Bioscience Reports, Vol. 17, No. 3, 1997
REVIEW
Respiratory Protection of Nitrogenase
Activity in Azotobacter vinelandii—Roles of
the Terminal Oxidases
Robert K Poole1 and Susan Hill2
Received December 17, 1996
Nitrogen fixation by aerobic prokaryotes appears paradoxical: the nitrogen-fixing enzymes—
nitrogenases—are notoriously oxygen-labile, yet many bacteria fix nitrogen aerobically. This review
summarises the evidence that cytochrome bd, a terminal oxidase unrelated to the mitochondrial and
many other bacterial oxidases, plays a crucial role in aerotolerant nitrogen fixation in Azotobacter
vinelandii and other bacteria by rapidly consuming oxygen during uncoupled respiration. We review
the pertinent properties of this oxidase, particularly its complement of redox centres, the catalytic
cycle of oxygen reduction, the affinity of the oxidase for oxygen, and the regulation of cytochrome bd
gene expression. The roles of other oxidases and other mechanisms for limiting damage to nitrogenase
are assessed.
KEY WORDS: Cytochrome bd; oxygen utilisation; nitrogen fixation; bacterial oxidases.
1. INTRODUCTION
"... must protect its own existence as long as such protection does not conflict
with the First or Second Laws" Isaac Asimov. I, Robot (1950) 'Runaround'
Biological N2 fixation is an anaerobic process requiring a large amount of
ATP and a source of low potential electrons.
N2 + 8H+ + 8e~ + 16ATP = 2NH3 + H2 + 16ADP + 16Pi
(1)
This process is catalysed by the nitrogenases; they differ in their metal content,
but all contain Fe and are composed of two proteins. The most common and
extensively studied type of nitrogenase contains Mo. The others contain either V
or Fe alone and appear to be alternatives in some organisms (Bishop &
Premakumar, 1992). They are more O2-sensitive and more energy-demanding
than the Mo enzyme and their quantitative contribution may be slight.
The ability to fix N2 is restricted to prokaryotes, but is widely distributed,
1
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology,
The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, and 2Nitrogen Fixation
Laboratory, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK.
303
0144-8463/97/0600-0303512.50/0© 1997 Plenum Publishing Corporation
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Poole and Hill
occurring in obligate anaerobes, facultative anaerobes and obligate aerobes
(Young, 1992). The principal input of fixed N2 into the biosphere is from
phototrophs and from heterotrophs in symbiosis with plants (Sprent & Sprent,
1990). The majority of these prokaryotes are obligate aerobes. In addition, a wide
range of free-living heterotrophs fix N2. Clearly, the ability to carry out an aerobic
catabolism is beneficial for the high ATP requirement for N2 fixation, but it could
be detrimental since the presence of oxygen may inactivate nitrogenase. A range
of strategies have evolved to cope with this paradox (Sprent & Sprent, 1990).
These provide, to differing degrees, tolerance towards O2 during diazotrophy. For
example, N2 fixation in the free-living facultative anaerobe Klebsiella pneumoniae
occurs anaerobically, but an O2 limitation is beneficial, when measured by the
amount of N2 fixed per unit of carbon and energy source consumed (Hill, 1992).
Under these conditions, the maximum dissolved O2 concentration tolerated is
about 30 nM. In contrast, the free-living obligate aerobe Azotobacter can fix N2
over a wide range of O2 concentrations. It can grow diazotrophically in an
air-saturated medium (approx. 225/iM O2) (Poste et al, 1983a) as well as
effectively scavenge traces of O2. Under conditions of O2 limitation, growth is
accompanied by the deposition of polyhydroxybutyrate, which acts as an electron
sink and store of carbon and energy (see Hill, 1992 and references therein).
Azotobacters are found in soil and fresh water (Thompson & Skerman, 1979),
where supplies of carbon and energy, nitrogen, water, and O2 are likely to
fluctuate.
It is now 40 years since Lenhof and Kaplan suggested a branched respiratory
pathway in bacteria. In 1967, Harrison and Pirt proposed that the high,
"uncoupled" rate of respiration in Klebsiella aerogenes at low dissolved oxygen
tension was a result of a change to an alternative pathway of electron flow to
oxygen (for references, see Harrison, 1976). However, the concept that uncoupled
respiratory activity might prevent O2 inhibition of nitrogenase activity in
Azotobacter was formulated by Dalton & Postgate (1969a) to explain its
particular behaviour when fixing N2. Azotobacter has the ability to adjust its
respiratory rate to match a wide range of O2 supplies (Drozd & Postgate, 1970,
Poste et al, 1983a). It has one of the highest rates of respiration known,
particularly when fixing N2. The high uncoupled rate of respiration as the rate of
O2 input is increased is accompanied by a decline in the amount of N2 fixed per
unit of carbon energy consumed, giving rise to the high maintenance requirements typical of Azotobacter (Dalton & Postgate, 1969b, Kuhla & Oelze, 1988).
Here, we review the present knowledge of the respiratory chain of Azotobacter
sp. and its role in diazotrophy.
2. ORGANISATION OF THE RESPIRATORY CHAINS OF
A. VINELANDII
The respiration of Azotobacter has been extensively studied (see Haddock &
Jones, 1977; Robson & Postgate, 1980; Yates & Jones, 1974; Yates, 1988). The
respiratory system is located in the extensively invaginated membrane and
Protection of nitrogenase
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Fig. 1. Scheme for the respiratory chains of A. vinelandii with special
reference to the oxidases. Dehydrogenases for NAD(P)H, malate and
other substrates are shown collectively transferring electrons to a pool of
ubiquinone-8 (UQ). During diazotrophy, the major electron flux to
oxygen occurs via the noncoupled pathway that terminates in cytochrome
bd, encoded by the cydAB genes and which has a Km in vivo of about
5 /iM. The alternative branch to oxygen is not required for a diazotrophy
and consists of a cytochrome o-like oxidase in the haem-Cu superfamily
and/or the products of the ceo operon. Two higher affinity states are
measured in vivo, but assignment to particular oxidases has not been
made.
consists of a rich complement of redox carriers. A revised version of the branched
chain proposed by Haddock & Jones (1977) is shown in Fig. 1.
A. vinelandii is an obligate aerobe and no reductases for anaerobic utilisation
of alternative electron acceptors exist. Haddock & Jones (1977) proposed three
routes for electron transfer to oxygen. These were (i) a major route via
cytochromes b and d, (ii) a route via cytochromes c4 and cs to a "cytochrome
oai" complex, and (iii) cyanide-insensitive autooxidation of cytochrome b. This
scheme was based on studies of membranes and the sensitivities of the terminal
branches to CO and cyanide, photodissociation spectroscopy of the CO complexes, and the strikingly different oxidation-reduction kinetics of the b- and
c-type cytochromes. Recent studies have necessitated a reinterpretation of some
of these data (e.g. the role of "cytochrome flj"). Other aspects of respiratory
chain organisation, such as the roles of quinones and the basis of cyanide
insensitivity, have not been followed up.
2.1. Low Potential Segments of the Respiratory Chain
The branched respiratory system of A. vinelandii comprises flavin-dependent
dehydrogenases (NADH, NADPH, succinate, and malate), donating electrons to
a pool of ubiquinone-8. The dehydrogenases are fed by the tricarboxylic acid
(TCA) and glyoxylate cycles of central metabolism. Moderate increases in
oxygenation appear to cause increases in the levels and activity of NAD(P)H and
malate dehydrogenases (Poste et al, 1983b) as well as of enzymes associated with
the TCA cycle (see Hill, 1992 for references). The importance of an active TCA
cycle has been shown by characterisation of mutants of Azotobacter chroococcum
that are unable to fix N2 in air when provided with sugars (Fos~ mutants) as a
Poole and Hill
306
result of defects in enzymes that replenish TCA cycle intermediates (Ramos &
Robson, 1987).
During diazotrophy, the flux of reducing power for nitrogenase activity
represents only a small diversion of electron flow away from the respiratory chain
(only 1-10%) (Yates, 1988). The electron input to nitrogenase is from flavodoxin
hydroquinone, although in Azotobacter the pathway of electron transfer from a
carbon intermediate to flavodoxin is unclear. A scheme for respiratory-linked
reversed electron flow has been proposed (Haaker & Klugkist, 1987). A
proportion of the electron donation to nitrogenase (a minimum of 25%) appears
as hydrogen (see equation 1), which, in Azotobacter, is recycled through the
respiratory chain by an uptake hydrogenase. Whether this recycling is beneficial
remains controversial (Yates, 1988; Linkerhagner & Oelze, 1995a).
2.2 Cytochrome bd.
Evidence for involvement of the cytochrome bd-type oxidase in respiratory
protection of nitrogenase in A. vinelandii is threefold (for references, see D'mello
et al, 1994a).
(1) The level of this oxidase is increased when the supply of oxygen to a
culture is increased.
(2) Under such conditions, consumption of carbon and energy sources is
partially uncoupled from anabolism and there appears to be preferential electron
flux to this oxidase, rather than to cytochrome o-like oxidase(s) (Fig. 1). By
analogy with E. coli, cytochrome bd in A. vinelandii is indeed anticipated not to
be a proton pump; this allows electron transport to proceed without being limited
by ADP. An alternative oxidase identified by Leung et al. (1994), on the basis of
gene sequence similarlity with E. coli cyoB, encoding the haem-copper subunit of
cytochrome bo', is anticipated to be a proton pump.
(3) The demonstration that Cyd~ mutants cannot fix nitrogen in air was the
first genetic evidence for the essential role of this oxidase in respiratory
protection.
The suggestion by Haddock and Jones (1977) that "the cytochrome b-+d
branch thus resembles the terminal limb of... E. coli" has been fully vindicated
by molecular studies. The structural genes for cytochrome bd (cydAB) were first
cloned (using the E. coli cydAB operon for probing a genomic DNA library) by
Kelly et al. (1990), allowing construction of Tn5 mutants affected in cytochrome
bd synthesis and regulation. Subsequent sequencing of the cydAB operon
(Moshiri et al, 1990) revealed striking similarities with the Escherichia coli
cytochrome fed-type oxidase, in accord with spectral studies. The deduced amino
acid sequences of the CydA and CydB subunits have extensive homology with
those of the corresponding subunits in E. coli and indistinguishable hydropathy
profiles. Amino acid residues identified as ligands to the haems and a quinonebinding site are preserved, but the amino acid(s) that are ligands to the d-type
haem are unknown in both organisms. The A. vinelandii oxidase can be expressed
in an E. coli Cyd" mutant under the control of the lac or tac promoter (Moshiri et
al., 1991). Although the presence of the A. vinelandii oxidase reconstitutes
Protection of nitrogenase
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NADH oxidase activity in these membranes, it does not support lactate or
succinate respiration. This surprising observation may suggest compartmentalisation of electron flow from particular dehydrogenases and could have important
implications for respiratory protection but appears not to have been studied
further.
One class of Tn5 mutants has proved to be particularly useful in elucidating
the structure and function of the cytochrome bd-type oxidase. Mutations
upstream of the cydAB operon in cydR (Wu et al, 1997) over-express this oxidase
and such mutants have been used to purify the oxidase (Kolonay el al, 1994;
Jiinemann et al., 1995; Jiinemann & Wrigglesworth, 1995). These studies have
reinforced the view that the E. coli and A. vinelandii cytochrome bd complexes
are similar in terms of their subunit composition, spectral properties and reactions
with ligands. Analysis of the redox centres by Jiinemann & Wrigglesworth (1995)
supports the view that there is one mole each of haem d, haem fo558 (low spin)
and fe595 (high spin). Electron transfer from quinol bound to CydA occurs via
haem 6558 to the other two haems, which may be in a common pocket in CydB,
where oxygen is reduced, as in E. coli (Hill et al., 1993). Despite these apparent
structural similarities with the E. coli enzyme, the kinetic behaviour in vivo and
the regulation of cydAB expression are markedly distinct (see below). Although
no studies of the topology of the CydA and CydB subunits within the membrane
have been made with A. vinelandii, it is probable that the organisation is the
same as in E. coli. It is tacitly assumed that the H+ for oxygen reduction in the E.
coli oxidase originate on the inside of the cytoplasmic membrane, whereas a
scheme for the A. vinelandii oxidase places the oxygen-reducing site on the
outside (Jones, 1977). Such a disposition is attractive if a major role of
cytochrome bd is to protect nitrogenase (on the inside) from oxygen penetrating
from the outside, and could also explain the different oxygen affinities measured
in cells and membranes (D'mello et al., 1994a). In E. coli, two further proteins
(the CydD/CydC ABC-type transporter) are required for assembly of the
cytochrome bd complex and probably translocate an unidentified substrate into
the periplasm (see Poole, 1994). Interestingly, we have failed to detect cydD and
cydC homologues in A. vinelandii (S. E. Edwards, S. Hill & R. K. Poole,
unpublished).
The catalytic mechanism of oxygen reduction by cytochrome bd has been
studied with both membranes and purified oxidase from a cytochrome bdoverexpressing cydR mutant. CO and NO, probes of the oxygen-binding site(s),
bind to haem d with high affinity and to reduced haem b595 with lower affinity
(Jiinemann & Wrigglesworth, 1995). These studies confirm the weak Soret band
(443 nm) of haem d in E. coli (for references, see Poole, 1983, 1994) and A.
vinelandii (D'mello et al., 1994b). CO binds to both haem d and 6595 and evidence
has been presented in both the E. coli and A. vinelandii oxidases that oxygen is
bound at this haem-haem binuclear centre (for references see Jiinemann &
Wrigglesworth, 1995). However, results with NO and cyanide binding suggest that
any cooperative interaction between haems d must be weak and that the close
distance exchange coupling found between haem a3 and CuB in the haem-copper
superfamily of oxidases is not present in cytochrome bd. A remarkable feature of
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Poole and Hill
the oxidase in both E. coli and A. vinelandii is the ability to form stable
compounds with oxygen and its reduction products. Poole et al. (reviewed in
Poole, 1994) used low-temperature ligand exchange techniques and resonance
Raman spectroscopy to demonstrate that E. coli cytochrome d forms a stable
oxygenated complex (d650) with ferrous cytochrome d. The 680 nm form of
cytochrome d obtained on reacting hydrogen peroxide with the oxidase (see
Poole, 1994) is now attributed in the case of the E. coli oxidase to the ferryl form
(Kahlow et al., 1991). Both spectral forms can also be observed in A. vinelandii,
although early studies failed to explain their identity (reviewed in Poole, 1983).
The oxy-form of A. vinelandii cytochrome d is generated readily in air-exposed
samples or by photolysis of the Co-ligated cytochrome d in the presence of
oxygen at sub-zero temperatures (Poole, 1983; D'mello et al., 1994b). Subsequent
reaction leads to decay of this species and thence a wavelength shift in the
absorbance due to cytochrome b595 suggesting that oxygen migrates between the
haems under these conditions (D'mello et al., 1994b). A model for the catalytic
cycle of oxygen reduction by A. vinelandii cytochrome bd (Jiinemann et al, 1995)
proposes that the oxygenated form (650 nm) is not a true intermediate but is
formed by reaction of oxygen with the haem d formed by reduction with a
reducing equivalent remaining in the complex after turnover in the absence of
further reductant. This proposal is supported by changes in the dMO form
independently of redox changes in cytochromes b in vivo (S. E. Edwards, S. Hill
& R. K. Poole, unpublished). Whether the very unusual formation of these stable
forms is directly related to the ability of cytochrome bd to participate in
respiratory protection is unknown.
2.3 Cytochrome o-like Oxidase(s)
Less is known of the properties of the oxidase(s) terminating alternative
route(s) to oxygen in A. vinelandii, although early work showed cytochrome(s) c
to be involved (Wong & Jurtshuk, 1984; Yang, 1986 and references therein). Km
measurements reveal, in addition to cytochrome bd, two high affinity oxidase
activities (D'mello et al, 1994a). Assuming that a second oxidase might belong to
the "haem-copper" superfamily of terminal oxidases (Poole, 1994), in which the
oxygen-reactive binuclear centre comprises a copper atom coupled with a haem
A, B, or O, we cloned (with PCR primers designed to amplify a gene in this
superfamily) a 0.55 kb fragment of such an oxidase gene from A. vinelandii
(Leung et al, 1994). Recombination of the fragment carrying a kanamycinresistance marker with the chromosome resulted in a mutant lacking this oxidase,
tentatively called Cyo. Since this mutant (cyo) remained aerotolerant when fixing
nitrogen, the role of this oxidase is clearly different from that of cytochrome bd
(Leung et al, 1994). The mutant over-expresses the latter oxidase, particularly at
lower oxygen tensions, presumably as compensation for the lack of the haemcopper type oxidase. Recently, a gene fragment from A. vinelandii has been
sequenced and shown to resemble fixN or ccoN encoding a cb-type cytochrome c
oxidase (Ceo) (Thony-Meyer et al, 1994). It is not known whether Ceo and Cyo
are distinct oxidases. Ng et al (1995) have cloned, sequenced and mutated the
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gene encoding cytochrome c4 of the A. vinelandii respiratory chain. Such mutants
grow and respire normally, even under nitrogen-fixing conditions, indicating that
cytochrome c4 is not essential for diazotropy.
2.4 Energy Conservation
Energy conservation (see Yates & Jones, 1974, Yates, 1988, for reviews) is
associated wtih NADH dehydrogenase, (site I), the ubiquinone segment (site II),
the branch associated with the cytochrome bo-type oxidase (site III) and possibly
with the hydrogenase (site IV). Increases in the O2 supply to cultures lead to loss
in respiratory control (the ability of ADP and phosphate to stimulate O2 uptake),
and to losses of energy conservation at sites I and III. Upon perturbations in
either O2 or N status, the level of intracellular ATP appears to be linked to
nitrogenase activity, and various mechanisms have been proposed to account for
these changes (Linkerhagner & Oelze, 1995b; see Hill, 1992 and references
therein). However, uncertainties remain concerning the precise balancing of
energy supply and demand during growth at high rates of O2 supply.
3. APPARENT AFFINITIES OF THE TERMINAL OXIDASES FOR
OXYGEN
Evaluation of the respiratory protection hypothesis requires knowledge of
the properties of the candidate oxidases. However, measurement of the Km values
for terminal oxidases is not a trivial task since oxygen concentrations of a few ^iM
cannot be reliably measured with membrane-covered polarographic electrodes, in
which the response at low oxygen tensions is limited by the "unstirred layer" at
the membrane. Nevertheless, early measurements with A. vinelandii suggested
that the affinity for oxygen was relatively low (for references, see D'mello et al,
1994a). A re-evaluation has been made of the apparent affinity of cytochrome bd
for oxygen in cells and membranes, exploiting (i) the availability of a mutant
defective in cytochrome bd synthesis—allowing the contribution of this oxidase to
be assessed—and (ii) a modified analytical method for oxygen consumption
employing continuous multi-wavelength recording of the deoxygenation of
oxyleghaemoglobin or oxymyoglobin as sensitive reporters of dissolved oxygen
concentration. A kinetic component with a Km of about 4.5 p,M O2 has been
assigned to cytochrome bd (D'mello et al., 1994a) based on the absence of such a
component in measurements of the Cyd" mutant. Subsequent measurements
using cytochrome bd purified from the over-producing cydR mutant have given
very similar values, whether measured with an oxygen electrode (5.7 /j,M,
Kolonay et al., 1994) or oxymyoglobin deoxygenation (4.1 /xM, Junemann et al,
1995). Measurements of the oxygen uptake kinetics of cells and membranes, in
which all oxidases are anticipated to be present, reveal two further components,
both of higher affinity (apparent Km values about 0.33 /xM and 0.016 /uM), which
have not been assigned to specific oxidases. Molecular evidence raises the
possibility that there are two oxidases other than cytochrome bd (see section
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Poole and Hill
2.3), but unequivocal assignment of these Km values to the oxidases has not yet
been made.
4. REGULATION OF OXIDASE EXPRESSION BY OXYGEN AND
NITROGEN STATUS
There is a remarkable difference between the E. coli and A. vinelandii
oxidases with respect to regulation of synthesis and the affinities of these oxidases
for oxygen. The E. coli cytochrome bd-type oxidase is synthesised maximally
microaerobically (Tseng et al, 1996). Expression of the cydAB operon comprising
the oxidase structural genes is affected by Fnr and ArcA/ArcB, although
dissection of the direct or indirect roles of these regulators requires further study
(Gennis & Stewart, 1996). Cytochrome bd levels are also evaluated at low oxygen
tensions in the diazotroph Klebsiella pneumoniae (Smith et al, 1990). In contrast,
the level of cytochrome bd in N2-fixing A. vinelandii oxidase is greatest under
high aeration (Drozd & Postgate, 1970; Jones et al., 1973; Haaker & Veeger,
1976), although one report describes the reverse situation (Ackrell & Jones,
1971).
Mutations caused by Tn5 insertions upstream of cydAB result in overexpression of cytochrome bd and, surprisingly, the inability of the organism to
grow microaerobically. Sequencing the mutated region revealed a new gene cydR.
The deduced amino acid sequence of the product, CydR, indicates that it is a new
member of the Fnr class of transcription factors (for reviews, see Becker et al.,
1996). CydR has 79.5% identity to Anr, an Fnr-like protein from Pseudomonas
aeruginosa and striking levels of identity to other bacterial proteins in this family
(Wu et al, 1997). Several features of the amino acid sequence of CydR are
noteworthy. CydR has three cysteine residues at the N-terminus as well as an
internal cysteine at virtually identical positions to those of Fnr but it lacks the
most N-terminal cysteine residue (C-16 in Fnr) which, in Fnr, has been shown not
to be essential for activity. CydR also contains a helix-turn-helix motif at its
C-terminus in a position equivalent to that of Fnr and Crp and which presumably
binds target promoter sequences. We suggest that CydR is a negative regulator of
cydAB expression. The cloned cydR gene complements anaerobic growth of E.
coli frir mutants and strongly enhances expression of a narG-lacZ fusion
(regulated by Fnr) in an E. colifnr mutant (Wu et al, 1997).
The influence of the rate of O2 supply to batch cultures on the contents of
cytochromes bd and "o" in NH^-grown Azotobacter vinelandii has recently been
re-investigated, exploiting the availability of these mutants and fusions (D'mello
et al, 1997). The content of cytochromes d and &595 in a wild type, and the
expression of /3-galactosidase encoded by a Tn5-B20 inserted in cydB so as to
monitor transcription, increased as in the O2 supply was raised, suggesting that
O2 regulates cydAB expression even in the absence of diazotropy. In a cydR
mutant, which over-produces cytochrome bd, the responses to O2 supply were
reversed. CO difference spectra of both the wild-type strain and the cytochrome
ftd-deficient mutant revealed a haemoprotein with spectral characteristics similar
Protection of nitrogenase
311
to cytochrome o, the levels of which increased as the O2 supply was raised. These
results indicate that the content of the cytochrome o-like haemoprotein, like that
of cytochrome d, is regulated by O2 supply in a positive manner (at least in a
cydB mutant). Earlier estimates (Ackrell & Jones, 1971; Haaker & Veeger, 1976)
of the content of a cytochrome o-like haemoprotein, showed that the level
increased when the O2 supply for growth was lowered. The difference between
these and earlier results may reside in the N source for growth. Understanding
the possible contributions of both nitrogen status and oxygen status to the
regulation of components of the branched respiratory chains of A. vinelandii will
require further study. Liu et al. (1995) have suggested that the levels of
cytochrome o are less responsive to oxygen tensions than those of cytochrome d.
However, cytochrome o levels are notoriously difficult to measure with precision
by CO difference spectroscopy in the presence of other CO-reactive oxidases.
Nevertheless it is interesting that galactose-grown A. vinelandii appear to contain
more cytochrome o than do glucose-grown cells which may explain why cell yield
is higher in the former cultures.
5. RESPIRATORY PROTECTION IN OTHER BACTERIA
Respiratory protection has been invoked to explain the prevention of
inactivation of oxygen-sensitive nitrogenase in the cytoplasm of Azotobacter (this
review) and of Rhizobium and Klebsiella under microaerobic conditions (Hill et
al, 1990; Witty et al., 1987). In A. vinelandii and K. pneumoniae, cytochrome bd
appears to terminate the respiratory protective electron transport pathway,
whereas in Rhizobium meliloti and Bradyrhizobium japonicum cytochrome cbb'
(or cbb3) is critically important (Batut et al., 1989; Preisig et al, 1993).
Azorhizobium caulinodans appears to be capable of synthesising five terminal
oxidases, two of which—cytochrome bd and cytochrome ebb'—act as oxidases
during growth at low oxygen tensions. Either seems to offer sufficient respiratory
protection to allow nitrogen fixation at 50% of wild-type levels when the
organism grows symbiotically (Kaminski et al, 1996). However, mutants defective
in both oxidases fail to fix nitrogen symbiotically, i.e. when the available oxygen is
maintained at around 10-20 nM by leghaemoglobin. Interestingly, in microaerobic culture, cytochrome bd is able to sustain growth and respiration only at or
above 3.6 ju,M oxygen whereas cytochrome ebb' does so at submicromolar oxygen
concentrations (Kaminski et al, 1996). It is therefore puzzling that cytochrome bd
appears to have a role in symbiosis when in vivo cultures suggest that this oxidase
is ineffective. Possibly, cytochrome bd has a high Vmax and assists the maintenance of the bacteroid oxygen tension well below that of external, oxygenunbuffered conditions; the cytochrome ebb' oxidase has a higher affinity for
oxygen but may have a lower Vmax. Alternatively, the kinetic behaviour of
cytochrome bd may be different in bacteroids from that observed in culture.
Recent experiments with E. coli suggest that a respiratory protection type of
mechanism does not affect the function of the global transcriptional regulator,
Fnr. Fnr activity may be directly influenced by cytoplasmic oxygen levels and
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their effect on a labile iron-sulfur cluster in Fnr. Indeed, diminished respiration
rates, achieved experimentally by mutations in cydAB or ubiA (ubiquinone
biosynthesis) increased the extracellular oxygen concentration required for
activation (= "oxygen damage"?) of Fnr activity, assayed by expression of an
Fnr-regulated 3>(frdA-lacZ) fusion (Becker et al., 1996). However, interpretation
of these data and of all experiments that control or measure extracellular oxygen
concentrations is frustrated by the lack of any direct measurement of intracellular
concentration and by the assumption that oxygen diffuses readily into the
cytoplasm and remains available for oxidative damage if not consumed.
Finally, in examining the cytochrome composition of bacteria to identify
potentially oxygen protective oxidases, it is salutary to consider the warning of
Harrison (1976): difference spectroscopy of cells or membranes is not generally
sensitive enough to detect oxidase concentrations that can support substantial
oxygen uptake rates! For example, the turnover number for A. vinelandii
cytochrome bd is about 1000s"1 (Jiinemann et al., 1995). If the molar absorption
coefficient in a CO difference spectrum is about 14mM~ 1 cm~ 1 (see D'mello et
al, 1997) and if the practical limit of detection is AA = 0-001 for a bacterial
sample containing 10 mg protein ml"1 (much less optimistic than the value given
by Harrison, 1976), then such an oxidase level could support a respiration rate of
480nmol O2 mg"1 min"1. This exceeds the rates measured for membranes from a
wild-type strain of A. vinelandii (Kelly et al, 1990). Experimental data supporting
the concept that bacteria have excess terminal oxidase for observed respiration
rates has been obtained with several bacteria (see Harrison, 1976) including A.
vinelandii (Liu et al, 1995).
6. OTHER WAYS OF PREVENTING O2 FROM DAMAGING
NITROGENASE
A respiratory chain terminating in the cytochrome id-type oxidase is clearly
crucial for preventing O2 damage to nitrogenase because a cydB mutant lacking
this oxidase fixes N2 only under micro-aerobic conditions (Kelly et al, 1990,
Linkerhagner & Oelze, 1995b). However, other mechanisms exist in Azotobacter
which provide protection to nitrogenase (for reviews, see Robson & Postgate,
1980; Yates, 1988; Hill, 1992). The short-term reversible inhibition by O2 of
nitrogenase activity in intact Azotobacter chroococcum was first described by
Postgate and colleagues (Dalton & Postgate, 1969a; Drozd & Postgate, 1970;
Yates 1988) and was referred to as "conformational protection" because the
enzyme was protected from O2 damage and inactive towards reducible substrates.
A similar behaviour has been described and studied in A. vinelandii wild type
(Dingier & Oelze, 1985) and the cydB mutant (Linkerhagner & Oelze, 1995b).
Nitrogenase in crude extracts of Azotobacter occurs in an insoluble O2tolerant complex composed of the two nitrogenase proteins with a third iron
sulphur redox protein called the Shethna, FeS II or protective protein (Robson &
Postgate, 1980; Yates, 1988; Moshiri et al, 1994). Purified preparations of the
latter from either A. chroococcum (Robson & Postgate, 1980) or A. vinelandii
Protection of nitrogenase
313
(Moshiri et al., 1995 and references therein) when added to the nitrogenase
proteins in approximate equimolar ratios in the presence of Mg2"1" confer
protection from O2 inactivation. The mechanisms regulating reversible complex
formation in vivo are unknown, but may involve changes in electron flux to the
nitrogenase proteins and their state of reduction (Robson & Postgate, 1980;
Yates, 1988; Moshiri et al., 1995). The characterisation of an A. vinelandii mutant
lacking the FeS II protein has shown that this protein also specifically prevents
nitrogenase polypeptide degradation in vivo when cells are exposed to an O2
stress during energy limitation (Moshiri et al., 1994). A single gene (the fesll
locus), which has been sequenced, encodes the FeS II protein in A. vinelandii
(Moshiri et al., 1994). Its expression and the synthesis of the FeS II protein are
constitutive, so this protein may have other functions, unconnected with N2
fixation, although up-regulation occurs during diazotropy (Moshiri et al., 1994).
Prevention from O2 damage to nitrogenase may also occur by a phenomenon
termed "autoprotection". The purified Fe protein component of the nitrogenase
from A. chroococcum, in the presence of either ATP or ADP, can reduce O2 to
H2O (via H2O2) without loss of activity provided that the protein is in a four-fold
molar excess over O2 (Thorneley & Ashby, 1989). How significant this is in vivo
is unclear, but Linkerhagner & Oelze (1995b) suggest that it could account, in
part, for the reversible inhibition by O2 of nitrogenase activity. In addition, spatial
and morphological factors may help to separate O2 from nitrogenase, as the cell
size and extent of the intracytoplasmic membrane increased when the dissolved
oxygen concentration is raised in C-limited chemostats of A. vinelandii (Poste et
al., 1983b). Altogether, a combination of temporal, spatial, and morphological
factors probably contribute to aerotolerant diazotrophy in Azotobacter.
7. CONCLUSIONS AND OUTLOOK
Maintenance of the steep O2 gradient between air-saturated medium and
nitrogenase in the cytoplasm of A. vinelandii requires an arsenal of protective
mechanisms, of which respiratory protection is now recognised as a key element.
In A. vinelandii, cytochrome bd serves this role and the regulation of its synthesis,
energy coupling and kinetic characteristics appear consistent with this. Many
questions remain unanswered.
1. How are the syntheses of components involved in the branched respiratory systems regulated? CydR is a negatively-acting transcriptional regulator of
cydAB, but is it a global regulator, as is Fnr?
2. How do the cytochrome bd-type oxidases in E. coli and A. vinelandii
exhibit such different Km values for oxygen in vivo, given the striking similarities
in their protein sequences? Is oxygen penetrating from the external medium
intercepted in the periplasm in either or both of these bacteria?
3. Do the unique oxygen-binding properties of cytochrome d play a role in
respiratory protection? In E. coli, cytochrome bd activity appears to be inhibited
by high oxygen concentrations (D'mello et al., 1996), but such a mechanism might
be superfluous in A. vinelandii given the oxygen-induced regulation observed.
4. During diazotrophic growth, how is electron flow in the cytochrome
314
Poole and Hill
W-terminated pathways regulated so as to achieve uncoupling throughout the
respiratory chain? Are there uncoupled dehydrogenases specific to diazotrophic
respiration?
5. At what extracellular oxygen concentrations are other oxidases in A.
vinelandii effective, or not, at protecting nitrogenase? A mutant in cyo, encoding
an oxidase in the haem-Cu superfamily is unaffected in diazotrophic growth in
air-saturated media, but is there a third oxidase? Do globin-like proteins, now
identified in many bacteria (Poole, 1994), play a role in intracellular oxygen
buffering?
6. How does Azotobacter cope when fixing N2 by the Mo-independent
nitrogenases? Diazotrophic growth of A. chroococcum with V-dependent nitrogenases appears to be more O2 sensitive than when using Mo-dependent
nitrogenase (Eady et al, 1987) and the FeS II protein appears not to protect the
V-dependent nitrogenase from O2 damage in A. chroococcum (Eady el al., 1987)
or in A. vinelandii (Moshiri et al., 1994).
ACKNOWLEDGEMENTS
Work in the authors' laboratories was supported by the Biotechnology and
Biological Sciences Research Council, in association with the Nitrogen Fixation
Laboratory. We are grateful to Dr G Wu and Ms S Edwards for unpublished
results and to Dr G Sawers and Dr C Appleby for many stimulating ideas.
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