyperthermophilic archaea was extended by studies with Ther-
15
mococcus litoralis. This anaerobic heterotrophic was
isolated from a shallow submarine thermal spring
and grows up to 98°C in a complex medium by
peptide fermentation [143]. Cell-free extracts were
found to contain high formaldehyde-oxidation activity, and purification of the responsible enzyme
showed that it was yet another W-containing ironsulfur protein. This was termed formaldehyde ferredoxin oxidoreductase (FOR) [33]. In contrast to AOR,
FOR has a very narrow substrate range and catalyzes
the oxidation of only C1-C3 aldehydes. Although
FOR was purified by its formaldehyde oxidation
activity, this is thought not to be its physiological
function. This is suggested by the low affinity (high
apparent K m value) of FOR for formaldehyde and
its low activity (low apparent Vm value) in catalyzing formaldehyde oxidation (see below). However,
whether an aldehyde per se or a metabolic product
formed from amino acid degradation is the true
substrate for FOR remains to be established.
Although 7". litoralis grows well in the maltosecontaining medium routinely used to grow P. furiosus, growth of the latter organism results in a decrease in the medium pH due to acetate production,
whereas there is no dramatic pH change upon the
growth of T. litoralis, consistent with peptide fermentation being its primary metabolic route. Conversely, P. ,furiosus will grow, although not as well,
on peptides in the absence of sugars. The differences
in the metabolic properties of these two organisms
appear to be related to the activities of their W-containing enzymes. For example, T. litoralis also contains AOR and GAPOR (Mukund, S. and Adams,
M.W.W., unpublished), although the specific activities of both are about 10% of those in P..furiosus.
Similarly, P. furiosus contains low concentrations of
a W-containing iron-sulfur enzyme whose molecular
and catalytic properties are virtually identical to those
of the T. litoralis FOR [144]. Thus, P. furiosus and
T. litoralis contain different amounts of three different types of aldehyde-oxidizing tungstoenzyme, all
three of which have been purified from P. furiosus
[32,34,144].
The relative activities of GAPOR and FOR in P.
furiosus are consistent with their proposed roles in
carbohydrate and peptide catabolism, respectively.
The question then becomes, what is the physiological
role of AOR? Insight into this came from studies
16
A. Kletzin, M. W. W. Adams/FEMS Microbiology Reviews 18 (1996) 5-63
with another species of Thermococcus, strain ES-1.
ES-1 was isolated from the gut of a tube worm
(Paralvinella sp.; [128]), which lives in close vicinity to deep sea hydrothermal vents. ES-1 grows up to
91°C by peptide fermentation and does not utilize
carbohydrates. Nevertheless, cell-free extracts contain high aldehyde-oxidation activity, and the molecular and catalytic properties of the enzyme responsible are virtually identical to those of P. furiosus
AOR [145]. Like the P. furiosus enzyme, ES-1 AOR
oxidized a wide range of aliphatic and aromatic
aldehydes, but those with the highest apparent
kcat/Km values ( > 10 /zM -I s -1) were acetaldehyde, isovalerylaldehyde and phenylacetaldehyde
( K m < 100 /zM), which are the aldehyde derivatives
of transaminated amino acids. The pure ES-l enzyme also catalyzed the reduction of acetate (apparent K m = 1.8 mM) below pH 6.0 but at much less
than 1% of the rate of the oxidative reaction. It was
proposed, therefore, that ES-1 AOR functions to
oxidize aldehydes generated during amino acid
catabolism, although it could not be ruled out that it
could generate aldehydes from organic acids produced by fermentation (see Fig. 2; [145,146]). The
same applies to the AORs of T. litoralis and P.
furiosus, although it is not clear why the specific
Amino acid
TA
2-Keto acid
• """
Aldehyde
~
Fd~ + CoASH
Acyl CoA + CO2
F ~ A C S
~ A D P + Pi
A! d~.ATP + CoASH
Fdred
Fig. 2. Proposed role of AOR in the peptide-utilizing hyperthermophilic archaea. The abbreviations are: TA, transaminase;
KAOR, 2-keto acid ferredoxin oxidoreductase; ACS, acetyl-CoA
synthetase; AOR, aldehyde ferredoxin oxidoreductase; Fd, ferredoxin. The KAORs include those specific for pyruvate [178],
aromatic 2-keto acids [179l and branched-chain 2-keto acids [177].
Modified from 1145,146].
activity of the enzyme is much higher in P. furiosus
than it is in T. litoralis, while the opposite is true for
FOR. Perhaps these two W-containing enzymes have
a complementary and perhaps overlapping role in
peptide fermentation. AOR has also been purified
from the deep sea isolate, Pyrococcus sp. ES-4, and
this also contains low FOR activity (Mukund, S. and
Adams, M.W.W., unpublished). ES-4 grows up to
110°C and, like P. furiosus, utilizes both peptides
and carbohydrates as growth substrates. It was isolated from the solid material of vent flanges, a
unique porous structure from deposits of smoker
vent fluids [77].
The presence of W-dependent and Mo-dependent
isoenzyme forms of FMDH in the thermophilic
methanogens [27-29] prompted a study to determine
if this was also the case with the three types of
tungstoenzyme in P. furiosus [147]. In addition, the
effects of vanadium (V), which has chemical properties similar to those of W, were also investigated.
Such a study was complicated by the fact that, unlike
the methanogens, P. furiosus is routinely grown on a
complex medium (containing yeast extract, tryptone,
etc.). Thus, in the absence of added tungstate (to 10
/zM), the medium contained W at a concentration of
approximately l0 nM. P. furiosus was grown in
media containing added Mo (100 /zM, but no W or
V), added V (as vanadate, 5 /zM, but no Mo or W)
or added W (10 /zM, no Mo or V). AOR was
purified from V-grown cells (V-AOR) but such cells
did not contain measurable FOR or GAPOR activity.
V-AOR had 15-fold lower catalytic activity than
AOR purified from W-grown cells (W-AOR) and
contained 0.2 g-atoms/mol (rather than the expected
2.0 g-atoms/tool) of W. V could not be detected in
purified V-AOR. AOR and FOR were purified from
Mo-grown cells but these cells also lacked GAPOR
activity. Both the specific activities and the W-contents of Mo-AOR and Mo-FOR were similar to those
of the enzymes purified from W-grown cells, and
neither of them contained detectable Mo. Hence, on
a peptide-maitose medium containing a 10000-fold
excess of Mo over W, P. furiosus is able to scavenge sufficient W to synthesize both FOR and AOR,
and these are expressed in deference to GAPOR. On
the other hand, vanadate appears to inhibit the uptake
a n d / o r incorporation of W into the three tungstoenzymes, and that which is available is preferentially
A. Kletzin, M.W.W. Adams/FEMS Microbiology Re~'iews 18 (1996) 5-63
utilized tbr AOR synthesis. Moreover, these results
show that P. furiosus uses exclusively W to synthesize AOR, FOR and GAPOR, and that Mo (or V)
isoenzymes are not expressed [147].
At present it is not known if the dependence of
the Thermococcales (which includes both Pyrococcus and Thermococcus) upon W and their unique
possession of three different types of tungstoenzyme
is indicative of a more general W metabolism in
other hyperthermophilic archaea. Some preliminary
evidence in support of this comes from the identification of a putative GAPOR-encoding gene in the
hyperthermophilic chemolithoautotroph, Pyrodictium occultum, which is a member of the Pyrodictales (Kletzin, A. and Adams, M.W.W., unpublished
data; see below). A second piece of evidence in
favor of a more general W-dependence for hyperthermophiles is the depletion of Mo and enrichment
of W in hydrothermal waters and the W deposition
in deep-sea vents and in terrestrial hot springs. Thus,
the high densities of archaea that have been found
near 100°C in the porous SiO2/metal sulfide-based
matrix of chimney walls and vent flanges ([76] and
Baross, J.A., unpublished]) appear to be exposed to
W-rich but Mo-poor environments. Interestingly, one
of the hyperthermophiles isolates from a deep sea
flange is the W-dependent and AOR-containing Pyrococcus sp. ES-4 [77]. On the other hand, FMDH
has been purified from the hyperthermophilic archaeon, Archaeoglobus fulgidus [148]. It should be
noted that this organism is not a methanogen but it
contains several methanogenic cofactors and related
enzymes which are involved in its C-1 metabolism
[149]. By analogy with methanogens, the FMDH of
A..fulgidus was assumed to be a molybdoenzyme
[148] but this was not proven, and it is possible that
the enzyme contains W. The growth dependence of
Archaeoglobus sp. on varying W or Mo concentrations has been reported, and it is not known if
species of this unusual genus express both W- and
Mo-containing FMDH isoenzymes, like the thermophilic methanogens.
Acidophilic hyperthermophiles belonging to the
Sulfolobales potentially offer another avenue for W
metabolism at extreme temperatures. For example,
the acidophilic, facultatively aerobic and facultatively autotrophic Sulfolobus acidocaldarius (Topt =
80°C), together with some related hut less ther-
17
mophilic organisms, are able to couple the reduction
of molybdate to the oxidation of S° under
chemolithoautotrophic but not under heterotrophic
conditions ([150], reviewed in [ 151 ]). The mechanism of Mo reduction was proposed to involve the
formation of isopolymolybdate anions in acidic solution (pH < 6). These undergo facile reduction to
generate molybdenum blue dyes wherein Mo is in a
mixed oxidation state (V and VI). Similarly, some
mesophilic organisms such as Pseudomonas guillermondii, Micrococcus sp. [152] and Thiobacillus ferrooxidans [153] are able to catalyze the reduction of
Mo(VI) and in some cases this is coupled to S°
oxidation. As yet there have been no reports of
analogous metabolic reactions with tungstate serving
as a terminal electron acceptor in place of molybdate. However, it seems reasonable to suggest that
some microorganisms, either hyperthermophiles or
mesophiles, might be able to overcome the typically
very low redox potential of the W(VI)/(V) couple
by the stepwise reduction of para or metapolytungstates [38], particularly in acidic media.
In addition to the effects of W on hyperthermophilic archaea, there have been two reports on W
and hyperthermophilic bacteria, both of which involve Thermotoga sp. The type strain, T. maritima,
was isolated from a shallow submarine thermal spring
and grows up to 90°C by carbohydrate fermentation
[154]. While the elements V, Mo, Se, Cs, F, Pb, and
Rb did not affect growth of the organism, the addition of W (10/.,M) was reported to increase both the
cell yield and the specific activity of hydrogenase in
cell-free extracts [154]. However, since the purified
hydrogenase did not contain W and no W-containing
protein has been identified in T. maritima, the physiological relevance of this result remains to be elucidated. Indeed, when the related organism Thermotoga sp. strain ZB was examined for ~85W incorporation during studies with methanogens, no incorporation was found (Table 3; [90]). In addition, extracts
of T. maritima cells grown in the presence of W do
not contain detectable AOR or FOR activities
(Mukund, S. and Adams, M.W.W., unpublished
data).
Although not a hyperthermophile, we also include
here the report [155] of an unnamed extremely thermophilic (Toot = 75°C) and acetate-producing anaerobe that was isolated in a complex medium contain-
18
A. Kletzin, M, W. W. Adams / FEMS Microbiology Reviews 18 (1996) 5-63
Table 4
Effects of W and M o on the expression of FDH and C A R in purinolytic and other clostridia and other bacteria
Strain
Element
required
for FDH a
FDH
(U/rag) b
CAR
(U/rag) b
Other W proteins ~
Reference
11.3
8.3
1.8
0.6
3.9
5.7
4. I
5.6
1.7
3.8
7.3
4.7
n.r. d
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
n.r.
+
+
n.r.
n.r.
n.r.
n.r.
n,r.
n.r.
n.r.
n.r.
n.r.
n.r.
[169]
[165,170,172]
[ 166]
[171]
[171]
[171]
[171]
[171]
[171]
[171]
[171]
[171]
2.3
2.7
3.9
4.8
0.4
1.2
2.9 f
< 17.0
1.8
7.6
19.6
0
3.4
8.5 g
n.r.
2.1
4.1
6.2
0.1
1.1
0.4
0.4
0.6
n.r.
0.13
0.02
4,5
n .r.
n.r.
n.r.
n.r.
0.18
0.9
1.6
n.r.
n.r.
n.r.
n.r.
0.3
0.3
0.9
n.r.
n.r.
0.11
0.9
1.0
9
n.r.
[171]
[161 ]
n.r.
[ 1,2,8]
+
n.r.
[171]
[161,167]
n.r.
[3,168]
n.r.
n.r.
[174]
[ 161 ]
n.r.
n.r.
n.r.
[164]
[ 175]
[161]
n.r.
[161]
n.r.
[161]
+
[35]
Purinolytie ciostridia
C. acidiurici
C. cylindrosporum HC-1
C. purinolyticum W A - 1
AAM-I
AAM-2
AC-I
AC-3
MBJ-2
MJ-2
MJ-6
NOA-I
NOA-2
O t h e r ciostridia
C. thermoaceticum
C. formicoaceticum
C. aceticum
C. pasteurianum
C. thermoautotrophicurn
W > Mo
Mo > W
Mo > W
Mo > W
W = Mo
W > Mo
W = Mo
W = Mo
W > Mo
Mo > W
W > Mo
W > Mo
W > Mo
Neither
10/.tM M o ~
10/zM W ~
Neither
100 g,M Mo e
100/xM W e
W > Mo
Neither
10/xM Mo e
10/xM M W e
Neither
100 p,M M M o e
100/zM M W e
W > Mo
Neither
I 0 brM M o e
10/zM W e
Mo > W
Mo
1 /xMMo ~
10/xM Mo e
1 0 / x M W e,h
Other bacteria
Bu~ribacterium
thermoautotrophicum
Eubacterium limosum
Eubacterium acidaminophilum
Neither
0.3
0
1 0 / z M Mo e
10 brM W e
Neither
10/zM Mo ~
10 ~ M W e
Neither
10/zM Mo
0.1 / x M W
0.6
6.0
0.4
1.3
0.1
0.1
0.5
1.1
0
0.06
0
0
0.1
0
0.05
0.6
A. Kletzin, M. W. W. Adams / FEMS Microbiology Ret~iews 18 (1996) 5-63
ing yeast extract, tryptone, and inositol. It is a nonspore-forming and Gram-negative rod which has cellulolytic activity that is absolutely dependent on the
presence of W in the growth medium. Although the
major fermentation product was acetate, it was not
reported if the organism is a homoacetogen. Nevertheless, these tantalizing data suggest that W may be
involved in as yet undiscovered metabolic pathways.
4.3. Gram-positive bacteria
The first definitive evidence for the positive influence of W on the growth of a microorganism was
reported in 1973 and this concerned the acetogenic
Gram-positive bacterium, Clostridium thermoaceticum [1]. It was shown that its growth was
dependent upon W and that the element was incorporated into formate dehydrogenase (FDH, [1]), an
enzyme that was finally purified a decade later [8]. In
1989, a second type of W-containing enzyme, carboxylic acid reductase (CAR), was purified from the
same acetogenic bacterium [156]. In fact, this was
the second type of tungstoenzyme to be isolated
from any source. Even more recently, a second type
of Gram-positive organism, Eubacterium acidaminophilum, was shown to incorporate W into at
least three different proteins [35], suggesting perhaps
additional roles for W in this class of bacterium. The
history of tungstoenzymes in the Gram-positive bacteria therefore parallels that in the methanogens,
where the early discovery of a W-containing enzyme
(in both cases, FDH) prompted many related studies,
but it was only later that a second type of tungstoenzyme was discovered (FMDH in methanogens and
CAR in the acetogens). Consequently, in the following we will attempt to interpret results obtained in
19
the intervening period in light of the presence of at
least two rather than one tungstoenzyme in these
Gram-positive organisms.
4.3. I. Clostridium
Homoacetogenic clostridia are strict anaerobes that
can grow either heterotrophically, by the fermentation of organic substrates with acetate as the sole
product, or autotrophically, on H 2 / C O 2 or other C-l
compounds including CO, formate and methanol (reviewed in [157,158]). Unlike methanogens, acetogens reduce CO 2 to formate rather than to the formyl
derivative, and both autotrophic and fermentative
acetogens ultimately convert formate to acetate as
the end product. The first step in the conversion of
CO 2 to the methyl group of acetate is catalyzed by
FDH. This generates formate, which is subsequently
activated and reduced by a series of enzymes using
tetrahydrofolate as the C-l carrier [157,158]. In the
early 1970s, it was shown that the addition of W, as
well as Se, Mo and Fe, stimulated the growth of the
acetogens C. thermoaceticum [l] and C. formicoaceticum [3]. These elements also increased the
specific activity of FDH in cell-free extracts, and
~85W was incorporated into the enzyme, and into a
second protein with a M r of about 60 kDa ([4];
reviewed in [5-7,159]). It was later shown that FDH
from C. thermoaceticum contained W, Fe and Se
[2,4-8] and the FDH from C. formicoaceticum had
similar properties [3]. These data enabled rationalization of the results from the growth studies (except
for the Mo effect, see below) and also showed the
essential role of FDH in acetogenic metabolism
[ 157-159]. Similarly, the purification of W-containing CAR from C. thermoaceticum in 1989 explained
the double labelling results [156]. Namely, CAR was
Notes to Table 4"
Indicates the concentration of W or Mo, or whether W (W > Mo) or Mo (Mo > W) led to the highest specific activity of FDH, or if were
equally effective (W = Mo).
b Highest FDH and CAR activities observed.
c Indicates whether W-containing proteins other than FDH and CAR were identified.
d n.r., not reported,
The medium also contained 100 /xM Fe and 1 p,M Se.
f Additional l0 /xM Mo gave a specific acitivity of 4.6 U/rag.
Additional 100 /xM Mo gave a specific activity of 8.3 U / m g
n The medium also contained I /xM Mo.
20
A. Kletzin, M. W. W. Adams/FEMS Microbiology Reviews 18 (1996) 5-63
shown to incorporate W when cells were grown on
labelled tungstate and the enzyme corresponded to
the previously identified 60-kDa protein [4].
CAR was identified in acetogens by its ability to
catalyze the reduction of non-activated carboxylic
acids to the corresponding aldehyde using a viologen
dye as the electron donor [156]. The enzyme was
later purified from C. formicoaceticum [160]. However, in contrast to FDH, the physiological role of
CAR is still not known. CAR also catalyzes aldehyde oxidation, which is a much more thermodynamically favorable reaction than acid reduction (see
below). The reactions catalyzed by CAR are therefore the same as those catalyzed by AOR from the
hyperthermophilic archaea [31-33,156,160-162]. In
fact, more recent studies of CAR refer to it as an
aldehyde oxidoreductase, e.g. [161-163], but in the
following we will adhere to the original designation
to distinguish the acetogenic enzyme from similar
enzymes in other organisms.
Acetogens vary considerably in their responses to
the addition of W and Mo (as well as Se) to their
growth media (see Table 4). Part of the reason lies
with the fact that in some species FDH appears to
contain Mo, as in C. pasteurianum [164], C. cylindrosporum [165] and C. purinolyticum [166], while
in others, such as C. thermoaceticum [1] and C.
formicoaceticum [3], FDH activity is dependent upon
W. In the latter two organisms, and presumably in
some other acetogens, the situation is made even
more complicated by the presence of CAR. Although
W-containing CARs have been purified from both
species, C. formicoaceticum has recently been shown
to produce a second distinct CAR and this contains
Mo (see below, [162,167]). Earlier growth studies
[3,168] showed that optimal growth and FDH activity of C. formicoaceticum in a medium containing
fructose and bicarbonate as substrates required the
addition of Se (optimal 0.1 ~M, 1.0 ~M was inhibitory) and W. The cytoplasm accumulated W by a
factor of 100 as compared to the concentration in the
medium (0.1 /zM), and this was only slightly affected by a large excess of Mo (100 /zM, [169]).
Cells grown in the presence of 0.1 # M W and 10
/zM Mo incorporated W into two protein fractions
with M r values corresponding to 88 and 5.5 kDa.
The 88-kDa protein contained 80% of the label and
this corresponded to CAR, while the smaller protein
is presumably a W-processing or storage protein.
Curiously, only minor amounts of W were incorporated into the 25-kDa fraction (corresponding to
FDH), so the stimulatory effect of W on cellular
FDH activity observed in the earlier studies [3,168]
remains unexplained.
A similarly confusing effect of W and Mo
metabolism has been observed in the purinolytic
clostridia, which grow by the fermentation of purines
as their sole carbon and energy sources [166,169172]. For example, it was initially reported that C.
cylindrosporum and C. acidiurici could be differentiated by their W and Mo requirements [165]. C.
acidiurici required W for optimal growth and maximal FDH-specific activity in cell-free extracts while
the same was achieved with C. cylindrosporum only
when Mo was present in the growth medium [165].
However, in a later study, both organisms where
shown to incorporate similar amounts of 185W and in
both of them the radiolabel was in three different
protein fractions: one eluted as a high molecular
mass complex and the second co-eluted with the
FDH activity in both organisms. The extent of labelling of the FDH peak was unaffected by the
presence of Mo in the growth medium [169]. The
third fraction had an M s value of 90 kDa in C.
cylindrosporum and 33 kDa in C. acidiurici. However, it is not known if either of these organisms also
contains CAR. Both species possess a Mo-containing
xanthine dehydrogenase [165,172], but this enzyme
was not labelled with ~85W in either of them [169]
showing that both organisms contain an effective
mechanism for discriminating between Mo and W.
The two organisms do differ, however, in their ability to transport the oxyanions of these elements. C.
cylindrosporum cells accumulated tungstate by a factor of 10-1000-fold depending on the concentration
provided in media (1 nM-10 /zM) and W uptake
was unaffected by the presence of Mo [169]. However, with C. acidiurici, Mo at concentrations above
0.1 /zM decreased the uptake of W to near the
equilibrium point of diffusion when 1 /zM tungstate
was provided, indicating competitive inhibition [ 169].
4.3.2. Eubacterium
Like clostridia, Eubacterium sp. are phylogenetically diverse, Gram-positive bacteria which are defined largely by their metabolic properties, fermenta-
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reviews 18 (1996) 5-63
tion patterns and, in contrast to clostridia, an absence
of endospores. E. acidaminophilum can grow utilizing amino acids such as glycine or serine as the sole
carbon and energy sources, and formate can be used
as an electron acceptor [176]. Both W and Mo have
been shown to stimulate the growth of this organism
on a formate/betaine medium. Compared to a
medium lacking both elements, the addition of 1 nM
W resulted in an approximate doubling of the cell
yield, while at least 10 /xM Mo was required to
observe any effect. The addition of [18~W]tungstate
(1 nM) to the medium resulted in the labelling of
two proteins. One was shown to be FDH, while the
other catalyzed aldehyde oxidation [35]. Bearing in
mind the results with the acetogenic clostridia, this is
presumably a CAR-type enzyme and herein will be
so designated. Interestingly, higher W concentrations
( > 10 nM) resulted in the labelling of two additional
proteins with M,. values of 40 and 5 kDa, but these
were not further investigated. The presence of Mo in
the medium did not affect the ~85W-labelling efficiency. The highest specific activity of FDH observed in cell-free extracts was from cells growing in
the presence of both Mo and W (each 0.1 /zM), and
this was approximately twice that of extracts from
cells grown with either W or Mo. These results
suggest the presence of two different FDHs, one
dependent on W and the other on Mo. In contrast,
the CAR activity (measured by aldehyde oxidation)
appeared to be W-dependent. The highest specific
activity was observed in cells grown with 0.1 /~M
tungstate (higher concentrations decreased the specific activity). This decreased by 70% when molybdate (0.1 /xM) was also present, and CAR activity
was barely detectable in cells grown with molybdate
(10 /.tM) in the absence of tungstate. The amount of
CAR activity in cell-free extracts also depended on
the carbon source. It was highest in cells from a
serine/formate medium and this decreased to 50, 31,
25 and 6% using media containing serine only,
s e r i n e / f o r m a t e / b e t a i n e , formate/betaine, and
glycine only, respectively [35]. Hence, the expression of CAR must be carefully regulated depending
on substrate availability, as well as that of Mo and
W.
The pattern of CAR expression in E. acidaminophilum suggests that this enzyme participates
in amino acid fermentation [35]. Although this is
21
consistent with the proposed role of the analogous
enzyme AOR in the proteolytic hyperthermophiles
[ 145,146], the inability of the acetogenic clostridia to
catabolize amino acids suggests either two very different metabolic functions for these aldehyde-oxidizing enzymes, or that they catalyze a reaction that is
common to several fermentative pathways. We recently arrived at the latter conclusion based on the
properties of AOR in the hyperthermophiles and
CAR in the acetogens; both enzymes were proposed
to function in oxidizing aldehydes that are generated
from 2-keto acids [145,146]. Pyruvate would be the
predominant 2-keto acid in saccharolytic- and acetogenic-based metabolisms, whereas the products of
amino acid oxidation would be source of the 2-keto
acids during growth on peptides or amino acids. It
was also proposed [146] that, in the hyperthermophiles, aldehydes could be generated as side-products of the reaction catalyzed by coenzyme A-dependent 2-keto acid ferredoxin oxidoreductases [177179]. However, further work is obviously required to
substantiate this hypothesis since with the data currently available, it is not possible to unambiguously
define the physiological role of the AOR/CAR enzymes in species of Eubacterium, Clostridium or in
hyperthermophilic archaea (see below).
4.4. Sulfate-reducing bacteria
A systematic survey [30] of various sulfate-reducing bacteria for the effects of W and Mo during
growth on ethanol showed that Desulfovibrio gigas
NCIMB 9332, D. vulgaris strain NCIMB 8303,
Desulfocibrio sp. HDv, Desulfovibrio sp. 20020, D.
baculatus strains HL21, Norway 4 and X, and
Desu!fobulbus propionicus lpr3, all required the addition of either Mo or W to the medium for optimum
growth and in most cases both elements were required. On the other hand, D. gigas DSM 496, D.
fructosovorans J J, D. carbinolicus EDK82, D. alcoholovorans SPSN, D. desulfuricans ATCC 27774
and Desuljbbacterium sp. PM4 were not significantly affected by the presence of either element.
However, the media used in this study contained
yeast extract (0.1%, w/v), which supplies 1.4 nM
Mo and 0.3 nM W [161]. The actual Mo or W
dependence of these organisms is therefore in doubt.
Only one l~SW-incorporation study with sulfate-re-
22
A. Kletzin, M. W,W. Adams/ FEMSMicrobiology Reviews 18 (1996) 5-63
ducing bacteria has been reported and this was with
Desulfovibrio sp. MP47. This organism was used as
a bacterial reference strain in the W-incorporation
study with methanogens (Table 3; [90]). Unfortunately, the results were not conclusive as strain
MP47, while growing with lactate as the carbon
source, incorporated only 6.3% of the added label
(compared to > 70% for many of the methanogens).
It was speculated that the organism might contain a
W-dependent FDH but more recent work with D.
gigas NCIMB 9332 ([30]; see below) suggests that
an aldehyde-oxidizing enzyme was the site of W
incorporation. W was also reported to have a positive
effect on the anaerobic degradation of benzoate by
Desulfosarcina variabilis and Desulfococcus multivorans, but no details were given [180].
Desulfovibrio gigas NCIMB 9332 utilizes H 2,
formate, alcohols or carboxylic acids as energy
sources (for review, see [181]). Its growth was initially shown to be Mo-dependent and a Mo-containing aldehyde oxidase has been purified and extensively characterized, and the sequence of its gene has
been determined ([ 182-186]; reviewed in [ 187]). This
enzyme catalyzes the oxidation of various aldehydes
and the reduction of the redox dye DCPIP, but its
physiological electron carrier and metabolic function
remain unknown. It has also been shown, however,
that during growth on ethanol in the presence of 0.1
/zM tungstate, a second aldehyde-oxidizing enzyme,
termed aldehyde dehydrogenase, is present in cellfree extracts, and this enzyme uses benzyl viologen
(BV) as an electron acceptor [30]. Cell growth was
significant only if at least 1 nM W was added to the
medium, and no growth was observed after two
transfers if W was replaced by Mo. In contrast,
growth on lactate was unaffected by the omission of
either Mo or W from the medium. When D. gigas
cells were grown on lactate in the presence of Mo
(0.12 /zM), only the DCPIP-reducing aldehyde oxidase was detected in cell-free extracts but, surprisingly, if the growth medium contained both Mo and
W (each 0.12 /zM), only the BV-reducing aldehyde
dehydrogenase was present. Cells grown on ethanol
in the presence of Mo contained high aldehyde oxidase activity, but the addition of W caused a dramatic increase in the activity of the BV-reducing
aldehyde dehydrogenase (to twice the specific activity of lactate-grown cells), whereas the aldehyde
oxidase activity was barely detectable [30]. Thus, the
presence of W results in the repression of the Mo-dependent aldehyde oxidase, and expression of the
aldehyde dehydrogenase. This enzyme presumably
converts acetaldehyde to acetate during growth on
ethanol, although its role during growth on lactate is
not clear. Aldehyde dehydrogenase was recently purified from D. gigas and was shown to be a W-containing iron-sulfur protein [188]. As discussed below, the molecular properties of this aldehydeoxidizing enzyme are very similar to those of AOR
from the hyperthermophilic archaea. Indeed, we
speculate that the role of this aldehyde dehydrogenase during growth on lactate might involve the
oxidation of aldehydes generated by pyruvate decarboxylation, as proposed for AOR in the archaea
[145,146].
4.5. Nitrogen-fixing bacteria
The antagonistic effects of W on the ability of a
variety of microorganisms to assimilate N 2 has been
known for many years, and in some cases this has
proved to be a valuable tool for understanding the N 2
fixing process. The best studied in this regard is the
aerobic soil bacterium, Azotobacter vinelandii, which
expresses three different nitrogenase enzyme systems, depending on the metal supply. In addition to
Fe, one contains Mo, one contains V, and the third,
which is synthesized only under conditions of Moand V-limitation, contains only Fe ([189]; reviewed
in [190]). The three nitrogenase systems are encoded
by different structural genes and, to a large extent,
have different genes for regulation and for metal
cofactor synthesis. The first reports on the effects of
W on A. vinelandii appeared in the 1950s when two
research groups independently found that W inhibits
growth when N, and also NO 3, but not NH~-, were
the N sources []91-193]. In fact, from even earlier
studies it was concluded that W had a beneficial
effect on the growth in Mo-deficient media of various Azotobacter spp, as well as on the growth of
Clostridium butyricum [12,195] and species of Anabaena and Nostoc [196]. However, these effects
were later attributed to biologically significant
amounts of Mo that contaminated the W-sources
[197].
More recent studies have now established that W
A. Kletzin, M. IV,W. Adams/FEMS Microbiology Reviews 18 (1996) 5-63
is incorporated by A. t'inelandii into the same enzymes that Mo is, namely nitrate reductase and
nitrogenase [24,192,198]. It was suggested that the
inhibitory effect of W on cell growth occurs at the
level of Mo uptake [199], but it is now clear that
tungstate derepresses the synthesis of both nitrogenase [200-202] and nitrate reductase proteins
[203,204]. While these are assumed to be inactive
apo- or W-substituted proteins, several studies have
indicated the formation of active W [205] or mixed
W-Mo-nitrogenases [206]. The first report of this
type was in 1974 [207] when cells of the photosynthetic bacterium Rhodospirillum rubrum were shown
to grow both in Mo-free and in W-supplemented
media at about half the rate of cells in a Mo-sufficient medium. Furthermore, the addition of W to
Mo-starved cells stimulated the rate of N 2 fixation to
about half that obtained by Mo addition. That the
effects of W were due to contaminating Mo appeared
unlikely as cell-free extracts contained a 200-fold
excess of W over Mo, and the intracellular W-concentration was comparable to the Mo-concentration
in Mo-grown cells. In addition, like Mo-grown cells,
W-grown ceils showed net H 2 production activity,
indicating the presence of an active nitrogenase.
Unfortunately, the nitrogenase from W-grown cells
has not been characterized [207].
W- rather than Mo-dependent growth has also
been reported for a mutant strain of A. vinelandii
(WN 101; [205]). Although whole cell acetylene
reduction, a convenient measure of nitrogenase activity, was less than 10% of that of Mo-grown wild-type
ceils, the activity of the mutant strain was inhibited
by CO to the same extent as the wild-type. Moreover, ethane formation was observed from acetylene,
which is a characteristic of the V- and Fenitrogenases, suggesting one of these enzymes might
be modulated by W in the mutant strain. Other
W-resistant mutants of A. vinelandii are known that
are able to grow in the presence of either W or Mo.
In this case the Mo-nitrogenase is thought to incorporate both elements in a 1:1 ratio [189,206,208],
again suggesting that a W-substituted nitrogenase
can be functional. W-resistant strains can be generated easily by exposing the wild-type strain to media
containing high concentrations of tungstate for prolonged periods [189,206,208]. The growth rates of
the W-resistant strains are directly related to the
23
concentration of tungstate added to the medium over
the range of 0.1-10 mM. However, it is not clear
whether this effect depends on the concentration of
W or can be attributed to Mo impurities in the
tungstate.
Like Azotobacter, the cyanobacterium Nostoc
mucosum requires the addition of Mo to the medium
when grown on N 2 or NO 3 as the sole N source and
W (and also chromium, Cr) inhibit growth: [209].
Mutant strains requiring W (or Cr) for both N 2
fixation and NO~- reduction have been isolated and
the addition of Mo to such strains resulted in :growth
inhibition analogous the effect of W on the Mo-dependent wild-type stain. The presence of both nitrate
and molybdate repress the formation of heterocysts
in the wild-type, but in a Mo-free medium heterocysts develop as in N2-fixing cells. Similar effects
have also been observed with the W- (and Cr-)
dependent strains [209]. Unfortunately, neither nitrogenase nor nitrate reductase has been purified from
the W- (or Cr-) dependent organisms.
Species of Azotobacter have been shown to have
a high affinity for both Mo and W, and the ability to
accumulate high intracellular concentrations
[191,192]. In A. einelandii, Mo accumulation did
not appear to be regulated with nitrogenase synthesis
[210,211]. Accumulated Mo was bound mainly to a
Mo-containing storage protein which could be used
as a supply for nitrogenase synthesis under conditions of Mo depletion. In the presence of tungstate,
the W-analog was synthesized, in addition to inactive
W-containing nitrogenase. The Mo storage protein
has been purified and is a heterotetramer of approx.
100 kDa which binds at least 15 Mo atoms/tool
[2111.
4.6. Pelobacter
Pelobacter acetylenicus is a strictly anaerobic
bacterium which obtains energy for growth by the
oxidation of acetylene, acetoin, ethanolamine and
glycerol with acetate as the end product ([212];
reviewed in [213]). Ethanol and acetaldehyde are
known intermediates of acetylene metabolism. When
the organism was grown with acetylene as sole
carbon and energy source, the cell yields were dependent on the addition of either W or Mo to the
medium. Growth was very slow in media free of
24
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reviews 18 (1996) 5-63
both elements [214], but when either W (0.1 mM) or
Mo (0.1 mM) was added, the growth rate dramatically increased and the bacteria entered a exponential
growth phase without a lag period. When both elements were added at the same time, there was a 7-h
lag phase before exponential growth, but the maximum cell density was two-fold higher than those
obtained when Mo or W were added alone, indicating that both metals are required for optimal growth
[214]. The enzyme responsible for the first step in
acetylene metabolism is acetylene hydratase which
forms acetaldehyde from acetylene and water. The
specific activity of this enzyme in cell-free extracts
was much higher in W-grown cells than in cells
grown with Mo. Moreover, acetylene hydratase was
recently purified from W-grown P. acetylenicus and
was shown to be a W-containing iron-sulfur protein
[214]. This was a very unexpected result, and it
reveals a completely new catalytic function for Wcontaining enzymes, namely, hydration. This is in
stark contrast to the reduction-oxidation reactions
catalyzed by all other tungstoenzymes and, indeed,
by all molybdoenzymes. In fact, prior to this result,
one would have predicted that the W/Mo-dependence of the growth of P. acetylenicus resided with
W- and Mo-isoforms of an aldehyde-oxidizing enzyme which catalyzed the conversion of acetaldehyde to acetate, analogous to the situation in some
acetogenic clostridia and sulfate-reducing bacteria.
The nature of the enzyme that does catalyze acetaldehyde oxidation in P. acetylenicus has not been
reported, but an additional role for W in this organism in acetate production is not unreasonable.
4.7. Aerobic and facultatively aerobic bacteria
Proteus mirabilis is a facultatively anaerobic heterotroph that can use various terminal electron acceptors for anaerobic respiration [215]. Four terminal
reductases are known and each is expressed only
under certain growth conditions. These are nitrate
reductase (NR), chlorate reductase (CR), tetrathionate reductase (TR, which reduces tetrathionate,
trithionate and thiosulfate) and fumarate reductase
(FR, [216]). The synthesis of all four is repressed
during aerobic growth. Under anaerobic conditions,
NR is produced only in the presence of nitrate, while
the three other reductases are expressed simultane-
ously in the absence of nitrate. The addition of W to
the growth medium results in a concentration-dependent decrease of NR activity, and the same was true
for CR and TR, but not for FR, in cells grown
without nitrate [216]. Reactivation of the W-repressed enzymes could be accomplished by the addition of molybdate to cells in the presence of the
translation inhibitor chloramphenicol, suggesting that
the Mo-deficient apoenzymes accumulate in Wgrown cells. Interestingly, in W-containing media,
cells contained more apo-NR when grown in the
absence of nitrate than they did when grown in its
presence, indicating a transcriptional regulatory effect of tungstate on the structural NR genes. Similar
observations have been made for the assimilatory
Mo-containing NR of plants (see below, [204,217219]). P. vulgaris contains another Mo-containing
enzyme, (2R)-hydroxycarboxylate-viologenoxidoreductase (HVOR) [220]. Interestingly, HVOR
is phylogenetically related to the family of W-containing aldehyde oxidoreductases as judged N-terminal amino acid sequence comparisons (see below).
However, no W-containing enzymes have been reported in P. vulgaris.
Methylotrophic bacteria obtain energy for growth
by the aerobic oxidation of C-I compounds and
some of them, termed methanotrophs, are able to
oxidize methane [221,222]. The C-I substrates are
oxidized to formaldehyde, some of which is used for
biosynthesis. The rest is oxidized first to formate and
then to CO 2 via NAD-dependent enzymes for aerobic respiration. FDHs have been purified from several methylotrophs, and, like the FDHs of many
other obligate aerobes [223,224], they do not contain
any prosthetic groups [225,226]. The situation may
be different, however, in the facultative methylotroph Methylobacterium sp. RXM. When this
organism was grown using methanol as the sole
carbon source, the addition of Mo or W to the
growth medium led to a two-fold increase in the cell
yield [227]. In addition, the specific activity of FDH
in the cell-free extract increased five-fold when Mo
was added and 22-fold if W was present, and in the
absence of either element, formate accumulated in
the medium [227]. These results clearly suggest that
Methylobacterium sp. RXM contains either W- and
Mo-dependent FDH isoenzymes or one type of FDH
which can use either element.
A, Kletzin, M.W.W. Adams / FEMS Microbiology Reciews 18 (1996) 5-63
The effects of W and Mo on the growth of this
organism have also been investigated using continuous cultures [228]. Using methanol as the sole carbon
substrate in a nitrogen-limited chemostat, the removal of Mo (4 /zM) from the feed resulted in a
30% increase in the rate of methanol consumption, a
23% decrease in cell density and a decrease in the
carbon conversion factor from 40 to 31%. If either
Mo or W (each 4/~M) were then added to the feed,
all of these parameters reverted to their original
values. These effects of Mo and W are now attributed to a formaldehyde-oxidizing enzyme that is
analogous to the Mo-containing aldehyde oxidase of
D. gigas described above (Girio, F., personal communication). At this point it is not clear if this
organism contains two such aldehyde-oxidizing enzymes, or whether Mo and W are incorporated into
the same enzyme. Based on what is known about the
W and Mo metabolism of some methanogens, acetogens and sulfate-reducers, the presence of two distinct isoenzymes seems more likely. Remarkably,
these preliminary data suggest that this obligately
aerobic Methylobacterium species metabolizes aldehydes and formate in a manner that is identical to
that of some obligately anaerobic acetogens, namely,
by two different Mo- and W-dependent isoforms of
FDH and by two different Mo- and W-dependent
aldehyde-oxidizing isoenzymes.
The effects of W and Mo on the metabolism of
aerobic mycobacteria have also been reported. The
methylotroph Mycobacterium caccae 10 can grow
using methanol as the sole carbon source and under
such conditions two distinct NAD-dependent FDHs
can be induced [229]. If Mo is also present in the
growth medium, the predominant FDH (I) has a M r
value of 440 kDa. FDH I has not been purified, but
is characterized by its resistance to azide in in vitro
assays. If W is added to the growth medium instead
of Mo, another enzyme, FDH II, is preferentially
induced and to very high concentrations such that it
represents 10% of the cytoplasmic protein. FDH I! is
a homodimer of M r 90 kDa, is sensitive to inhibition
by azide, and has no detectable cofactors. Addition
of molybdate to W-grown cells in the presence of
chloramphenicol led to full restoration of the activity
of FDH I, suggesting that the FDH I apoenzyme is
synthesized in the presence of W but that W is not
incorporated [229]. To date, this represents the only
25
example where the function of a Mo-dependent enzyme (FDH I) is taken over by a cofactor-less isoenzyme (FDH II) when cells are grown in the absence
of Mo and presence of W. In fact, studies of the Mo
and W effects on the growth of another mycobacterium, Mycobacterium sp. INAI represent the typical scenario. This organism is able to utilize isonicotinate as a sole source of carbon, nitrogen and
energy. The first step in the degradation pathway is
catalyzed by isonicotinate dehydrogenase, a Mo-containing iron-sulfur flavoprotein. Growth of cells in
the absence of Mo is inhibited by 0.! /~M W,
although it is not known if W is incorporated into the
dehydrogenase [230,23 I]. A similar effect of W was
reported with Arthrobacter oxidans, which obtains
carbon and energy for growth by the aerobic degradation of nicotine. The initial reaction is catalyzed by
the molybdoenzyme, nicotine hydroxylase [232]. In
this case, the organism does not grow in a medium
containing W and Mo at a ratio above 100:1 and in
such cells the activity of nicotine hydroxylase cannot
be detected.
Tungstate has also been shown to inhibit the
growth of the aerobe Pseudomonas putida strain
Ful, This organism was isolated on a medium containing 2-furoic acid and it is also capable of degrading other heterocyclic compounds aerobically. The
addition of W to the medium slowed down the
growth of this organism in a concentration-dependent manner and it was shown that 2-furoyl-CoA
dehydrogenase, which is a molybdoenzyme, was inhibited [233]. P. putida contains another molybdoenzyme, xanthine dehydrogenase, and both it and 2furoyl-CoA dehydrogenase were labelled when cells
were incubated with [185W]tungstate (0.1 /.tM).
However, the labelling also required the presence of
0.01 p~M molybdate (or 0.1 nM molybdate in the
presence of 10 nM tungstate). This requirement indicates a Mo-specific and W-discriminating regulatory
mechanism for the synthesis of both of these molybdoenzymes, whereas the incorporation of W or Mo
into their apoproteins appears to be non-specific
[234].
From the perspective of the W metabolism by an
organism that contains molybdoenzymes but not
tungstoenzymes, the best studied example is Escherichia coli. This organism expresses two major
molybdoenzymes when it is grown anaerobically in a
26
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reviews 18 (1996) 5-63
nitrate-containing medium, FDH and NR. If W is
also present, inactive forms of two enzymes are
produced, but these can be converted to their active
Mo-containing forms by incubating cell suspensions,
but not cell-free extracts, with molybdate and chloramphenicol [235]. Radiolabelling studies with
[99Mo]molybdate have shown that Mo is incorporated into the demolybdo forms of the enzymes in
cells grown in the presence of W, but this does not
occur with cells grown in the presence of Mo
[106,235].
5. Tungsten and eukaryotes
Very few studies have been carried out to examine either the effects of W on the growth of eukaryotes or on the W contents of their cells. Such reports
deal almost exclusively with higher plants or mammals, and the W metabolism of other organisms is
virtually unexplored. The exceptions are the marine
sapropelic ciliate Metopus contortus, which harbors
the endosymbiontic methanogen Methanoplanus endosymbiosus [95], the pioneering studies of W / M o
antagonism in rats and chickens [236], and in nitrate
reduction by the ascomycete Aspergillus niger
([237]; see below). Similarly, we have been unable to
find literature that deals with resistance mechanisms
against the inhibitory effects of W on the molybdoenzymes of plants growing on W-rich soils, nor to
physiological or molecular mechanisms that might
explain the etiology of the W-related 'Hard Metal
Disease' in humans. In the following we present a
brief overview of W metabolism and accumulation
in plants, W toxicity in mammals, and the use of W
salts as substrate analogs for some enzymes of medical importance.
5.1. Plants
The metabolism of W by plants has been studied
almost exclusively with reference to its effect on two
molybdoenzymes of high agricultural significance:
assimilatory nitrate reductase (NR) in the plants
themselves and nitrogenase in symbiotic rhizobia
[13,24,189,200,207,238]. In fact, it is in this context
that some of the earliest, and often most contradictory references to the biological effects of W can be
found. As early as 1938, Arnon [131 reported a
beneficial effect on the yield of asparagus and green
lettuce that were watered with defined liquid medium
containing W, Mo, Ti, V, Cr, Co, and Ni (each at a
concentration of 0.01 ppm). However, which of the
elements that was responsible for this effect was not
investigated. In an another example, Davies [239]
found that the application of a phosphate solution
containing either W or Mo had the same stimulatory
effect on the yields of New Zealand pastures, suggesting a replacement of Mo by W in the N 2 fixation
process. More detailed data are available on the
effects of W on various plants due the inhibitory
effect of W on NR, and these are discussed in a
subsequent section. Here we focus on the W-content
of plants and on the cellular accumulation of W.
Not surprisingly, the total W-content of plants
depends largely on the origin of the soils that they
grow in. One study [240] found that the W contents
of crops and wild plants (corn, rice, pineapple, etc.)
in an area of Central America with no obvious W
source ranged up to 0.04 ppm (dry product), with
coconuts (0.35 ppm) and some banana plants (0.12
ppm) being exceptions. In fact, the total metal content of plants growing on soils in areas of metal
enrichment has been used for geobotanical ore
prospecting for a considerable time [241] and it is
now known that W can be accumulated to very high
amounts. For example, up to 0.15% (W, dry weight)
has been reported in Pinus sibirica, Salix sp., and in
mosses and lichens growing on W-rich fissures
[10,46,242-245]. The W content of a herb (mother
wort) growing in the vicinity of a W-processing
plant was found to decrease from 600 ppm to 50
ppm (dry weight) with increasing distance from the
pollution source [242], and the leaves of the tree
Robinia pseudoacacia contained 58 ppm W (dry
weight) in the vicinity of a light bulb factory [245].
On the other hand, tomato plants discriminate against
W when grown in the presence of W and Mo in
varying ratios [246]. In contrast to some nuclear
fall-out products that have been studied, including
239pu and 2J°Pb, crop plants such as barley readily
take up and accumulate 185W from various soil types
[247], and various plants growing near nuclear cratering tests concentrate very high levels of radioactive
W in their roots [248].
These findings suggest that W-accumulation by
A. Kletzin, M.W.W. Adams / FEMS Microbiology Ret'iews 18 (1996) 5-63
plants does not lead to detrimental effects. This is
supported by studies [249,250] in which various crop
plants were fertilized with sludge wastes that contained up to 186 ppm W. Symptoms of phytotoxicity
were not observed, and the pigs that were fed the
plants accumulated W in the kidneys (see below) but
were otherwise unaffected. In contrast, a much earlier study [11] showed W is toxic to plants at very
low concentrations (5 ppm). Clearly, much is to be
understood about the effects of this element, particularly on agricultural crops in W-rich environments.
The most relevant question is: how do plants that
have accumulated high amounts of W protect against
W inhibition of assimilatory NR? As yet there have
been no reports that address this issue.
5.2. Mammals
Since the 1950s it has been known that W and its
salts are moderately toxic to mammals including
humans. Since the literature on this topic up to 1988
has been summarized [45,251-255], only a short
overview will be presented. Diets containing tungstic
oxide or ammonium tungstate at a concentration of 5
m g / g do not kill rats or rabbits, although their
growth rate is decreased, and higher levels are lethal
[251]. The presence of 5 m g / m l tungstate in the
drinking water of rats over a life time had no effect
on tumor induction, lifetime and body weight [254].
Tungstate has been shown to inhibit the transport of
sulfate, molybdate, sulfite and thiosulfate in the gut
[255] and it inhibits xanthine oxidase in lactating
animals, but the milk yield was unaffected [256]. In
rats, dietary tungstate rapidly decreases the activities
of xanthine dehydrogenase and sulfite oxidase in the
liver, lungs, kidneys and intestine. More than 90% of
radiolabelled tungstate administered with the diet
was excreted within hours, although that which was
accumulated had a half-life of 44 days in the spleen
and about 3 years in the bone [251]. In humans, the
natural dietary intake of W is estimated to be 8-13
/xg/day [45,251,252]. Interestingly, ammonium
tungstoantimoniate (HPA23) reportedly has some
protective effect on tumor induction in mice [45].
The predominant health hazard of W to humans
relates to those in the tungsten carbide industry
(reviewed in [45,251]). The production of so-called
'Hard Metal', an alloy of WC 2 with cobalt (Co),
27
which is used for making cutting, sawing, and grinding tools, leads to high concentrations of fine Hard
Metal dust in the air of the production facilities. This
has been know since the 1940s to induce various
lung and skin diseases, some of which could be fatal
if exposure was not terminated [45,251], as well as
neuropsychological problems [257]. Although these
effects were originally attributed to the cobalt contents in the alloy [251,252], more recent research has
shown that it is the Hard Metal itself rather than Co
or WC 2 that is the major cause of toxicity [258-26 l].
The mechanisms of the disease at the molecular and
cellular levels are not understood, although reactive
oxygen species have been excluded as possible mediators [260].
Tungstate has also been used as a substrate analog
for phosphodiesterases, enzymes which have significant medical implications. The action of insulin,
growth factors, and oncogenes involves phosphotyrosine protein kinase activity, and phosphotyrosine protein phosphatases, which inactivate the kinases and
oppose their action, play an important role in these
biological processes. Tungstate and other transition
metal oxyanions such as vanadate, chromate and
molybdate, have been investigated for their inhibitory effects on various phosphatases and related
enzymes [262-264]. Although the inhibition constants depended on reaction conditions and particularly pH, tungstate was the most efficient competitive inhibitor for both acid and alkali phosphatases
but not with an aryl phosphatase. As tetrahedral
species in solution, these group Via dianions are
capable of accepting two additional ligands such as
an active site serine hydroxyl and a water molecule
to form octahedral structures. Such structures can be
viewed as analogs of a pentacoordinate transition
state of the phosphatases [265]. The K~ values of the
group six oxyanions decrease with the atomic number, an observation which can be explained by the
fact that the formation of octahedral isopolytungstates in water is more favorable than the formation of isopolymolybdates [38], and such structures
are unknown for chromates in aqueous solution. The
assumption that tungstate replaces the true substrate
pyrophosphate in the active site of phosphatases was
confirmed very recently with the phosphotyrosine
protein phosphatase of Yersinia pestis, a pathogenic
bacterium that enters and suppresses host immune
A. Kletzin, M. W. 14,"Adams / FEMS Microbiology Reviews 18 (1996) 5-63
28
cells [266]. Co-crystallization of the protein with
tungstate gave crystals where tungstate occupied the
catalytic site in a manner expected for pyrophosphate
[266]. Binding of tungstate to the phosphate-binding
loop triggered a protein conformational change which
trapped the oxyanion and caused the important catalytic residue A s p 356 to move by about 6 A into the
active site. The same anion-binding loop is also
found in the enzyme rhodanese (thiosulfate sulfur
transferase [267]). Tungstate may therefore be useful
in rationalizing the mechanism of action of a variety
of enzymes that use polyanionic substrates.
6. Tungstoenzymes
Four distinct types of tungstoenzyme have been
purified from various microbial sources. These are
formate dehydrogenase (FDH), formylmethanofuran
dehydrogenase (FMDH), acetylene hydratase (AH),
and aldehyde-oxidizing enzymes such as carboxylic
reductase (CAR) and aldehyde ferredoxin oxidoreductase (AOR). FDH has been purified from a species
of Clostridium [1,2,8] and partially purified from
Methanococcus vannelii [89] and Eubacterium acidaminophilum [35], while W-containing FMDHs
have been obtained only from some thermophilic
methanogens [27-29,99]. Both FDH and FMDH are
specific for CO 2 as a substrate. Members of the
AOR/CAR family catalyze the oxidation of various
types of aldehyde, and they include AOR [31,32,145],
formaldehyde ferredoxin oxidoreductase (FOR;
[33,144]) and glyceraldehyde-3-phosphate dehydrogenase (GAPOR [34]) from the hyperthermophilic
archaea, and CAR [156,160] from mesophilic and
moderately thermophilic clostridia. In this group, we
also include the so-called aldehyde dehydrogenase
(ADH) of the sulfate-reducer Desulfovibrio gigas
[188]. The fourth type of tungstoenzyme known, AH
of Pelobacter acetylenicus, is the most recent to be
Table 5
Origins and reactions catalyzed by purified tungstoenzymes
Enzyme
Organism
Substrate/
product
Physiological
electron
carrier
Mo-isoenzyme a
Reference
FDH
FDH
FMDH
Clostridium thermoaceticum
Eubacterium acidaminophilum
Methanobacterium wolfei
NADPH
NADPH
Unknown
Related
Related
Yes
[ 1,2,8]
[35]
[27,99,102]
FMDH
CAR
CAR
AOR
AOR
AOR
FOR
FOR
ADH
GAPOR
AH
Methanobacterium thermoautotrophicum
C. thermoaceticum
C. formicoaceticum
Pyrococcusfuriosus
Thermococcussp. ES-1
Pyrococcus sp. ES-4
T. litoralis
P. furiosus
Desulfovibrio gigas
Pyrococcusfuriosus
Pelobacterace~.'lenicus
CO 2 + [2H]/HCOOH
b
CO 2 + MFR ~"+ [2H]/
CHO-MFR ~
~
RCOOH d / R C H O + 2[H]
b
RCHO a / R C O O H + 2[H]
b
b
~g
bg
b
GAP h / 3 P G + 2[H]
C2H 2 + H 2 0 / H 3 C - C H O
Unknown
Unknown
Unknown
Fd c
Fd
Fd
Fd
Fd
Unknown
Fd
None
Yes
Related
Yes
No
No
No
No
No
Yes
No
(Yes)
[28,29]
[ 156,161,163,273]
[ 160,167]
[31,32]
[145]
f
[33]
[ 144]
[ 188]
[34]
[214]
Indicates whether a Mo-containing isoenzyme is known in: yes, the same species; related, a related species; (yes), in the same species but
has yet to be characterized; no, is not present in the same or a related species.
b The reaction is the same as for the other members of this enzyme type.
c MFR and CHO-MFR represent methanofuran and N-formyl methanofuran, respectively.
d R includes a wide range of both aliphatic and aromatic groups.
e Fd, ferredoxin.
f Mukund, S. and Adams, M.W.W., unpublished data.
g R is limited to C1-C3 groups.
h Glyceraldehyde 3-phosphate (GAP), which is hydrolyzed to 3-phosphoglycerate (3PG), is the only known substrate.
A. Kletzin, M. W. W. Adams / FEMS Microbiology Ret'iews 18 (1996) 5-63
discovered and also the least studied [214]. So far, it
is the only tungstoenzyme that catalyzes a hydration
reaction rather than one involving reduction-oxidation. All of the others catalyze a two electron transfer
reaction and at a redox potential that is very low for
a biological system. That is, all are equivalent to or
more negative than the hydrogen electrode ( E ° ' =
- 4 2 0 mV. pH 7.0, 25°C). The values at 25°C and
pH 7.0 for the carboxylic acid/aldehyde, CO2/formylmethanofuran, and CO2/formate couples are
- 5 8 0 mV, - 4 9 7 mV ( - 5 3 0 mV at 60°C, [268])
and - 4 3 2 mV, respectively [269,270].
Table 5 gives an overview of the four types of
tungstoenzyme known, the organisms they have been
purified from, and the reaction that they catalyze.
Most of the tungstoenzymes have analogous Mocontaining counterparts in the same or in a closely
related organism, with the hyperthermophilic enzymes being the notable exception. The best characterized tungstoenzyme is the AOR of the hyperthermophile, Pvrococcus furiosus [31,32]. Its gene has
been sequenced [271] and the crystal structure of the
enzyme has been determined [272]. The structure of
the enzyme also provided the first structure of the
pterin cofactor which binds the tungsten atom. Pterin
cofactors are found in all Mo-containing enzymes
(with the exception of nitrogenase) and, although
structurally uncharacterized in a molybdoenzyme, it
is termed molybdopterin, or MPT [15-18]. In the
following, to avoid any confusion invoked by referring to molybdopterin in a tungstoenzyme (which
lacks Mo), we will use the term pterin cofactor when
referring to the pterin moiety in both types of enzyme.
6.1. Formate dehydrogenase
Formate is a ubiquitous metabolite in virtually all
life forms and in most cases its production or consumption involves the enzyme formate dehydrogenase (reviewed in [6,7,157-159,225,226]. FDH catalyzes the reversible two-electron oxidation of formate according to Eq. (3).
CO 2 + 2 H + + 2 e - ~ H C O O -
(E"'=-432mV)
(3)
29
Although there are some exceptions [274,343], the
FDHs that have been purified from aerobic microorganisms do not usually contain metals or other cofactors (see Table 5; [225,226]). NAD(H) is the usual
physiological electron carrier for these enzymes and
in most cases they function to oxidize formate. In
contrast, the FDHs of anaerobes catalyze the first
step in CO 2 assimilation, although in some
methanogens, such as Methanobacterium formicicum, FDH oxidizes formate in a formate hydrogen
lyase system [275]. More importantly, and in contrast
to most FDHs from aerobes, FDHs from anaerobes
contain metal cofactors. For example, as early as
1954 it was recognized that Mo is an integral part of
the FDH of E. coli and related organisms [276].
Virtually all FDHs of anaerobes that have been
characterized since then have been shown to be
Mo-containing iron-sulfur proteins, and some also
contain flavin, heine or Se (Table 5). There are two
notable exceptions. First, the FDHs that contain W
instead of Mo, and second, the FDH of the hyperthermophilic archaeon Thermococcus sp. ES-I. This
was recently found to lack both Mo and W, although
it did contain iron-sulfur clusters and an unusual
flavin group (Heider, J., and Adams, M.W.W., unpublished data).
Growth studies have indicated that W-containing
FDHs are present in some acetogenic clostridia, including C. thermoaceticum [1,5], C. fi~rmicoaceticum
[3,5] and C. acidiurici [165,169], although not in
others, such as C. cylindrisporum [165,169] and C.
pasteurianum [ 164,175]. Only the W-containing FDH
from C. thermoaceticum has been extensively characterized, in fact, it was the first tungstoenzyme to
be purified [1,2,4,8]. This enzyme is extremely oxygen-sensitive and uses NADP as the electron acceptot. The apparent K m values for NADP and formate
are 117 and 109 /xM, respectively, and the reaction
was shown to be fully reversible [8]. C. thermoaceticum FDH has a c~2/32 subunit structure and
contains 2 W and 2 Se per holoenzyme [811 Radiolabelling with 75Se and electrophoretic analyses indicated that Se is present as selenocysteine and that
this is located in the c~-subunit. Fluorescence analysis of material extracted from acid-denatured FDH
showed that a pterin-type cofactor was present, similar to that found in molybdoenzymes (see below),
suggesting that W was present as a monotmclear site
30
A. Kletzin, M. W. W. A d a m s / F E M S Microbiology Reviews 18 (1996) 5-63
[8]. By analogy with Mo, such a site would be
expected to be W(VI) and W(IV) in the oxidized and
reduced states of the enzyme, respectively. Only the
intermediate W(V) state has an unpaired electron,
and only this state would be detected by electron
paramagnetic resonance (EPR) spectroscopy. A W(V)
EPR signal was not observed in dithionite-reduced
FDH [277,278], but when samples were poised at an
intermediate potential, a weak EPR signal (0.07
spin/mol) with g values of 2.101, 1.980 and 1.950,
was observed at temperatures up to 200 K. This was
proposed to originate from a small amount of the
W(V) form of the enzyme [277,278]. Hence, the
enzyme as purified in a dithionite-reduced state must
contain a reduced W(IV) center.
The nature of the W site in FDH was 91so examined by X-ray absorption spectroscopy. This tech-
nique gives information on nature, number and distance of ligands to the W atom. EXAFS (extended
X-ray absorption fine structure) of the dithionite-reduced enzyme indicated a coordination sphere containing at least 2 W-O or W-N at 2.13 ,~, and at least
2 W-S at 2.39 ,~ [279]. Inclusion of a W-Se interaction at 2.6 ,~ improved the fit analysis, but there was
no evidence for terminal oxo ( W = O ) bonds, in
contrast to what was reported with the Mo-containing FDH from C. pasteurianum [279]. Thus, for the
W-containing FDH, selenocysteine is thought to be
one of the W ligands, together with two S that are
assumed to be provided by the dithiolene group of
the pterin (see below), together with at least 2 WO / N bonds. As discussed below, this W site differs
from that in the hyperthermophilic tungstoenzyme,
AOR, as this has four S ligands provided by the
Table 6
Properties of formate dehydrogenases from various sources ~
Organism
Growth
conditions
Clostridium thermoaceticum
Anaerobic
Eubacterium acidaminophilum
Holoenzyme
Mr
Subunits
( M r, kDa) b
K m for
formate
(raM)
Cofactors
(mol/mol
native)
Electron
acceptor
References
340
a 2 (96)
/32 (76)
0.2
NADP
[1,2,4,8,277,278]
Anaerobic
155
c~2 (95)
0.16
NADP
[35]
Clostridium pasteurianum
Anaerobic
118
1.7
Ferredoxin
[164,175,283]
Methanobacterium formicicum
Anaerobic
177
a
/3
a
/3
F420
[285,286]
Escherichia coli (FDHN)
Anaerobic
590
W-pterin
2W, 2Se
20-40 Fe, (S) "
W-pterin
23-28 Fe, (W, S)
Mo-pterin, 2 Mo
24 Fe, 28 S
Mo-pterin
1 Mo, 2 Zn, 1 FAD
21-24 Fe, 25-29 S
Mo-pterin
4 cytochrorne b, 4 Mo
4 Se, 56 Fe, 52 S
Mo-pterin, 2 Mo,
19Fe, 18S
(Flavin d, Fe, S)
Quinone
[287]
Quinone
[288]
(kDa)
(76)
(34)
(85)
(53)
O~4 (l 10)
0.6
0.03
/34 (32)
e4 (20)
Wolinella succinogenes
Anaerobic
Thermococcus sp. ES- 1
Anaerobic
Pseudomonas oxalaticus
263
a 2 (110)
1.5
Aerobic
315
13.5
1 FAD, (Fe, S)
NAD
[274]
Achromobacter parvulus
Aerobic
80
a (85)
/3 (53)
cH (100)
/32 (59)
a 2 (46)
15
None
NAO
[223]
Candida rnethanolica
Aerobic
82
c~2 (43)
3.0
None
NAD
[224]
Compiled from Ref. [226].
b The M r values for the individual subunits are indicated.
c Parentheses indicate that the particular moiety has not been accurately quantitated.
d An unknown deazaflavin-type group has been identified.
e Heider, J. and Adams, M.W.W., unpublished data.
Ferredoxin
31
A. Kletzin, M. W. W. Adams / FEMS Microbiology Ret'iews 18 (1996) 5-63
dithiolene groups of two pterin molecules [272].
Interestingly, the chemistry of the W site of FDH can
be mimicked with W-containing model compounds.
The complex [w~Vo(s2c2(CN)z)2] 2
has been
shown to reduce CO 2 or HCO- to formate and
[wVlO(SzC2(CN)2)2] 2- [280]. However, in contrast
to W in FDH, the complex also reacts with molybdate in a displacement reaction which yields the
homologous Mo compound [281].
The nature of the iron-sulfur clusters in the FDH
of C. thermoaceticum are less well defined. Chemical analyses indicate that it contains between 20 and
40 Fe atoms per molecule [8,277,278]. An EPR
study of a sample poised at intermediate redox state
showed the presence of both [2Fe-2S] and [4Fe-4S]
clusters (for a review on iron-sulfur clusters in
proteins see. for example [282]). Spin quantitations
indicated that the enzyme contained two types of
each cluster, but this accounts for only 12 Fe
atoms/tool [277,278]. The nature of the remaining
Fe is not clear but presumably much, if not all, of it
is present at additional 2Fe- and 4Fe-clusters that
have very low redox potentials such that they are not
reduced, and therefore not observable by EPR spectroscopy, under the conditions used so far.
W-containing FDHs have been partially characterized from two other organisms, C. formicoaceticum
and Eubacterium acidaminophilum. Growing C.
fi)rmicoaceticum cells incorporate 185W, even in the
presence of a 10000 fold excess of Mo, into FDH
(subunit M,, 88 kDa) and into an as yet uncharacterized protein of M r 5.5 kDa. The FDH is extremely
oxygen-sensitive but readily loses activity even under anaerobic conditions [3,168]. The FDH of E.
acidaminophilum is similarly sensitive to oxygen
and this has been obtained in a partially purified
form. It is a homodimeric protein with a subunit M r
of 95 kDa and contains 23-28 Fe and 6W atoms/mol
(but less than 0.6 Mo atoms/mol). The enzyme had
apparent K,,, values for formate and NADP of 160
and 65 ~M, respectively, and was sensitive to inhibition by cyanide. Spectroscopic analyses of this FDH
have not been reported [35].
The analogous Mo-containing FDHs that are present in C. thermoaceticum and C. fi~rmicoaceticum
have not been well studied, due mainly to their
extreme oxygen sensitivity. The only FDH to be
characterized from a related species is the Mo-con-
taining FDH from C. pasteurianum [164,175,283],
The molecular properties of this enzyme, such as
subunit size and metal content, are very similar to
those of the W-containing FDH from C. thermoaceticum (see Table 6). The methanogen Mc.
~'annielii harbors two different Mo-dependent FDHs.
In Mo-grown cells, one is a 108-kDa protein which
contains Mo, Fe and S while the other is a high
molecular mass complex with the same cofactors. In
W-grown cells, the latter FDH is the predominant
form, but it contains W rather than Mo. Both the
Mo- and W-forms of the enzyme utilize 8-hydroxy5-deazaflavin as the physiological electron carrier;
they do not reduce NAD(P). However, an additional
protein, a 8-hydroxy-5-deazaflavin NADPH 0xidoreductase, is also necessary for formate oxidation in
vivo ([87-89]; reviewed in [284]). The Mo-containing FDH from another methanogen, Mb. fi)rmicicum,
has been well studied and some of its properties are
also listed in Table 6.
The W-containing FDH of C. thermoaceticum
therefore remains the best characterized of this enzyme type, in spite of the fact that all of the analyses
on it were carried out more than a decade ago.
Unfortunately, the genes for the subunits of the
enzyme have yet to be cloned, and N-terminal sequences for the subunits have not been reported.
Direct comparisons with the other tungstoenzymes
are therefore not possible. Hopefully, the results
from additional studies of the C. thermooceticum
enzyme will be reported in the future.
6.2. Formylmetham?furan dehydrogenases
The
reversible
formation
of
N-formylmethanofuran from CO 2 and methanofuran
(MFR) catalyzed by FMDH is the first step in CO 2
utilization by methanogens (Eq. (4)):
CO 2 + MFR ++ H + + 2 e - ~ C H O - MFR + H 2 0
( E" = - 497 mV)
(4)
Although the reaction was known in principal for
many years, it is only recently that the enzyme
systems involved have been characterized [101].
FMDHs, like FDHs, are specific for CO2 as the
carbon substrate. While NADP is the physiological
electron carrier for FDH, that of FMDH is not
A. Kletzin, M. W. W. Adams / FEMS Microbiology Retqews 18 (1996) 5-63
32
known, and there is evidence for the presence of an
as yet unknown low-molecular mass electron carrier
[268]. FMDH is found only in methanogens, with the
notable exception of the hyperthermophilic sulfate
reducer, Archaeoglobusfulgidus ([148]; reviewed in
[149]). During methanogenesis, the formyl group
generated by FMDH is sequentially reduced to
methane by a complex series of enzymes and cofactors most of which are unique to these organisms
(for reviews, see [85,284,289-293]). FMDH was
found to be a molybdoenzyme in the first
methanogens that were examined [101,294-297], but
two W-containing FMDHs have recently been purified. These are from the moderate thermophiles,
Methanobacterium wolfei [99] and Mb. thermoautotrophicum (Marburg) [28], both of which grow
optimally near 60°C. However, as discussed above,
considering the large number of methanogenic
species that exhibit W-dependent growth and incorporation of W (see Table 3), a much wider distribution of W-containing FMDHs seems likely.
Remarkably, both Mb. wolfeii and Mb. thermoautotrophicum contain two FMDHs: one (FMDH
II) contains W [28,29,99,102] while the other (FMDH
I) contains Mo [28,29,102,268]. The two organisms
differ, however, in their responses to the addition of
W and Mo to the growth medium. When Mb. wolfei
is grown in the presence of Mo, it expresses exclusively Mo-containing FMDH I, while in the presence
of W, both FMDH I and FMDH II are expressed and
both contain W [27]. Exactly the opposite is observed with Mb. thermoautotrophicum. Cells grown
with Mo contain two distinct FMDH activities while
in W-grown cells only the W-containing FMDH II is
expressed [28]. The catalytically active, Mo-substituted (W-)FMDH II isoenzyme from Mb. thermoautotrophicum and the W-substituted (Mo)FMDH I isoenzyme from Mb. wolfei have both
been purified and characterized separately [27,29].
These are the only examples known so far where Mo
and W can be interchanged to yield active enzymes.
The molecular and catalytic properties of these various forms of FMDHs I and II are summarized in
Table 7.
In Mb. wolfei, Mo-FMDH I and W-FMDH II
have the same subunit composition and the subunits
Table 7
Structural and catalytic properties of W- and Mo-containing FMDHs
Property
Methanobacterium wolfei a
Mb. thermoautotrophicum b
Ms. barkeri
W
Mo
W-subst.
W
Mo
Mo-subst.
Mo
HoloenzymeMr(kDa)
SubunitMr(kDa)
130
64
51
35
130
64
51
31
130
64
51
31
160
65
53
31
15
110
65
45
35
n.r.
200
65
5O
37
34
29
17
Fe (S) content/mol
Vma~ ( U / r a g )
Topt (oC)
pHop t
K m (MFR, /.zM)
K,~ (formate, mM)
Pterin type d
Inactivation by cyanide ~
EPR signal at 77 K f
Reference
2-5
11
65
7.4
13
inactive
MGD
r
[27,29,102]
7
37
70
7.9
13
35
MGD
s
+
[29,102]
n.r.
27
70
7.4
13
1100
n.r.
n.r.
+
[27,29]
8 (8)
15
80
8.0
30
n.r.
MGD
r
[28,29]
4 (4)
70
70
8.0
30
n.r.
M(G/A/H)D
s
+
[29,100,101,268,294,295]
n.r. ¢
n.r.
n.r.
n.r.
30
n.r.
n.r.
s
n.r.
[28,29]
n.r.
The properties of the W-substituted form of Mo-FMDH are also given.
b The properties of the Mo-substituted form of W-FMDH are also given.
n.r,, not reported.
a The abbreviations for the dinucleotide forms of molybdopterin are: G, guanine; A, adenine; H, hypoxanthine.
e Indicates whether the enzyme is sensitive (s) or resistant (r).
f Indicates whether an EPR signal from W(V) or Mo(V) is observed ( + ) or not ( - ) .
28 (28)
175
n.r.
n.r.
0.02
1700
MGD
s
+
[29,101,103,294- 297]
A. Kletzin, M.W.W. Adams/FEMS Microbiology Rel:iews 18 (1996) 5-63
are of comparable size, while in Mb. thermoautotrophicum the W-isoenzyme has an additional small
subunit (Table 7). Between the two organisms, the
N-terminal amino acid sequences of the large subunits (M r approx. 65 kDa) of all four isoenzymes are
virtually identical (Fig. 3). The same is true for the
next largest subunit (M r approx. 50 kDa), with the
exception of Mo-FMDH I of Mb. thermoautotrophicum, which shows almost no similarity with its
counterparts. Excluding this subunit, the two larger
subunits of the Mo and W isoenzymes from both
organisms also show a high degree of similarity to
the Mo-containing FMDH of the mesophile
Methanosarcina barkeri [29,101,295-297]. However, note that the mesophilic enzyme contains six
subunits (Table 7). The relationships between the
smaller subunits in the W- and Mo-containing enzymes are less obvious. Three N-terminal sequences
are known for the 31-35-kDa subunit of the Mo-isoenzymes, but these show no similarity to each other,
and attempts to sequence the equivalent subunit from
the W-isoenzymes were not successful. On the other
hand, the N-terminal sequence of the smallest (I 5
kDa) of the four subunits in W-FMDH II from Mb.
thermoautotrophicum does show some similarity to
the smallest (17 kDa) of the six subunits present in
Mo-FMDH from Ms barkeri (17 kDa, [29]).
In spite of their physical similarities, the W- and
Mo-isoenzymes of FMDH do differ in their chromatographic behavior and in their catalytic properties [29]. Mo-FMDH I of Mb. wolfei, but not W-
33
FMDH II, uses N-furfurylformamide and formate as
pseudosubstrates, and they also differ in their specific activities in oxidizing N-formylmethanofuran
and in their affinities for the artificial electron acceptor, methyl viologen (used in the routine assay of
these enzymes). They also differ in their pH and
inhibition properties for N-formylmethanofuran oxidation (Table 7). Interestingly, the W-substituted
form of (Mo-)FMDH I also uses N-furfurylformamide as a substrate, albeit with lower activity and
lower affinity. On the other hand, W-substituted
(Mo-) FMDH I has a pH optimum more similar to
that of FMDH II rather than Mo-FMDH 1. In addition, all of the Mo-containing forms, which include
Mo-FMDH I enzymes from Mb. wo![eii and Mb.
thermoautotrophicum, the Mo-FMDH from Ms.
barkeri [101] and the Mo-substituted (W-) FMDH II
from Mb. thermoautotrophicum, are reversibly inactivated by cyanide [29]. In contrast, the W-containing
FMDH II enzymes from both of the Methanobacterium species are resistant to cyanide.
The extent to which the W- and Mo-containing
isoenzyme forms of FMDH differ in the nature of
their redox-active cofactors is unclear at present. In
their purified forms, FMDHs I and II typically contain less than 0.5 atoms of W (or M o ) / m o l and less
than 0.01 atoms of Mo (or W)/mol. However, these
enzymes are to a greater or lesser extent oxygen-sensitive and typically lose substantial amounts of activity during purification. For example, the recovery of
activity of W-FMDH II from Mb. thermoautotroph-
otm,~n sax~ lst,n~ym~ ~
~Da)
/~b.
/49.
/~2.
/vb.
Ms.
th.
th.
w.
w.
b.
65
65
64
64
65
W
M3
W
Mo
bid
/~9.
Mb.
~.
/v~.
th.
w.
w.
b.
53
51
51
50
W
W
14o
M3
~.
. MEYI
IKNGFVY
. PLN(N)VDGE
. MD(I)
. M R Y I I K N G F V Y
. PLN(N)
. MEYI
IKNGFVY?PLNGVDGE?M(D)
. MEYI
IKNGFVY
. P L N G V D G E
. MDI(I)V
(AG)TIAVKNGYVFDPLNEI(N)?
? IM(D)
.(M)EYV.
. MEYV
. MEYV
. MIY?
KN
. KN
. KN
. KN
.
.
.
.
.
.
.
.
VV.
PFGTLD
VV
. PFGTLDDI
VV
. PFGTLDDI
IV.
PVGA(A)
.(I I)
I(F)KV
I{I)KVEGN
th.
45
Mo
?(S)F V L V P ( T S D ) F Q I G L E A(D)
/vb. th.
bls. b.
Ms. b.
35
34
37
l~b
14D
Mo
M K F G I E F V P N E
P I E K I V K L V K L A E
. MEGVLINENE(F)VESLAVI
PG
MQEDM.
VLSVFAEKKD(K)QLIYSPEE
/~9. t2~.
Ms. b.
15
17
W
MD
?(M)VILNTG
. TI
. QGQAI
E(S)G
MEVLL
I S G S T I N E G ?
LARGG
Fig. 3. N-terminal amino acid sequences of FMDHs, Methanobacterium thermoautotrophicum, Mb. wolfei and Methanosarcina barkeri
FMDH N-terminal amino acid sequences aligned by subunit size and amino acid similarity. Residues conserved between the 64/65- and
51-53-kDa subunits are shown in boldface (upper two panels), as are the residues conserved between the 34/35-kDa and the smaller
subunits of Ms. barkeri and Mb. thermoautotrophicum (modified from [29]).
34
A. Kletzin, M. W. W. Adams / FEMS Microbiology Retqews 18 (1996) 5-63
icum after purification was less than 1% [28]. Presumably, at least part of the activity decrease is due
to loss of W (or Mo). In addition, FMDH I and II
appear not to contain the same number of iron-sulfur
clusters, although this differs in the two organisms.
Thus, purified F M D H I from Mb. thermoautotrophicum contains half the amount of Fe of FMDH II,
whereas in the enzymes from Mb. wolfei the situation is reversed (Table 7). Moreover, the Fe content
of all four of these enzymes is about 4-fold less than
that in the M o - F M D H of Ms. barkerii. Notably,
W - F M D H II from Mb. thermoautotrophicum contains both 4 more Fe a t o m s / m o l and an additional
small subunit (15 kDa) compared to Mo-FMDH I
(Table 7), and a similar subunit is found in Ms.
barkeri FMDH. Therefore, perhaps this subunit contains one or more ferredoxin-like iron-sulfur centers.
On the other hand, M o - F M D H I of Mb. wolfei has a
similar Fe content to W - F M D H II from Mb. thermoautotrophicum but it lacks this smaller subunit.
Both the Mo- and W-forms of FMDH from the
thermophilic methanogens contains the guanine dinucleotide form of the pterin cofactor (see below),
although preparations of M o - F M D H I of M. thermoautotrophicum also contains small amounts of
other dinucleotide forms (Table 7).
Accepting some loss of metals during purification, the W and Mo-containing FMDHs from Mb.
wolfei and Mb. thermoautotrophicum are predicted
to contain a mononuclear W or Mo center and one or
two iron-sulfur clusters per holoenzyme. This is
confirmed to a large extent by spectroscopic studies.
For example, the reduced forms of the M o - F M D H
enzymes from Mb. wolfei, Mb. thermoautotrophicum and Ms barkeri all exhibit an EPR spectrum at
77 K accounting for 0.05-0.1 spins/tool. That this
originated from a S = 1 / 2 Mo(V) species was confirmed by the observation of hyperfine splitting in
the spectrum of the Mb. wolfei enzyme enriched in
97Mo (I = 5 / 2 ) [29]. The three enzymes gave similar rhombic-type signals with gz,y,× values of 2.004,
1.984 and 1.951, and the signals were lost when the
enzymes were exposed to air. Reduced M o - F M D H I
from Mb. wolfei also gave rise to EPR signals that
were assigned to two [2Fe-2S] clusters and one or
more [4Fe-4S] clusters [102]. In contrast to the Mocontaining enzymes, the W - F M D H II enzymes from
Mb. wolfei and Mb. thermoautotrophicum were
2.1
2.0
B
--
VALUE
Fig. 4. EPR spectra of W-substituted (Mo-) FMDH I from Mb.
wol]ei. The samples were: A, air-oxidized; B, computer simulation
of experimental EPR spectrum; and C, the difference spectrum.
The reduced enzyme exhibited at 55 K an isotropic signal (g =
2.003) and a second signal with g values of 1.925 and 1.875
which was attributed to a reduced [4Fe-4S] cluster. The latter
signals disappeared upon air oxidation and were replaced with
spectrum A (gzyx = 2.0488, 2.0122, and 1.9635;spin quantitation,
0.21 spin/tool of enzyme and 0.63 spin/tool of W). Spectrum B
is the result of a computer simulation of the experimental rhombic
spectrum and depicts the sum of a S = 1/2 signal without hyperfine interaction and the same signal interacting with a nuclear spin
of I = 1/2 according to the natural abundance (14.4%) of 183W.
Taken from [27].
EPR-silent at 77 K in their as purified reduced forms
[29]. However, the W-substituted (Mo-)FMDH I enzyme of Mb. wolfei exhibited, upon air oxidation, a
complex rhombic-type signal with gz,y,x values of
2.049, 2.012 and 1.963 (Fig. 4; [27]). This spectrum
represented over 60% of the W in the enzyme (0.3
a t o m s / m o l ) and it was assigned to a S --- l / 2 W(V)
site, the complexity being due to hyperfine interactions from the presence of the nuclear spin isomer
183W ( I = 1/2; see Fig. 4). However, one assumes
that exposure to oxygen also inactivated the enzyme
so the observed W(V) EPR signal is probably from
an inactive form of the catalytic site. The reduced
enzyme gave rise to an EPR signal that was assigned
to a reduced [4Fe-4S] cluster, although it was observed at 55 K which is unusual for a cluster of this
type.
These preliminary results therefore suggest that,
by analogy with Mo-containing enzymes, the W site
in W-substituted (Mo-) FMDH I is in the reduced
(IV) state as the enzyme is purified, and that this is
oxidized predominantly to the W(V) state by expo-
35
A. Kletzin, M. W. W. Adams/FEMS Microbiology Ret,iews 18 (1996)5-63
sure to oxygen. This is p r e s u m a b l y true for the
W - F M D H II e n z y m e s , and, as described above, for
the W - c o n t a i n i n g F D H from C. thermoaceticum. O n
the other hand, M o - F M D H e n z y m e s as purified contain some Mo(V), Clearly, in addition to the nature
o f their i r o n - s u l f u r clusters, m u c h r e m a i n s to be
understood about the differences b e t w e e n the W - and
M o - c o n t a i n i n g sites in the various F M D H e n z y m e s .
As yet they have not b e e n subjected to potentiometric analysis ( u n d e r anaerobic conditions to m a i n t a i n
e n z y m e activity), nor have they b e e n e x a m i n e d by
X-ray absorption spectroscopy to determine how the
coordinating ligands compare in the two types o f
catalytic site. In addition, the complete a m i n o acid
sequences of the e n z y m e s should provide important
clues as to the overall structural similarity b e t w e e n
these W - and M o - c o n t a i n i n g isoenzymes.
6.3. Aldehyde-oxidizing enzymes
As discussed above, m a n y o f the k n o w n
t u n g s t o e n z y m e s catalyze the oxidation of aldehydes
of one type or another, and they have b e e n isolated
from diverse microorganisms. They are all purified
by coupling aldehyde oxidation to the reduction of a
redox dye which in most cases is a v i o l o g e n deriva-
tive. The exception is carboxylic acid reductase
(CAR), which was discovered by its ability to catalyze the reduction to aldehydes of non-activated
carboxylic acids [156]. C A R s also readily catalyze
the reverse reaction, aldehyde oxidation, and although only one of the other aldehyde-oxidizing
t u n g s t o e n z y m e s , A O R from T h e r m o c o c c u s strain
ES-1, has been shown to do the same [145], this
should be possible for all of the aldehyde-oxidizing
e n z y m e s (Eq. (5)):
C H 3 C H O + H 2 0 ~-* C H 3 C O O - + 3 H + + 2 e ( E:, = - 5 8 0 m V : [ 2 6 9 ] )
(5)
The p r o b l e m lies with the extremely low potential of
the reaction, such that aldehyde production is very
unfavorable t h e r m o d y n a m i c a l l y ( A G ° approx. 8 kJ
m o l - l ; [269]). For example, from Eq. (5), equilibrium concentrations can be calculated for acetate and
acetaldehyde of 100 m M and 1.5 g M , respectively,
w h e n the electrons are used to form H 2 (at p H 7.0,
25°C, 1 atm. H2). Hence, in the case of CARs, low
potential viologen derivatives were used to obtain
significant rates of acid reduction, but these rates are
typically m u c h less than 10% o f rate o f oxidation of
the corresponding aldehyde [156].
Table 8
Structural properties of tungsten-containing aldehyde-oxidizing enzymes and related molybdoenzymes
Organism and enzyme
Holoenzyme
M~ (kDa)
Subunits
Subunit
M~ (kDa)
Mo/W
content "
FeS or cluster content b
Reference
C. thermoaceticum CAR form I
C. thermoaceticum CAR form II
C. Jbrmicoaceticum CAR
C. formicicum Mo-AOR
P. furiosus AOR
P. furiosus FOR
P. furiosus GAPOR
T. litoralis FOR
Thermococcus sp. ES-I AOR
Pvrococcus sp. ES-4 AOR
D. gigas ADH
D. gigas AOX
P. culgaris HVOR
86
300
134
170-300
136
281
63
280
135
135
132
200
600 (80)
c~/3 c
c~3/333, c
c~2 c
ot~_3
ot2 c
oq c
ot ~
o~4 c
c~2 c
~2 c
c~2 c?
a2
a8~
64, 14
64, 14, 43
67
100
67
69
63
69
67
67
65
97
80
1W
3W
2W
I Mo d
2W
4W
1W
4W
2W
2W
2W
2 Mo
1 Mo d
~ 29 Fe. ~ 25 S
~ 82 Fe, ~ 54 S (2 FAD)
~ 11 Fe, ~ 16 S
~ 7 Fe, ~ 7 S 2 [Fe~S2] d
2 [Fe4S4] + 1 Fe
4 [Fe4S4]
~ 6Fe
4 [Fe4S4]
2 [Fe4S4] + 1 Fe?
2 [Fe4S~] + 1 Fe?
~ 10 Fe
4 [Fe2S2]
4 Fe, 4S '~
[156,161,163,273]
[156,161,163,273]
[160,167]
[162,167]
[31,32]
[ 144]
[34]
[33]
[145]
a Expressed as an integer value per tool of holoenzyme.
b Cluster content is based on EPR spectroscopy or crystallography. See text for details.
Indicates enzymes with N-terminal amino acid sequence similarity in their ot subunit.
d Expressed per subunit.
Mukund, S. and Adams, M.W.W., unpublished results.
[188]
[182-187]
[220]
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reciews 18 (1996) 5-63
36
In the following, to avoid confusion, we will
retain the names of the aldehyde-oxidizing, W-containing proteins as they were originally designated.
Thus, CAR is from acetogens; aldehyde, formaldehyde and glyceraldehyde-3-phosphate ferredoxin oxidoreductases (AOR, FOR and GAPOR, respectively) are from the hyperthermophilic archaea; and
aldehyde dehydrogenase (ADH) is from sulfate-reducing bacteria. However, it will become evident
that the molecular and catalytic properties of these
apparently diverse enzymes are, for the most part,
very similar, and that they all fall within the same
enzyme family.
6.3.1. Carboxylic acid reductases from acetogens
CAR was the second tungstoenzyme to be purified and was obtained from the thermophilic acetogen Clostridium thermoaceticum in 1989 [156]. Two
years later, the enzyme was purified from the
mesophilic acetogen, C. formicoaceticum [160]. As
discussed above, there was substantial evidence for a
second W-containing enzyme, in addition to FDH, in
both organisms prior to the discovery of CAR
[3,168,169]. The C. thermoaceticum enzyme is extremely oxygen-sensitive (half-life in air, < 2 min)
and occurs in two distinct forms which differ in
chromatographic behavior, size, subunit structure,
Pf
T1
AOR
AOR
AOR
FOR
FOR
Ot
CA~64
Pf
ES4
ESI
MYGNWGRFI
MY~x-w~,q4IL
MFGYHGKIL
MYGWWGRIL
MKGWWGKIL
RVNLSTGDIK
~SDGTIK
RVN/,/I~
~ L A K
GXTADXK
RV~L~XV~
RVDLTNNKVW
KWLGSRGLAIY
LLLKEMDPTV
DPL
VQ~pF~rA
VQEYSPEVAK
NFIGGRGLAAW
ILWNE.AKNV
DPL
RVNLSN
RVENTTLKVT
XTEX. E V P A K
.Y A G
KLDVGKREVE
AQEIERE...
DIFGVVDYGI.
Dg
HVORMo
ADH
M~I~QLL
.... MNKFI
MKFSVL
MINGMTGNIL
MDKIL
Ct
CARy43
MRYLI IGNSA A G V A
Mt
MW
FMDH65
FMDH64
MEYIII~-GFV Y . P ~ E
~YII~FV
Y X P ~ B
Pa
AH
Dg
Cf
AOX~o
AOR~
Cf
CAR64
P f GAPOR
Pv
ASKKHVVCQ
and in their catalytic properties [156,163]. CAR I is a
heterodimer ( a / 3 ) of M r 86 kDa with subunits of
M r 64 and 14 kDa. CAR II is a large complex of
three different subunits with a proposed o~3 [337 stoichiometry. Its a and /3 subunits are identical to
those of CAR I, while the third subunit is of M r 43
kDa. CAR I contains approximately 1 mol of pterin,
1 W and 30 Fe and acid-labile sulfide atoms per
heterodimer, while CAR II contains about three times
as much (Table 8). As yet the EPR properties of
neither enzyme have been reported so the number
and nature of iron-sulfur clusters and the redox state
of W are not known. However, the pterin in CAR I
was recently shown to be the non-nucleotide form
(see below; [273]). CAR II also contains 2 F A D / m o l
of holoenzyme, which are located in the 7 subunit,
and CAR II, but not CAR I, will reduce NADP using
reduced viologen as the electron donor. Both forms
of the enzyme form high-molecular mass aggregates
as determined by gel filtration chromatography, and
the precise nature of the physiological complex is
not known. Based on recovery of activity during
purification, the amount of CAR I was more than
twice that of CAR II. The maximal specific activities
in aldehyde oxidation of CARs I and II were similar
(approx. 500 U / m g ) when assayed using butyraldehyde as substrate ( K m = 8 /zM) and l,l'-
RINLTTGAIS
RII)VGAEGGP
]344 ]
[33]
[16 3 ]
[163 ]
MR/4NE.XRTY
EVN
]34]
[22 o ]
KLTTLPVG..
EYA
[188]
[163]
[29]
[29]
.MDI
~
CCDINCWEA
MI.QKVIT VNGIEQNLFV
MKMLXKKGLL V~I
[32]
[a]
[145]
[214]
DAEALLSDVL
RQQ
[186]
[162]
Fig. 5. N-Terminal amino acid sequences of related tungsto- and molybdoenzymes. For proteins with more than one subunit, the Mr value
of the relevant subunit is indicated (see Table 8). Mo-containing enzymes are indicated (Mo). Proteins with N-terminal amino acid
similarities are grouped together, and identical positions and conservative exchanges are shown in boldface. The sources of the enzymes are:
P f Pyrococcus furiosus, ES-4, Pyrococcus sp. strain ES-4; ES-1, Thermococcus sp. strain ES-1; TI, Thermococcus litoralis, Ct, Clostridium
thermoautotrophicum, Cf Clostridium formicicum, Pv, Proteus t,ulgaris, Dg, Desulfo~,ibrio gigas, Mr, Methanobacterium thermoautotrophicum, Mw, Mb. wolfei, Pa, Pelobacter ace~lenicus. References are given in parentheses, a Mukund, S. and Adams, M.W.W., unpublished
data. Modified and extended from [271 ],
A. Kletzin, M.W.W.Adams/ FEMSMicrobiology Ret~iews18 (1996) 5-63
carbamoylmethylviologen ( K m = 50 ~ M ) as electron acceptor. The maximal specific activities of both
forms in the reduction of propionate ( g m = 10 mM)
were about 5% of the reverse reaction. A range of
both aliphatic and aromatic aldehydes and acids serve
as substrates for both forms of the enzyme
[156,163,273].
The properties of W-containing CAR from C.
formicoaceticum are quite distinct from those of the
C. thermoaceticum enzymes [160,167]. This is suggested by the fact that antibodies raised against CAR
from C. thermoaceticum showed strong cross reactivity with cell-free extracts of the closely related C.
thermoautotrophicum but there was virtually no reaction with extracts from C. formicoaceticum (nor with
C. aceticum, [161]). CAR from C. formicoaceticum
is very oxygen sensitive (half life in air, 5 min) and
was purified as a homodimeric enzyme with a subunit M~ of 67 kDa [160,167]. Each subunit contains
approximately 1 mol of pterin, 1 W, 5-6 Fe and 8
acid-labile sulfide atoms. The pterin is present as the
non-nucleotide form. EPR analysis of the reduced
enzyme (as purified in buffer containing sodium
dithionite) showed two different and very weak signals at 30 K with gz,y,x values of 2.035, 1.959 and
1.899 (A), and 2.028, 2.017 and 2.002 (B). These
represented approx. 0. I spin/tool and were assigned
to a W(V) species [167]. However, no evidence was
presented to support this, and signal (B) is highly
unusual as a gay value of < 2.0 would be expected
for W(V) (see [278]). At 16 K the reduced enzyme
exhibited an EPR spectrum typical of a single, magnetically isolated, reduced [4Fe-4S] cluster [167].
Quantitation of the spectrum was not reported, but in
light of the metal analysis (approx. 5 Fe/subunit) it
appears that the enzyme contains one [4Fe-4S] cluster per subunit.
The N-terminal amino acid sequence of the single
subunit of C. formicoaceticum CAR showed high
sequence similarity to the large subunit of CAR from
C. thermoaceticum (Fig. 5). N-terminal sequence is
not available for the small /3-subunit of C. thermoaceticum CAR, while the N-terminus of the ysubunit of CAR II showed no similarity to any
protein in the databases [163]. The specific activity
of the C. formicoaceticum enzyme in aldehyde oxidation (approx. 50 units/mg) was only about 10%
that of CAR from C. thermoaceticum, although like
37
the latter enzyme, it catalyzed the reduction of a
range of both aliphatic and aromatic carboxylic acids.
The apparent K m values were in the range of 1-20
mM for acids, and initial rate studies showed that
they were reduced by a bi uni uni bi mechanism
[167]. The affinity of CAR for aldehydes was much
higher than for acids, with apparent K m values for
propionaldehyde and butyrylaldehyde of 3 and 14
/xM, respectively. Although these data suggest that
aldehyde oxidation is the physiological role, the
precise function of CAR in vivo is presently unknown, as is the physiological electron carrier for the
enzyme. Nevertheless, CAR represents about 4% of
the total cytoplasmic protein in C. formicoaceticum.
The CARs of C. thermoaceticum and C, formicoaceticum also show a different response to Mo.
The amount of CAR protein present in cell-free
extracts of C. thermoaceticum, as determined by
immunological reaction, remained the same when
cells were grown on media containing Mo (10 /xM)
instead of W (10 /xM), but the specific activity of
the enzyme decreased by a factor of 60. The CAR
activity decreased about 25-fold when Mo (1 mM)
was included in the medium in addition to W (10
/zM) [161]. Thus, W is not required for the expression of CAR apoprotein, but is required for aldehyde-oxidation activity. In contrast, the addition of
Mo (1 mM) to the growth medium (containing 10
/xM W) for C. formicoaceticum had no effect on the
aldehyde-oxidizing activity of cell-free extracts, and
if W was omitted, the activity was about 15% of that
in cells from a W-sufficient medium [160]. Consequently, two distinct aldehyde-oxidizing enzymes
were purified from cells grown in the presence of 1
mM Mo and 15 /xM W. About half of the activity in
the cell-extract resided with W-containing CAR. The
remainder was catalyzed by another enzyme which
was purified and was shown to contain Mo. This was
termed a reversible, Mo-containing aldehyde oxidoreductase (Mo-AOR) [162]. In cells grown in the
absence of Mo, the Mo-AOR accounted for only 2%
of the total aldehyde oxidation activity.
Like CAR, Mo-AOR from C. formicoaceticum
contains only a single type of subunit (M r 100 kDa)
although it is not clear whether the holoenzyme is
dimeric or trimeric. However, the two enzymes
showed no similarity in their N-terminal amino acid
sequences [162]. Mo-AOR is less oxygen-sensitive
38
A. Kletzin, M. W. W. Adams / FEMS Microbiology Rer,iews 18 (1996) 5-63
than CAR (half-life in air, 1 h) but similar in cofactor content, except that Mo replaces W (Table 8).
The enzyme contained 0.6 Mo ( < 0.05 W) and 7 Fe
and acid-labile sulfide atoms/subunit. Fluorescence
spectroscopy of the denatured Mo-AOR showed the
presence of a pterin cofactor, but the intensity was
10-fold less than with CAR and the nature of the
cofactor in Mo-AOR is unclear. EPR analysis of
Mo-AOR has not been reported so the nature of the
Mo site and the iron-sulfur cluster(s) are also not
known. Like CAR, Mo-AOR catalyzed the oxidation
of aldehydes and the reduction of carboxylic acids
and exhibited broad substrate specificity. Its specific
activity in aldehyde oxidation was about 10-fold less
than that of CAR, although like CAR, the Mo-containing enzyme also showed a high affinity for aldehydes (apparent K m for butyrylaldehyde, 2 /xM)
and low affinity for acids (apparent K m for propionate, 7 mM). The two enzymes showed some differences in their preferences for various redox dyes
but Mo-AOR did not utilize NAD(P)(H) and it physiological electron carrier is also not known. Mo-AOR
and CAR did differ in their sensitivity to cyanide. A
concentration of 70 mM was required to inactivate
Mo-AOR (50% activity loss after 1 rain incubation),
but only 3 mM cyanide had the same effect on CAR
[162,167]. Based on cyanide sensitivity of various
molybdoenzymes [298], this result was interpreted
[167] as arising from a difference in coordination of
the W / M o centers, such that W in CAR, but not Mo
in AOR, was coordinated to a terminal sulfur that
could be removed as tbiocyanate by cyanide treatment (a so-called 'cyanaolyzable' sulfur). However,
this conclusion was not warranted since reactivation
by sulfide was not attempted and molybdoenzymes
exhibit a spectrum of sensitivities to cyanide in a
redox-sensitive manner (for review, see [225]).
6.3.2. Aldehyde ferredoxin oxidoreductases from hyperthermophilic archaea
From a historical perspective, the third type of
tungstoenzyme, after FDH and CAR, was discovered
in 1990 in the hyperthermophilic archaea. It had
been shown previously that the growth of Pyrococcusfuriosus, which was isolated in 1986 and grows
up to 105°C [127], was stimulated by the presence of
W in the growth medium, and a Ni-containing hydrogenase [126] and the redox protein ferredoxin had
been purified from this organism [299-301]. During
the purification of these proteins, significant amounts
of a red-colored fraction were noticed, purified, and
the responsible protein was shown to contain W [3 l].
Its EPR and redox properties were extensively analyzed (see below) and, in the absence of any catalytic
property or biological function, it was termed 'red
tungsten protein' (RTP). Two redox centers were
identified in RTP. These had extremely low potentials and transferred electrons to P. furiosus ferredoxin, suggesting that RTP was an oxidoreductasetype enzyme catalyzing a low potential reaction.
Accordingly, it was shown in 1991 that RTP was a
virtually inactive form of an extremely oxygen-sensitive enzyme that catalyzed the oxidation of aldehydes and reduced P. furiosus ferredoxin [32]. The
enzyme was termed aldehyde ferredoxin oxidoreductase (AOR), and very similar enzymes have now
been purified from the closely related organisms
Thermococcus sp. ES-I [145] and Pyrococcus sp.
ES-4 (Mukund, S. and Adams, M.W.W., unpublished). The properties of these AORs are summarized in Table 8. The Pyrococcus enzyme is now the
best characterized of all tungstoenzymes. It has been
the subject of numerous spectroscopic investigations
(see below), its gene sequence is known [271], and
its crystal structure has been determined to 2.3A
resolution [272].
A second type of tungstoenzyme has also been
purified and characterized from the hyperthermophilic archaea. It was discovered in 1993 in Thermococcus litoralis [33]. Cell extracts of this organism contain very low crotonaldehyde-oxidation activity, which is the usual assay for AOR, but high
formaldehyde-oxidation activity. The enzyme responsible for the latter activity was purified and was
termed formaldehyde ferredoxin oxidoreductase
(FOR). FOR oxidized only C1-C3 aldehydes and
reduced T. litoralis ferredoxin [300], but not NAD(P)
[33]. 7". litoralis also contains AOR, but at very low
concentrations and the enzyme has not been purified.
Conversely, P. furiosus contains significant FOR
activity and this enzyme has been purified [144]. The
properties of the two FORs are given in Table 8. The
third type of tungstoenzyme from the hyperthermophilic archaea was discovered in 1995. P. furiosus was found to contain a new type of enzyme,
which catalyzed the oxidation of glyceraldehyde 3-
A. Kletzin, M. W, W. Adams / FEMS Microbiology Reciews 18 11996) 5-63
phosphate (GAP) coupled to the reduction of ferredoxin. The protein responsible for this activity (GAP
ferredoxin oxidoreductase, or GAPOR) was purified
and, very surprisingly, was shown to be a W-containing, iron-sulfur protein (Table 8). In the following,
we first summarize the properties of AOR, followed
by a comparative description of those of FOR and
GAPOR.
6.3.2.1. Aldehyde ferredoxin oxidoreductase. AOR
from P. furiosus catalyzes the oxidation of a range
of aliphatic and aromatic aldehydes, and reduces
ferredoxin, the proposed physiological electron acceptor, but not NAD(P). It does not oxidize glucose,
glucose 6-phosphate, glyoxylate, or phosphorylated
aldehydes such as GAP, nor does it use coenzyme A.
The enzyme has an apparent K m of 40 /xM for
crotonaldehyde, a substrate which is stable at 80°C,
the temperature used to routinely assay the enzyme,
and the Vm value (with methyl viologen as the
electron acceptor) was 70 units/mg. The optimal
temperature for activity is > 95°C [31,32]. Extensive
kinetic analysis of the AOR from Thermococcus
strain ES-I have shown that the most efficient aldehyde substrates are those that correspond to the
oxidation products of amino acids (acetaldehyde,
phenylacetaldehyde, indolacetaldehyde and isovalerylaldehyde) [145,146]. This led to the proposal
that the hyperthermophilic AORs function in vivo to
oxidize aldehydes generated from 2-keto acids, which
are key intermediates in both peptide and carbohydrate catabolism. Both ES-1 and P. furiosus AORs
were sensitive to inhibition by iodoacetate, arsenite
and cyanide [32,145].
AOR from P. furiosus was originally proposed to
be a monomer of M r 85 000, as estimated by electrophoretic and chromatographic analyses [31,32].
On this basis, it contained approx. 1 W and 6-7 Fe
atoms/mol. However, its gene was recently isolated
and this codes for a protein of approx. 66 kDa [271],
and crystallographic analyses, while confirming the
deduced amino acid sequence, showed that AOR is a
homodimer [272]. Thus, AOR exhibits anomalous
behavior when analyzed by SDS-gel electrophoresis
(M r 80 kDa) and gel filtration (M r 90 kDa). The
determined metal content, when based on the revised
M r value, is 1 W and 4-5 Fe atoms/subunit, which
is in good agreement with that shown by crystallog-
39
raphy (see below). The AORs of Thermococcus
ES-I and Pyrococcus ES-4 have a similar metal
content to the P. furiosus enzyme and they appear to
also behave in a similar fashion when analyzed by
electrophoretic (apparent M r values, 75 kDa) and
gel filtration techniques (apparent M r values, 100
kDa; [145] and Mukund, S., and Adams, M.W.W.,
unpublished data). The N-terminal sequences of the
three proteins are highly similar to each other (Fig.
5). The ES-1 and ES-4 enzymes are assumed to be
structurally analogous to P. furiosus AOR (see Table
8) although this has yet to be confirmed. Extraction
and characterization of the pterin cofactors from the
AORs of P. furiosus and Pyrococcus ES-4 AOR
have shown that they are of the non-nucleotide type
[302].
P. furiosus AOR in its dithionite-reduced exhibited a remarkable EPR spectrum at temperatures up
to 20K which spans almost 0.5 Tesla with resonances from g = 1.3 to g = 10. This spectrum is the
same as that seen from the inactive RTP form of
AOR and is unlike that seen from any other biological or synthetic FeS system [31,32,147]. Upon partial
oxidation of the enzyme, the EPR signal simplifies
and resonances from a center with a well characterized S = 3 / 2 state are then apparent (gcff 4:.75, 3.33
and 1.92), while the thionine-oxidized enzyme is
EPR silent. The species with the S = 3 / 2 ground
had an E m value of - 4 1 0 mV (20°C, pH 8.0). Thus,
upon reduction of the enzyme below - 4 2 5 mV, the
more complex EPR signal arises from the spin-coupling of this S = 3 / 2 center with a second paramagnetic species which has a lower E m value ( - 5 0 0
mV at 20°C, pH 8.0). Analyses of the enzyme in
various redox states by magnetic circular dichroism
(MCD) and resonance Raman (RR) spectroscopy
have unambiguously identified the S = 3 / 2 species
as a reduced [4Fe-4S] cluster (Conover, R.C., Crouse,
B., Mukund, S., Adams, M.W.W. and Johnson, M.K.,
unpublished results). X-ray absorption spectroscopy
(EXAFS) of the inactive form of AOR (RTP) showed
that the enzyme contains a mononuclear W site with
two terminal oxo groups at 1.74 A, three W-S at
2.38 A, and one W - O / N at 2.13 A [303]. The W site
was therefore proposed to be very similar to the Mo
site in enzymes such as sulfite oxidase. Moreover, it
was assigned a W(VI) oxidation state, as no dioxo W
sites are known for any other oxidation state (see
40
A. Kletzin, M. W. W. Adams / FEMS Microbiology ReLVews 18 (1996) 5-63
[303]). However, more recent EXAFS analysis has
shown that the W site in the dithionite-reduced form
of active AOR has increased S coordination compared to inactive RTP. The best fit to the experimental data were with a W center which contained one
terminal oxo group and 4-5 W-S interactions (George, G.N., Mukund, S. and Adams, M.W.W., unpublished data). Unfortunately, the X-ray absorption
technique does not distinguish between W(IV), W(V)
and W(VI), and the redox state of the W site in
active AOR is unknown (see below).
The dithionite-reduced forms of the AORs from
Pyrococcus ES-4 and Thermococcus ES-I also give
rise to an EPR signal from an S = 3 / 2 species,
which presumably also arise from a reduced [4Fe-4S]
cluster ([145] and Mukund, S., and Adams, M.W.W.,
unpublished results). However, both enzymes exhibit
a 'complex' EPR signal but they are of much lower
intensity than that seen in P. furiosus AOR, suggesting that the second paramagnetic center in these
enzymes has a lower redox potential. This second
paramagnet was originally proposed to arise from
another FeS cluster in P. furiosus AOR [32], but a
second cluster is not evident from the crystal structure ([272], see below). Accordingly, preliminary
MCD analyses indicate that dithionite-reduced form
of active AOR does in fact contain an as yet undetermined amount of a paramagnetic W(V) species
(Conover, R.C., Crouse, B., Mukund, S., Adams,
M.W.W. and Johnson, M.K., unpublished data). The
spin coupling of this center with the reduced, S =
3 / 2 [4Fe-4S] cluster might well preclude the observation of its EPR signal at high temperatures ( > 20
K). Further support for this observation comes from
the EPR properties of P. furiosus AOR obtained
from cells grown in the presence of vanadate. The
dithionite-reduced enzyme, which contained only 0.1
W atom/subunit but had a full complement of Fe,
exhibited only the S = 3 / 2 type EPR signal, suggesting that it is the W site that is the source of the
second paramagnet [147]. Clearly, further work will
be required to elucidate the various forms of the W
site in the active form of P. furiosus AOR.
The recent determination of the crystal structure
of P. furiosus AOR revealed several surprises, in
addition to the dimeric structure of the enzyme [272].
First, the two subunits, each of which contains a
W-pterin site with an adjacent 4Fe-cluster, were
bridged by a monomeric Fe site. This is assumed to
be low spin ferrous (S = 0) in the dithionite-reduced
enzyme and would not be seen byoEPR spectroscopy.
The Fe atom is situated about 25 A from the W sites,
and is thought not to participate in catalysis but
rather has a structural role. A second unexpected
aspect was the nature of the pterin site. The structure
of the pterin was virtually identical to that predicted
by Rajagopalan and co-workers from their extensive
studies of many molybdoenzymes ([17,18]; for review see [305]), with the exception that the pterin
nucleus was a three rather than two ring system (Fig.
6). Similarly, as had been predicted for Mo-containing enzymes, the W atom was coordinated to two
dithiolene S atoms of the pterin. However, two
pterins, rather than the expected one, were coordinated to the W to give a distorted square pyramidal
arrangement of one W and 4 S atoms. In addition,
the phosphate groups from each pterin were bridged
to a single Mg 2+ ion, with two H 2 0 molecules and
two peptide carbonyls completing the coordination
(Fig. 7). Thus, each subunit of the protein contains
one W atom, two pterins bridged by a magnesium
atom, and a single [4Fe-4S] cluster which is located
10 A from the W atom. The close proximity of the
W atom and the cluster is consistent with the spincoupling between them that is manifested in the EPR
spectrum of the reduced enzyme. Moreover, the W
and 4Fe-cluster in one subunit are approximately 50
from the same sites in the other subunit and so
intersubunit magnetic interactions can be excluded.
A diagrammatic representation of the enzyme is
shown in Fig. 8.
From the gene sequence it was deduced that AOR
contained 605 amino acids (Fig. 9) which corresponded to a protein with a molecular mass of
66 630 (Fig. 9) or 136 066 for the homodimer including cofactors. The single [4Fe-4S] cluster in each
AOR subunit is liganded by the cysteinyl residues at
positions 288, 290, 295, and 494. There are no
covalent bonds from the protein to either the W atom
or to the two pterins. Rather, the two pterins are
'held' by a H bonding network. The main interaction
comes two homologous Asp-X-X°Gly-Leu-(Asp or
Cys) sequences (338-343 and 489-494) where the
Asp carboxylate and the Leu carbonyi H bond to the
amino side group of each pterin. In addition, Asp34~
H bonds to the ring N (N-8) of one pterin and the
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reviews 18 (1996) 5-63
o
A.
.
I
I
H2N
RCHO
~
~
*~o
R¢ooI-I~
H
41
+ 2H ÷
R+C2OO~H~" ~
"~
41o mY /
f/pterin~P-'~ e" ~
o'1 I'~'.e-~.'~l
W
~ / " - - ' ~ '4 ~4j "--'~lg~_~Jl
,
\pterin-P,)
(~'
J
.//
H
Fig. 6. Structures of the pterin cofactor. (A) The proposed structure of the pterin cofactor in molybdoenzymes [15-18,305]. The R
group is -H in the unmodified pterin or -phosphate-ribose-base in
the pterin dinucleotide where the base is A, G, HX or C. (B) The
actual structure of the unmodified pterin in P, furiosus AOR
[2721.
Fig. 8. Representation of P, furiosus AOR based on its crystal
structure and the proposed mechanism of electron transfer from
the tungstopterin site to P. furiosus [erredoxin (Fd) during catalysis. The redox potentials and spin states of the 4Fe-clusters in
AOR and Fd are indicated. The oxidation state of W in active
AOR is not known. Modified from [32,272].
cluster-coordinating Cys494 H bonds to the ring N of
the other pterin. Cys494 therefore serves to link the
hemerythrin, ribonucleotide reductase, methane
monooxygenase, rubrerythrin and stearoyl-acyl desaturase [306,307]. However, the function, if any, of
the ExxH motif not involved in Fe coordination is
unknown.
As shown in Fig. 9, the subunit of AOR is
comprised of three domains of similar size. The
tungstodipterin site is buried deep in the protein and
'sits' on the N-terminal domain (I). Domains II and
III surround the other side of the cofactor such that
the W atom can be thought of as projecting up from
the four dithiolene S atoms towards these two domains, each of which contains a consensus pterin-binding motifs. Domains II and III also form a long
hydrophobic channel that leads from the W atom to
the exterior of the protein. The channel is wide
enough to accommodate even aromatic aldehydes,
consistent with the broad substrate specificity of
AOR [272]. The mechanism of substrate binding to
the W site is not known at present. In addition to the
four pterin S atoms, the W atom has at least two
other coordination sites, one of which, according to
EXAFS data, is occupied by a terminal oxo group.
From the crystal structure, the side chains of Glu 3~3
and His448 in domain Il are adjacent to the vacant
coordination sites of the W atom, and these are
assumed to participate in substrate binding and catalysis.
4Fe-cluster and one of the pterins. A second cluster
interaction with the same pterin involves the side
chain of Arg 76, which H bonds both the pterin ether
oxygen and an inorganic S atom of the 4Fe-cluster.
Other relevant residues indicated in Fig. 9 include
Asn93 and Ala)~ 3, the carbonyl moieties of which
coordinate the magnesium ion, and Glu332 and
His382, both located in separate ExxH motifs, that
coordinate the monomeric iron site [272]. In fact, the
AOR sequence contains three ExxH motifs. These
are characteristic of iron-containing proteins such as
_ . . ~ ..~fAla-tS3
(°
...J
Fig, 7. Structure of the W-dipterin site in P. furiosus AOR.
Modified from [272].
A. Kletzin, M. W. W. Adams / FEMS Microbiology Ret,iews 18 (1996) 5-63
42
By analogy with postulated mechanisms for
molybdoenzymes, the two electron oxidation of aldehydes by AOR presumably involves reduction of
W(VI) to W(IV), possibly with the generation of an
intermediate, EPR-detectable W(V) species, and the
transfer of electrons from the W site to the [4Fe-4S]
cluster. Although in molybdoenzymes the pterin
moiety is rarely considered to have an electron transfer role, the integral H bonding network between one
of the pterin molecules and the 4Fe-cluster in AOR
suggests that electron transfer from the W atom to
the cluster proceeds via the pterin ring system. Since
the 4Fe-cluster undergoes only a one electron redox
reaction, the reduced cluster must be oxidized by
electron transfer to ferredoxin, the physiological
electron acceptor, before it can accept a second
electron from the W site. The [4Fe-4S] cluster is
situated about 6~, from the surface of the protein,
much closer than the W site, which is consistent with
its electron transfer role to ferredoxin. Since the two
W atoms are approx. 50 ,& apart [272], it is assumed
that the two subunits are catalytically independent.
The proposed pathway of electron flow during catalysis by AOR is shown in Fig. 8. Note the redox
potential of the W site is not known for the active
form of the enzyme• Variable temperature kinetic
and spectroscopic analyses of active AOR are obviously required to determine the nature of the W site
during catalytic turnover and to substantiate the proposed mechanism.
Pfu A O R MYGNWGRFIRV•qLSTGDIKVEEYDEELAKk•WLGSRGLA•YLLLKEMI•PTVDPLSPENKLIIAAGPLTGTSAPTGGRYNVV
] Jl:::il:l
.... :.d:ll..l:ll.::i:i111
::I:.I
...lJrr:l•lll::l.li:.f
..I.II:
Tli FOR M K G W W G K I L R V D L T N N K V W V Q E Y S P E V A K N F I G G R G L A A W I L W N E . A K N V D P L G P K N K L V F A T G P F N G L P T P S G G K M V I A
80
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.
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Pfu A O R T K S P L T G F I T M A N S G G Y F G A E L K F A G Y D A I V V E G K A E K P V Y I Y I K D E H I E I R D A S H I W G K K V S E T E A T I R K E V G S E K V K I
IIl]li .. :I I . . . .
P: JlJfl:llIIll. JrlJlll.r:::.P
.I. :Ill.. III .: If: :..I
Tli FOR AKSPLTGGYGDGNLGTMATVHLRKAGYDALVVEGKAKKPVYIYIEDDNVSILSAEGLWGKTTFETEREL.KEIHGK•NVGI
160
i
158
Pfu A O R ASIGPAGENLVKFAAIMNDGHRAA(~aGGVGAVMGSKNLKAIAVEGSKTVPIA~KQKFMLVVREKVNKLRNDPVAGGGLPK
lill:lJll!l:I.::.::
lillJ.l:llllrll.lll:.:.l.].:l:l[ll:i:.
:
.I
:
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240
.
:.:
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Tli FOR LSIGPGGENLVKYAVVISQEGRAA(~.PGMGAVMGSKKLI~AwIKGTKEIPVA~KEKLKELSQEAYDAILNSP~GYPFWH~
237
# -#t
Pfu A O R YGTAVLVNI~NENGL~PVK~rFQTGVYPYAYE~SGEAMAAKYLVRNKPCYACP~GC6RVNRLPTVGETEGPEYESVWAL~A
-
II
.
I:
- -
.I]1:
-
.t.:ll
.I
-
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-
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-
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320
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Tli FOR QGTMAAVEWTNENSALPTRNFSDGSFEFARSIDGYTMEGM~KVKQRGCPYCNMPCGNV.VLDAEGQESELDY]~NVALLGS
315
I
.....
I
Pfu AOR NLGINDLASiIF`ANHMCDE~GLD,~IS~GGTLATAME~YEKGHI~DEELG6A~PFRWGNT~VLHYYIEKIAKREGFGDKLi
II1.,I..:
I::.11:111111
I..:.
.tl
[11
Ill::
.:
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Tli FOR N L G I G K L N E V S V L N R I A D E M G L D T I S L G V A I S Y V M E A K E K G I I K D D D A P E F G D F K K A K
::
:1:::1:
400
I
.... Q L A L D I A Y R R G E L G N L A A
Pfu AOR E G S Y R L A E S Y G H P E L S M ~ K K L E L P A Y D P R G A E G H G L G Y A T N N R G G C H I K N . Y M I S P E I L G Y P Y K M D P H D V S D D K I K . . .
JI
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rl:.:l:•
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Tli FOR EGVKV.MSEKLGAKDFAM}[gKGLEVSGYNCY~YPAMALAYGTSSIGA~HH`~EAWVI~WEIGTAP~EGEKAQKVEYK~TYDP
• ..# •
Pfu A O R
...MLILFQDL.TALIDSAGLC.L.FTTFGLGADDYRDLLNAALGWDFTTEDYLKIGERIWNAERLFIqLKAGLDPARDI~
Tli FOR
Pfu AOR
:1
I
.:1::
.
I
I
:...11:
[
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]KAAKVIELQRLRGGLF~LTACRLPWVEVGLSLDYYPKLLEAITGVKYTWDDLYKAADRVYALMRAYWV
]~L,.. PKRFT',EEPMPEGPNKGHTV. RLKE i. M L P R Y Y K L R G W T E D G K I P K E K L E E L G I A E F Y
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:
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Tli FOR E M D Y P P E R W F K E G L K S G P Y K G Q H L E K D K Y D A L L S E Y Y K L R G W D E R G I P K K E T L K E L N L . E F V I P E L E K V T K L E
621
Fig. 9. Pairwise alignment of the AOR and FOR amino acid sequences. The boxes indicate the three domains I (upper), II (middle) and IlI
(lower) of AOR [272]• Specific residues (where numbers refer to the AOR sequence) or groups of residues are denoted by: boldface type,
cofactor coordinating residues in AOR; underlined, ExxH conserved motifs of diiron oxo clusters; # , cysteine residues that coordinate the
[Fe4-S 4] cluster, C l_3 are located in a conserved Cys-cluster motif (CxxCxxxCG); •, pterin-coordinating residues, including two symmetric
D x x G L C / D x motifs. The fourth ligand of the FeS-cluster (Cys494) and Arg7~ bridge the FeS-cluster and the tungstopterin; the G l u ~ and
the His383 residues (indicated by thick vertical lines) in both subunits provide the ligands of the non-heme iron bridging the two subunits.
The carbonyl moieties from the peptide backbone of the Asn93 and Alals 3 residues ( + ) provide two of the six ligands to the magnesium
ion, while the side chains of Glu313 and His448 ('J') are in the vicinity of the substrate binding site. The function of the remaining cysteine
residues (", two in AOR, one in FOR) is not known. Modified from Ref. [271].
A. Kletzin, M. W.W. Adams / FEMS Microbiology Reciews 18 (1996) 5-63
6.3.2.2. Formaldehyde ferredoxin oxidoreductase.
FOR has been purified from P. furiosus [144] and T.
litoralis [33]. These enzymes also use ferredoxin as
the physiological electron carrier and are maximally
active at temperatures > 95°C. However, in contrast
to AOR, they oxidize only CI-C3 aldehydes and are
of much lower specific activity [33,144]. For example, with formaldehyde as substrate, the apparent
Vm~x value for T. litoralis FOR was 17 units/mg
(apparent K m, 62 raM) which compares with a value
of 300 units/mg (apparent K m, 0.7 raM) for P.
furiosus AOR. FOR was much less sensitive than
AOR to inhibition by arsenite and iodoacetate, although like AOR, FOR does not oxidize aldehyde
phosphates, utilize coenzyme A, or reduce NAD(P).
By biochemical analyses, the FORs of both species
are homotetramers with a subunit M r of 70 kDa.
Each subunit contains approx. 1 W and 4 Fe atoms
[33,144], together with a pterin cofactor which is
present as the non-nucleotide form [302]. EPR spectroscopy indicated the presence of a single [4Fe-4S]
cluster in the T. litoralis enzyme, which was unusual
in that the reduced cluster existed in a pH-independent S = 3 / 2 form and a pH-dependent S = 1 / 2
state.
A close structural relationship between FOR and
AOR was suggested by the high similarity in their
N-terminal sequences ([33]; see Fig. 5). This was
confirmed by the cloning and sequencing of the gene
for FOR from T. litoralis, the second tungstoenzyme
for which a sequence is available (Fig. 9; [271]).
This coded for 621 amino acids which corresponded
to a protein of M r of 68 941, in good agreement with
the results from biochemical analysis. The calculated
M, for the tetramer is 28! 264 (including cofactors,
see below). As shown in Fig. 9, the complete sequences of FOR and AOR shows a remarkable similarity in several of the features that have been shown
by X-ray crystallography to be important for AOR,
although there are also some interesting differences.
The two enzymes share 231 identical residues (38%;
with 59% similarity); however, a much higher sequence similarity was observed for residues 1-210
of AOR (48% identity), which coincides with domain 1 [272], while in domains 2 and 3 the identity
score was 33%. The four cysteinyl residues that
coordinate the [4Fe-4S] cluster in AOR are conserved in the FOR sequence, as are the magnesium-
43
coordinating Asn93 and Ala183 residues and most,
but not all, of the pterin-coordinating residues. Notably, the second DxxGL motif is missing in FOR,
although it is possible that Glua98 in FOR replaces
Asp489 in AOR, which is a bidentate ligand to the
pterin. Interestingly, Glu3j 3 and His48 s, which are
next to the vacant coordination sites of the W atom
in AOR, are conserved in FOR, suggesting perhaps
identical mechanisms for substrate binding and catalysis in these two enzymes [271,272].
The scheme given in Fig. 8 for catalysis and
electron transfer by AOR applies at least in principle
to FOR, although there are clearly differences between them. For example, even though the subunits
of FOR and AOR are very similar in size and
sequence, one major and obvious difference between
them is their quaternary structures; FOR is a tetramer
while AOR is a dimer. It is unlikely, however, that
FOR contains monomeric iron sites equivalent to
that which bridges the two subunits of AOR. The
iron analyses are consistent with a single 4Fe-cluster
per FOR subunit and the equivalent of the Glu33 ?
and His3s 2 residues that bind the monomeric iron
site in AOR are not present in FOR (Fig. 9). In fact,
FOR lacks all three of the ExxH motifs found in
AOR. This suggests that this type of subunit interaction is not present in FOR. At this point it is not
clear which particular differences in sequence between AOR and FOR reflect their different substrate
specificities. We have speculated [271] that the channel from the exterior of the protein to the inlerior W
site that is formed by domains II and II1 in AOR
may be narrower in FOR, such that only small
aliphatic substrates have access to the catalytic site.
Obviously the crystal structure of FOR will be required to confirm this, and such studies are in
progress. A variety of spectroscopic analyses, including EPR, MCD and X-ray absorption, are also necessary to probe the nature of the W site in FOR, to
assess to what extent it is the same as that in AOR.
6.3.2.3. Glyceraldehyde-3-phosphate ferredoxin oxidoreductase. The specific activity of the glycolytic
enzyme, glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), is low in cell-free extracts of P. furiosus
grown on maltose and peptides (0.07-0.09 U / m g
protein), but this increases about 10-fold when cells
are grown on pyruvate, suggesting a role for the
44
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reviews 18 (1996) 5-63
enzyme in gluconeogenesis [142]. The identification
of a modified Embden-Meyerhof pathway for glycolysis in P. furiosus ([138], reviewed in [308]),
prompted a search for the enzyme that might take the
place of GAPDH in glucose catabolism. This led to
the finding that P. furiosus contains high amounts of
a glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR, approx. 6 U / m g in cell-free extracts
[34]). GAPOR is a monomeric, moderately oxygensensitive protein of M r 63 kDa which contains approx. 1 W and 6 Fe atoms per mol. Fluorescence
analysis of material extracted from the denatured
protein indicated the presence of a pterin-type cofactor, although it is not known if it is the non-nucleotide form. The enzyme oxidized glyceraldehyde 3phosphate (GAP) at 70°C, with apparent K m and
Vmax values of 30 /zM and 350 units/mg, respectively, and it reduced P. furiosus ferredoxin (apparent gm, 8 /J,M) but not NAD(P). GAPOR did not
oxidize non-phosphorylated aldehydes such as
formaldehyde, acetaldehyde, crotonaldehyde or benzaldehyde, nor related compounds like glucose 6phosphate or glycerol 3-phosphate. However, the
product of GAP oxidation by GAPOR inside the cell
is not clear. In the analogous reaction, GAPDH
oxidizes GAP, reduces NAD(P) and, in the presence
of inorganic phosphate, produces 2,3-bisphosphoglycerate (BPG). However, BPG is extremely unstable
at 70°C and undergoes hydrolysis to 3-phosphoglycerate (3-PG). Although phosphate (200 mM) stimulated the activity of GAPOR about three-fold in
vitro, the reaction product was 3-PG not BPG. Due
to its instability, it cannot not be ruled out that BPG
is the actual product of the GAPOR reaction, but this
seems unlikely since sodium sulfate, sodium citrate
and potassium chloride (each 200 raM) led to a
similar enhancement of GAPOR activity. Hence, it
was tentatively concluded [34] that 3-PG is the product of the GAPOR reaction in vivo, although this
needs to be substantiated.
The closeness of the relationship between GAPOR
and the other tungstoenzymes of P. furiosus, AOR
and FOR, is not known at present. Although its size
is comparable to that of the other two enzymes, the
N-terminal amino acid sequence of GAPOR shows
only distant similarity to AOR and FOR (Fig. 5).
Preliminary EPR analysis of dithionite-reduced
GAPOR indicated the presence of a single [4Fe-4S]
cluster, and a sharp rhombic EPR signal (gav< 2.0)
is observed at 50 K (Mukund, S. and Adams,
M.W.W., unpublished data). However, isotopic enrichment a n d / o r MCD analysis will be required to
determine if the latter signal arises from a W(V) site
or from an additional FeS center in the enzyme.
Interestingly, GAPOR contains approx. 2 Zn
atoms/mol, an element not found in AOR or FOR.
This leads to the very speculative hypothesis that
GAPOR is structurally analogous to AOR, but that
the hydrophobic channel leading from the exterior
contains one or more Zn atoms near to the W site.
These could serve to bind the phosphate group of
GAP such that the aldehyde group is positioned near
the W atom. Obviously, a great deal of data will be
required to validate this postulate.
6.3.3. Aldehyde dehydrogenase from Desulfovibrio
gigas
The observation that the presence of W in the
growth medium of ethanol-utilizing D. gigas caused
a dramatic increase in cell-free extracts of viologencoupled aldehyde oxidation activity led to the recent
identification of the first tungstoenzyme from a
Gram-negative bacterium [188]. This so-called aldehyde dehydrogenase (ADH) was very oxygen-sensitive and was purified under anaerobic conditions. It
is a homodimer with a subunit M r of 62 kDa and
contains 0.6 W and approx. 5 Fe atoms/subunit. A
pterin-type cofactor was extracted from the enzyme
but is not known if it is the dinucleotide form. Pure
ADH had a specific activity in acetaldehyde oxidation of 38 units/mg, but it utilized a range of both
aliphatic and aromatic aldehydes using benzyl viologen as the electron acceptor. The apparent K m values for acetaldehyde, propionaldehyde and benzaldehyde were below 25 /.~M. The physiological electron
carrier for the enzyme is not known. ADH was
severely inhibited after prior incubation with cyanide,
arsenite or iodoacetate (each 5 mM). The enzyme as
purified exhibited a complex rhombic-type EPR
spectrum at 55 K with gay < 2 and this was assigned
to a W(V) species. The complexity of the signal was
proposed to arise from heterogeneity at the W site,
although the spin content of the spectrum was not
reported. This signal disappeared upon addition of
dithionite to the enzyme and was replaced by a broad
complex spectrum seen at 15 K. This was assigned
A. Kletzin, M. W. W. Adams / FEMS Microbiology Rel'iews 18 (1996) 5-63
to a reduced [4Fe-4S] cluster, with the complexity
arising from its spin-coupling to another paramagnetic species, possibly the reduced W(IV) site (S = 1)
[188]. Although the redox state of the as purified
form of ADH was not specified, it appears that its W
site is reduced by sodium dithionite (E,~, approx.
- 4 5 0 mV; [304]). The general properties of D.
gigas ADH, including substrate specificity, subunit
size and quaternary structure, and W and Fe content,
are therefore very similar to those of P. furiosus
AOR. In fact, they have analogous spectroscopic
properties, with the dithionite-reduced enzymes exhibiting a complex EPR signal apparently arising
from the spin-coupling of a reduced 4Fe-center and a
heterogeneous tungstopterin site. The main difference is that the P. furiosus enzyme contains a reduced 4Fe-cluster with a S = 3 / 2 ground state while
the reduced 4Fe-cluster in D. gigas ADH has a
S = 1 / 2 ground state.
The extent to which the aldehyde-oxidizing, Wcontaining ADH from D. gigas is similar to the
Mo-containing, aldehyde-oxidizing enzyme from the
same organism is not clear. The latter was originally
purified in 1976 as a Mo-containing iron-sulfur
protein (MOP; [182]). More than a decade later, it
was shown to catalyze aldehyde oxidation and was
termed aldehyde oxidase (AOX: [183]), although
more recent studies have referred to it both as an
aldehyde oxidoreductase [185] and again as MOP
[186]. AOX uses flavodoxin as the physiological
electron carrier but the function of the enzyme is not
well understood. The stimulation of cell growth on
ethanol by the addition of W to the medium and the
increase in ADH activity suggest that AOX and
ADH have different physiological roles. As shown in
Table 8, AOX, like ADH, is a homodimer, with each
subunit containing 1 Mo and 4 Fe atoms, but the
latter are present as two [2Fe-2S] clusters rather than
a single 4Fe-center. It contains the guanine dinucleotide form of the pterin. The enzyme has been
crystallized [185], although its structure has not yet
been reported. The sequence of the enzyme was
determined from the cloned gene and it shows high
similarity to other molybdoenzymes [186]. For example, it showed 52% sequence identity with xanthine
dehydrogenases from various sources. Obviously, the
sequence of W-containing ADH is urgently needed
to determine the relationship between it and Mo-con-
45
taining AOX, as this appears to be a 'typical' molybdoenzyme.
6.4. Acetylene hydratase
Acetylene hydratase (AH) converts acetylene to
acetaldehyde according to Eq. (6) [214].
H - C-= C - H + H 2 0 - - , H 3 C - H C = O
(6)
This hydration reaction has also been proposed to
take place in aerobic acetylene-oxidizing bacteria
such as Nocardia rhodochrous, Rhodococcus sp. A I
and Rhodococcus rhodochrous, but the responsible
enzyme has only been purified from the anaerobe
Pelobacter ace~'lenicus [214]. This enzyme is a
monomer of M,. 73 kDa and contains 0.4 W and 4-5
Fe and acid labile sulfide atoms/tool. Evidence for
the presence of a pterin cofactor was provided by
fluorescence spectroscopy of the denatured protein.
It had an apparent K m ['or acetylene of 14/~M. The
enzyme was somewhat oxygen-sensitive (50% loss
of activity after 2 days in air) but was purified
aerobically. However, oxygen inhibition was completely reversible, as AH was able to utilize acetylene only in the presence of strong reducing agents
such as sodium dithionite or titanium citrate. AH
therefore differs from all the other tungstoenzymes
(AOR, CAR, FMDH, FDH, etc.) in being reversibly
inhibited, rather than irreversibly inactivated, by
oxygen. AH activity was not affected by chelating
agents nor by ethylene (C z H 4), was relatively insensitive to cyanide (40% loss of activity after 15 rain
with 10 mM KCN), but was strongly inhibited by
Hg 2+ ions, CO, and NO.
The N-terminal amino acid sequence of AH (Fig.
5) shows no similarity to those of W- or Mo-containing proteins, nor to any other protein sequence in the
databases. Interestingly, the N-terminal did contain a
cysteine-cluster motif (CxxCxxxC), which typically
coordinate FeS-clusters. The overall properties of
AH are therefore highly reminiscent of the AOR
family (subunit M r, 1 W and 4 - 5 Fe atoms per
subunit), although it obviously catalyzes it completely different type of reaction, a difference presumably reflected in its N-terminal sequence. Although the presence of W or Mo in an enzyme
implies that it catalyzes a redox-linked reaction, this
is not necessarily the case with FeS-clusters. Several
46
A. Klet;in, M. W. W. A d a m s / F E M S Microbiology Reriews 18 (1996) 5 - 6 3
hydrolytic enzymes are known that contain 2Fe- or
4Fe clusters, wherein the cluster participates in catalysis. Aconitase, which contains a [4Fe-4S] cluster, is
the prototypical example of such an enzyme [214].
Alternatively, the AH reaction might involve a redox
reaction wherein the substrate (acetylene) is first
reduced, then hydrated, and then oxidized. This
would also explain the requirement for a strong
reducing agent by AH for acetylene hydration activity. However, spectroscopic analyses of AH have not
been reported, so the role of the W site and the
iron-sulfur cluster(s) in the catalysis of acetylene
hydration is as yet unknown.
6.5. The classOqcation of tungstoenzvmes
Since the complete amino acid sequence [271], as
well as the crystal structure [272], of the W-containing AOR from P. furiosus is known, we will use
this enzyme as the paradigm for comparisons with
molybdoenzymes and other tungstoenzymes. However, amino acid sequences with significant similarity to AOR were not found in database searches.
Likewise, no similarity was found to any of the
amino acid sequences available for Mo-containing or
pterin-containing enzymes, even after retrieving them
from the database and performing specific searches
or by using the PROSITE database [271]. In addition, no similarity was found with the AOR sequence
when it was specifically compared with signature
sequences that are reportedly involved in pterin-binding in molybdoenzymes [309]. The only exception
was the DxxGL pterin-binding motif found in AOR.
This was observed in some of the sequences of
molybdoenzymes, though not all, but none of them
contained two such motifs like AOR. Hence, AOR is
phylogenetically very different from any known
molybdoenzyme.
The high similarity of the AOR sequence with the
only other tungstoenzyme for which a complete sequence is available, FOR from T. litoralis (38%
sequence identity, [271]), shows that these two enzymes are closely related. Except as noted above for
AOR, FOR showed no sequence similarity to any
molybdoenzyme. N-Terminal sequences are available for all of the other tungstoenzymes listed in
Table 8, with the exception of C. thermoaceticum
FDH, and these are aligned in Fig. 5. As expected,
the sequences of FOR of P. furiosus and the AORs
of Thermococcus strain ES-l and Pyrococcus strain
ES-4 are also closely related to AOR. The third
tungstoenzyme from the hyperthermophiles, GAPOR,
shows low but significant similarity to AOR and
FOR, suggesting a more distant relationship. Turning
to the non-hyperthermophiles, AOR and FOR show a
much greater similarity to C. fi~rmicoaceticum CAR
(10/26 are identical in both cases) and to the o~-subunit (64 kDa) of C. thermoaceticum CAR ( 9 / 1 5 are
identical in both cases). In fact, in these four proteins
there is a highly conserved sequence (-L-R-V-N-LT-) beginning at residue 9 of AOR (Fig. 5). In
contrast, apart from the N-terminal methionine
residues, the /3-subunit (43000) of C. thermoaceticum CAR shows no similarity to AOR and FOR.
We therefore conclude that AOR, FOR, CAR and
GAPOR form a family of homologous enzymes, all
of which catalyze the reversible oxidation of aldehydes. Since the hyperthermophilic archaea are regarded as the most slowly evolving of all known
organisms [121], we assume that present day AOR
represents an 'early version' of a general aldehydeoxidizing enzyme. One can then speculate that during the course of evolution (i) FOR acquired a much
narrower substrate range and formed a tetrameric
structure; (ii) GAPOR acquired a specificity for aldehyde phosphates, perhaps in part due to the incorporation of Zn, and formed a monomeric structure: (iii)
CAR from C. thermoaceticum acquired a second,
smaller subunit that is unrelated to this family, therefore, its large subunit would be expected to contain
the W site; and (iv) the properties of AOR were
preserved in CAR from the bacterium, C. formicoaceticum, since its CAR and AOR are very similar
in subunit size, tertiary structure, and substrate specificity.
Surprisingly, the Mo-containing hydroxycarboxylate viologen oxidoreductase (HVOR) from the
mesophilic bacterium Proteus t,ulgaris [220] has an
N-terminal sequence (Fig. 5) that shows similarity to
the AOR family. As shown in Table 8, the subunit
size and metal content (exchanging W for Mo) of
HVOR matches that of AOR. Moreover, HVOR also
has a very broad substrate range, contains the nonnucleotide form of pterin, and is sensitive to cyanide
[220]. Interestingly, W was found to inhibit the
growth of P. z'ulgaris, whereas Mo had the opposite
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reciews 18 (1996) 5-63
effect. HVOR therefore appears not to be expressed
in alternative Mo- and W-containing forms, and this
'true' molybdoenzyme appears to be a member of
the W-containing AOR family. It is unclear whether
this family also includes the W-containing ADH of
D. gigas as its N-terminal sequence has only a short
stretch of tentative similarity to the AOR-type enzymes. On the other hand, in addition to CAR, C.
thermoaceticum possesses a second aldehyde-oxidizing enzyme that contains Mo which we refer to as
Mo-AOR (Table 8). By its N-terminal sequence and
subunit size, it bears a remarkable similarity to the
Mo-containing AOX of D. gigas and is clearly not
part of the W-containing AOR family (Fig. 5).
Thus, it can also be concluded that there are at
least two phylogenetically separate families of enzyme with aldehyde-oxidizing activity present in various organisms. One is the W-based AOR family
( F O R / C A R / G A P O R and maybe ADH) which includes Mo-based HVOR, and the other is Mo-based
A O R / A O X . Note that the AOR family shows no
N-terminal sequence similarity to any of the other
tungstoenzymes, including the subunits of FMDH lI
of methanogens (nor with those of the Mo-containing FMDH Is), nor with AH of P. aceO,lenicus.
Hence, for now, at least based on N-terminal sequences, there appear to be three families of
tungstoenzyme, and these are represented by AOR
( F O R / C A R / G A P O R ) , FMDH and AH. It remains
to be determined whether the other two types of
W-containing enzyme known, FDH and ADH, can
be included in any of these three groups. FDH
obviously has different catalytic properties from the
other three families, and it seems likely that it will be
the first member of the fourth group of tungstoenzyme.
6.6. The tungsten-pterin catalytic site
A pterin moiety has been found in all tungsto- and
molybdoenzymes that have been examined, with the
notable exception of nitrogenase. However, the crystal structure of P. furiosus AOR [272] provided the
first unequivocal evidence that the W atom is directly coordinated to the S atoms of a pterin-based
cofactor (or specifically, by four S atoms provided
by two pterin molecules). The structure also demon-
47
strated that the pterin was a three-ring system. Of
course, the association of the W atom with pterin
was obviously anticipated based on the voluminous
literature on the likely structure and function of
pterin in molybdoenzymes (for reviews, see [1522,305,309,310]). Such studies have established that
the pterin cofactor (Fig. 6) is unmodified or lacks a
terminal nucleotide in all known eukaryotic molybdoenzymes, while the prokaryotic version usually
contains a dinucleotide group. This is not true for the
tungstoenzymes, as the AORs of P. furiosus and
Pyrococcus strain ES-4, the FORs of T. litoralis and
P. furiosus, CAR l of C. thermoaceticum, and CAR
from C. formicoaceticum, have all been shown to
contain the unmodified pterin. The GDP dinucleotide
form is found in FMDH I1 of M. wo(fei and Mb.
thermoautotrophicum, while the type of pterin in C.
thermoaceticum FDH, D. ,eigas ADH and P.
acetvlenicus AH have not been determined.
That the nature of the pterin moiety in tungstoenzymes was the same as in molybdoenzymes was
demonstrated in the early 1980s when it was shown
that a mutant nitrate reductase (nit-I) lacking the
pterin cofactor could by activated in vitro with extracts of C. thermoaceticum FDH, but only in the
presence of additional molybdate [311]. This experiment was based on an earlier study in which a
W-substituted nitrate reductase was shown to reconstitute the activity of the nit-1 protein but only if
molybdate was present [312]. Further aspects of the
chemistry, biochemistry and biosynthesis of the pterin
cofactor of molybdoenzymes will not be discussed
here since these topics have been reviewed repeatedly (see above) and for more recent literature the
reader is referred to Refs. [313-317]. Moreover, in
the absence of any data, it is assumed that the pterin
biosynthetic pathway and its regulation is the same
for tungstoenzymes as it is for molybdoenzymes.
Similarly, there are many reports on synthetic compounds as models for Mo- [15,281,305,318,319] and
W-based [280,320-323] pterin cofactors. However,
the emphasis in this area might change somewhat in
the future, based on the finding that the W site in P.
.fi~riosus AOR is coordinated by four S atoms from
two pterin molecules.
The unexpected finding of a dipterin site in P.
furiosus AOR raises the issue of whether this is a
general feature of all tungsto- and molybdoenzymes?
48
A. Kletzin, M. W. W. Adams / FEMS Microbiology Ret'iews 18 (1996) 5-63
For those that contain a pterin dinucleotide, this
appears unlikely as preliminary crystallographic data
on the Mo-AOR of D. gigas indicates that the Mo is
bound by a single pterin molecule (Romao, M.J.,
personal communication). However, a dipterin structure for enzymes containing the unmodified pterin
would seem likely, although crystallographic data
from other enzymes is obviously needed. So, what
variation is there in the pterin- and W-containing
catalytic sites of the known tungstoenzymes, in particular, in the nature of the pterin and of the nonpterin ligands to the W atom? At present, C. thermoaceticum FDH is the only other tungstoenzyme
besides P. furiosus AOR for which there is some
structural information. EXAFS and inhibition analysis has shown that its W site is coordinated by 2
W - O / N , 2-3 W-S and one putative W-Se ligand
from selenocysteine [8,279]. These data suggest that
only a single pterin is coordinated to the W atom,
and that this site differs considerably from the W
center in AOR, for which there are 4-5 W-S interactions (with 4 S from the dipterin) and no coordinating protein residues. Thus, as might be expected, this
preliminary comparison suggests that the W sites in
tungstoenzymes are likely to show a range of properties. On the other hand, the W site in the reduced
forms of C. thermoaceticum FDH [278], D. gigas
ADH [188], and in the W-substituted form of Mb
wolfeii FMDH I [27], are similar in that all are
predominantly in the reduced W(IV) state. Thus,
partial oxidation of these enzymes was required to
induce a W(V) EPR signal. In contrast, the dioxocontaining W site in the RTP form of P. furiosus
AOR was predominantly oxidized W(VI) [303], although the redox state of the W site in active AOR,
which contains a single terminal oxo group, has yet
to be established. At present, from a structural perspective, we can only conclude that the range of
variants observed in the Mo sites of molybdoenzymes is quite likely to be reflected in the W sites of
tungstoenzymes.
6. 7. Tungsten-substituted molybdoenzymes
If the W sites of tungstoenzymes are structurally
analogous to the Mo sites in molybdoenzymes, e.g.
[323], one might expect that molybdoenzymes would
retain catalytic activity after substitution of Mo by
W. However, the only example of where Mo and W
can be interchanged to yield active enzymes is the
FMDH isoforms found in the thermophilic
methanogens (see above). Thus, the literature is replete with reports where the growth of organisms on
W leads to the formation of inactive, W-substituted,
molybdoenzymes. The best studied example is nitrate reductase (NR) which catalyzes the two electron reduction of nitrate to nitrite (reviewed, for
example, in [218,225]). Thus, when the fungus Neurospora crassa is incubated in a medium containing
tungstate, it synthesizes an enzyme that resembles
native NR in its behavior during purification, in its
thermal stability, sedimentation, inducibility and its
NADPH-cytochrome c oxidoreductase activity, but
the enzyme did not catalyze nitrate reduction [324].
Similar effects of W on NRs have also been described in other fungi [237], higher plants [203,217],
algae, [325] and bacteria [198,326], and direct incorporation of W into NR has been demonstrated
[198,219,326,327]. Thus, Azotobacter chroococcum
grown in the presence of 99Mo yields active radiolabelled NR, but in the presence of J8SW inactive
radiolabelled NR is produced [198]. On the other
hand, the ability to grow in the presence of W can be
used as a diagnostic tool for the identification of
regulatory NR mutants, and W has been used to
monitor the expression of NR in plants [203,204,328]
as well as in bacteria [105,193].
However, there have been no structural or spectroscopic comparisons of a native Mo-containing
nitrate reductase with its inactive, W-substituted
counterpart, and so it has not been established why
W-containing NR is inactive. Indeed, this issue has
only been addressed with a W-substituted form of rat
liver sulfite oxidase (W-SOX), and this was in a
pioneering study conducted in the mid-1970s
[25,26,329]. It was shown that upon treatment with
sulfite, inactive W-SOX exhibited a W(V) EPR signal analogous to the Mo(V) EPR signal of sulfitetreated, native Mo-SOX [26]. Their respective g,v
values were 1.97 and 1.89, consistent with electronic
properties of the two elements [278]. Moreover, experiments conducted in D20 showed that the W site
had a dissociable proton, just like the Mo site, and
that their pH dependence and anion-binding properties were similar. However, while dithionite readily
reduced the Mo(V) site in SOX to Mo(IV), the W(V)
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reciews 18 (1996) 5-63
site in SOX was not reduced. The inability of W-SOX
to catalyze sulfide oxidation was therefore attributed
to the much lower potential of the W ( I V / V ) redox
couple relative to the M o ( I V / V ) couple in the catalytic site of SOX. In fact, the initial reduction by
sulfite of the W(IV) site in oxidized W-SOX was
proposed to occur via intermolecular electron transfer from residual Mo-SOX in the W-SOX preparation [26]. Thus, W-substitution of Mo-SOX leads to
a catalytic site that accepts and donates electrons at
much lower potentials than those of the native enzyme, and at potentials that are not compatible with
the two electron oxidation of sulfite. Although the
reduction potentials of W- and Mo-sites within the
same enzyme have not been measured, the conclusions drawn from the results of SOX are presumably
applicable to other molybdoenzymes such as NR.
The effects of W-substitution have also been investigated with the one Mo-containing enzyme that
does not contain a pterin cofactor, nitrogenase. This
enzyme is highly conserved in all N2-fixing organisms. In brief, it consists of two proteins which
together catalyze the ATP-dependent six electron
reduction of N z to ammonia with concomitant two
electron reduction of two protons to form H 2 (reviewed, for example, in [190,330-333]). In the Modependent nitrogenase, the N2-reducing MoFe protein accepts electrons from the Fe protein (M r approx. 60 kDa), which contains a single [4Fe-4S]
cluster. The MoFe protein ( a 2/32, M r approx. 220
kDa) contains two 'P' clusters and two FeMo cofactors, the site of N 2 reduction. The structures of these
two sites were elucidated only recently with the
determination of the crystal structure of the MoFe
protein from Azotobacter cinelandii [334-336] and
Clostridium pasteurianum [337]. Each P cluster contains eight Fe atoms while the FeMo-cofactor has a
MoFe7S 9 stoichiometry and contains a MoFe3S 4
subcluster as well as an organic moiety, homocitrate.
The Mo atom is hexacoordinate with 3 inorganic S
ligands of the cluster, one N ligand from a histidine
side-chain, and two O ligands from homocitrate. In
the so-called alternate V- and Fe-only nitrogenases,
it is thought that the Mo atom is replaced by V and
Fe, respectively. Hence, the Mo site of nitrogenase is
distinctly different from that in all other molybdoenzymes.
It has been known for many years that W is a
49
competitive inhibitor for the expression of active
nitrogenase [191-193,193,199], that W is incorporated into the enzyme [24,200,201,207], and that
there are some W-resistant bacterial strains [208].
However, there is only one example of an active
W-substituted nitrogenase [206]. In contrast to Wsensitive strains and to the W-dependent Azotobacter cinelandii strain WN 101 [205], a W-resistant
strain of A. cinelandii was shown to produce a
nitrogenase which upon purification contained Mo
and W in a 1:1 ratio [206]. EPR analyses indicated
that it contained a W-substituted, FeMo-cofactor,
which gave rise to a S = 3/2-type signal analogous
to that of the native cofactor. However, while the
Mo-dependent EPR signal decreased upon enzyme
turnover, the putative W-dependent signal did not,
implying that the W-cofactor is inactive, as established by previous growth studies with W and its
effect on the N2-fixing process. It was postulated that
the active W-containing form of the enzyme arose
from protein molecules containing one FeMo-cofactot (active) and one 'FeW'-cofactor (inactive). Substitution of Mo by W in the FeMo cofactor gave rise
to a cluster with a much lower reduction potential
than the Mo-form, such that it could not be reduced
during the catalytic cycle and hence was inactive
[206]. The analyses of W substitution in both nitrogenase and pterin-containing molybdoenzymes therefore show that lack of catalytic activity is due, not to
the lack of incorporation of W, but to the differences
in the reduction potential of analogous W and Mo
sites. The tendency of synthetic W complexes to
have lower reduction potentials than their Mo counterparts [39,338,339] supports this interpretation.
7. Conclusions: is tungsten really a phylogenetically ancient redox cofactor?
With the exception of acetylene hydratase, all of
the tungstoenzymes known so far (Tables 7 and 8)
catalyze reactions of very low potential (_< - 4 2 0
mV), and they have all been purified from strictly
anaerobic organisms (although, as discussed, some
aerobes are known to exhibit W-dependent growth).
The majority of these organisms are also thermophilic or hyperthermophilic. The thermophilic organisms appear not to be obligately dependent upon
50
A. Kletzin, M. W. W. Adams / FEMS Microbiology Reciews 18 (1996) 5-63
W for growth as analogous molybdoenzymes are
synthesized if W is omitted and Mo is added to the
culture medium. The heterotrophic and hyperthermophilic archaea represented by P. furiosus are the
only known organisms that do not replace their
tungstoenzymes with Mo-containing analogs.
Whether they are the first organisms to absolutely
require W for growth remains to be established, as
the complex media that they are grown in contain
trace amounts of W, and it is not clear if the
organisms will grow in the complete absence of W.
The fact that they contain three different types of
tungstoenzyme (AOR, FOR and GAPOR) which are
proposed to function in their primary metabolic pathways would support the conclusion that they are
obligately W-dependent. The presence of a putative
GAPOR gene in Pyrodictium occultum (Kletzin, A.
and Adams, M.W.W., unpublished observations), a
hyperthermophilic archaeon only distantly related to
Pyrococcus and Thermococcus, suggests that W-dependency may be widespread in this group of organisms. It may not to be a universal property of the
hyperthermophilic archaea, however, as the FMDH
from the sulfate-reducing Archaeoglobus fulgidus
[148] might contain Mo, although this has not been
proven. Nevertheless, since 'hyperthermophily' is
regarded as an ancestral phenotype [116-121], one
could speculate that the earliest life forms were not
only hyperthermophilic but also W-dependent
[28,36,37,131]. These then evolved into the
mesophilic, Mo-dependent species that now overwhelmingly predominate. Consequently, a limited
number of moderately thermophilic and mesophilic
species exist that can use W or Mo. So, is there
evidence to support this hypothesis, and what advantages or disadvantages are there in using W rather
than Mo? The 'choice' of W or Mo obviously depends on the availability of the two metals and their
chemical properties, properties that may or may not
enable organisms to discriminate between them.
As discussed above, Mo (110 nM) is far more
abundant than W (1 pM) in oxic, marine environments, systems in which higher life forms are assumed to have evolved. In general, no element has
been found to be essential for life if its abundance is
less than that of cobalt (Co), and rare heavy metals
such as W are used only in exceptional cases; more
frequently, they are toxic [340]. Since the concentra-
tion of W in sea water is far below of that of Co (0.9
nM: [82]), there appears to be a fundamental obstacle
for its utilization in the open ocean. There is no
reason to believe that the rate of W-scavenging and
sedimentation from sea water was much different in
the primordial ocean and its W concentration is
assumed to be similar. Consequently, higher organisms, whether they inhabit sea, fresh water or land,
do not have to discriminate between W and Mo, and
they appear not to have that ability. Specific uptake
and incorporation mechanisms for W rather than Mo
appear to be limited in the anaerobic microorganisms
that can use one or both of the elements. However,
the situation is very different in marine sediments
and hydrothermal vents where, compared to the open
ocean, W is present in much higher concentrations
(Table 2). Thus, it may not be coincidental that life
has been proposed to have originated at extreme
temperatures in deep sea hydrothermal systems (for
recent reviews, see [341,342]), and that at least some
of the present-day marine hyperthermophiles appear
to be obligately W-dependent. Consequently, a potential factor in the present-day dominance of Mocontaining enzymes may well be the result of utilization of the more readily available of two elements
during the course of the evolution.
In addition to availability, a key factor in W
utilization appears to be its redox properties relative
to Mo. At least some W-substituted molybdoenzymes are inactive because of the lower reduction
potential of the W site with respect to the Mo site, in
accordance with the properties of synthetic complexes of the two elements [338,339]. Conversely, to
catalyze a reaction of extremely low potential, W
should be 'preferred' over Mo. This notion is supported by the fact that all known tungstoenzymes
catalyze reactions at potentials near or below the
standard hydrogen electrode, with the exception of
the hydration reaction catalyzed by acetylene hydratase. Nevertheless, for all of these tungstoenzymes, there is a molybdoenzyme known which
catalyzes the same chemical reaction. So, can we
assign a specific function to a tungstoenzyme that
cannot be performed by a Mo-containing counterpart? To date, the only reaction is aldehyde oxidation
at temperatures near and above 100°C, catalyzed by
AOR, FOR and GAPOR of the hyperthermophilic
archaea. This reaction has one of the lowest reduc-
A. Kletzin. M. W. W. Adams / FEMS Microbiology Reciews 18 (1996) 5-63
tion potentials in biological systems (carboxylic
acid/aldehyde, E ° ' = - 5 8 0 mV, pH 7.0, 25°C;
[269]), suggesting that only W, and not Mo, is able
to catalyze aldehyde oxidation and acid reduction at
the highest temperatures known to support life. It
remains to be determined whether the FMDHs present in other hyperthermophilic archaea, such as
methanogens and the sulfate-reducing A. fulgidus,
are W- or Mo-containing enzymes. Notably, the
FMDH reaction has a more positive reduction potential (CO2/formylmethanofuran, E °' = - 497 mV, pH
7.0, 25°C, [268]) than aldehyde oxidation, and both
Mo and W may be able to participate in catalyzing
CO 2 reduction near 100°C.
An absolute requirement of W may therefore be
limited to certain extremely low potential reactions,
such as aldehyde oxidation, but only when they are
carried out at extreme temperatures. In contrast, the
tendency of Mo-containing centers to have higher
redox potentials, and potentials that are more in the
biological range, makes it a much more versatile
element, particularly in oxic environments. Whether
W was preferred to Mo by ancestral microorganisms
such that W-containing enzymes are the evolutionary
precursors of all Mo-containing enzymes, or whether
present day hyperthermophiles have evolved to utilize W to catalyze a very specific reaction in a
unique environment, is a question that cannot be
answered at present. Parallel evolution of W- and
Mo-containing enzymes may have taken place once
a metal-pterin cofactor was 'invented', or alternatively, a tungstodipterin site of the type found in P.
furiosus AOR may have been the precursor to all
present day Mo-containing and monopterin dinucleotide-containing enzymes. Perhaps, in the future,
with the realization that W has a much more
widespread role in biological systems than was
thought even 5 years ago, new types of tungstoenzymes will be isolated. Hopefully, their properties
may enable some of these questions to be more fully
addressed.
8. Note added in proof
Hoaki et al. [344] reported W and Mo concentartions of approx. 0.8 g / k g and 35.1 m g / k g in shallow marine bydrothermal sediments inhabited by a
51
dense community of hyperthermophilic, sulfur-dependent heterotrophs off the coast of a Japanese
island (see Table 2). Hochheimer et al. [345] reported the cloning and sequencing of the W-FMDH
from the methanogen Methanobacterium thermoautotrophicum. The operon consisted of seven genes.
Four of them encoded the subunits of the enzyme
(see Table 7), two encoded ferredoxins and one
encoded polyferredoxin. The second largest subunit
of the FMDH (65 kDa) showed, in its N-terminal
half, sequence similarity to molybdopterin dinucleotide-containing enzymes including FDH, Rom~.o
et al. [346] reported the X-ray crystal structure of the
Mo-containing AOX from Desulfocibrio gigas. The
Mo atom was coordinated by three water ligands and
by two thiolene sulfur atoms from the guanine nucleotide form of a single pterin cofactor. The enzyme
also contained two [2Fe-2S] clusters, in accordance
with prior spectroscopic data.
Acknowledgements
We thank J,R. Andreesen, F. Girio, K. Granderath,
T.A. Hansen, M.J. Romao, B. Schink and R.K.
Thauer for providing results prior to publication, and
M.K. Chan, M.K. Johnson, S. Mukund, D.C. Rees
and R.K. Thauer for many helpful discussions. Research carried out in the author's laboratory was
supported by grants from the Department of Energy,
the Office of Naval Research, the National Institutes
of Health and the National Science Foundation.
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