FEMS MicrobiologyLetters 34 (1986) 313-317
Published by Elsevier
313
F E M 02423
A periplasmic location for the thiosulphate-oxidizing
multi-enzyme system from Thiobacillus versutus
Wei-Ping Lu
Department of Biology, Fudan Unwersity, Shanghai, People's Republic of China
Received 14 January 1986
Accepted 28 January 1986
1. SUMMARY
Evidence is presented to show that the thiosuiphate-oxidising multi-enzyme system from
Thiobacillus versutus has a periplasmic location,
and that the oxygen-binding site of the cytochrome oxidase ( a a 3 ) is on the inner surface of
the membrane. A scheme for the mechanism of
generation of a proton motive force during electron flow from thiosulphate to oxygen via cytochrome c and aa 3 is proposed.
2. INTRODUCTION
T. versutus is a facultative chemolithotrophic
bacterium which can grow on thiosulphate as its
sole source of energy. Thiosulphate oxidation by
the organism has been shown to be affected by a
soluble multi-enzyme system [1-3], which consists
of four major components: 'enzyme A', 'enzyme
B', cytochrome c551, and cytochrome c552.S, and
catalyses the following proton-yielding dehydrogenation process [3,4]:
$2O2- + 5H20 ---, 2SO2- + 10H + + 8 e-
(1)
The electrons released are coupled to the electron
transport chain through the c-type cytochromes
and finally reduce 0 2 by an aa3-type cytochrome
oxidase [3,5,6]. The fate of the protons liberated is
apparently dependent on whether the multi-enzyme system is on the cytoplasmic or periplasmic
side of the membrane. A resolution of this uncertainty is essential to understanding the mechanism
of energy conservation accompanying the oxidation of thiosulphate in terms of chemiosmotic
theory, as it has been demonstrated that ATP
synthesis in T. versutus is effected exclusively by
electron transport phosphorylation with a P / O
ratio of approx. 1 [3,5]. Although our preliminary
work using EDTA-lysozyme lysis of cells led to
the proposal of a cytoplasmic location of the
multi-enzyme system [7], the result was questionable, and a periplasmic location for thiosulphateoxidising systems in sulphur bacteria was proposed by analogy with the well-studied enzymes
responsible for the oxidation of inorganic and C I
compounds [8]. A re-examination has been undertaken, and evidence for a periplasmic location of
the multi-enzyme system in T. versutus and a
cytoplasmic location of the reaction of the cytochrome oxidase with azide (and hence with oxygen)
is presented in this paper. From these and previous findings a scheme for vectorial arrangement of
the multi-enzyme system, the terminal respiratory
chain and the mechanism of generation of a proton gradient accompanying the oxidation of thiosulphate is presented.
0378-1097/86/$03.50 © 1986 Federation of European MicrobiologicalSocieties
314
3. MATERIALS A N D METHODS
3.1. Growth of the organism
T. uersutus (formerly Thiobacillus A 2) was cultured in flasks shaken at 30°C essentially as described previously [9] in a medium containing
( g . l - 1 ) : N a 2 S 2 0 3 . 5 H 2 0 , 12.5; Na2H PO 4.
2H20, 7.9; KH2PO 4, 1.5; NH4CI, 0.8; MgSO 4.
7H20, 0.5; and 1 N NaOH, 15 ml; trace metal
solution [10], 10 ml; phenol red, 0.0003 g. 1-1. pH
was maintained manually at about pH 7.8 by
addition of 1 M NaHCO.~, using phenol red as the
indicator. Cells from about 5 1 of culture were
harvested after 4 days' growth and washed once
with 25 mM Tris-HCl, pH 7.5. Fresh cell preparations were used throughout the work.
3.2. Preparation of periplasmic, cytoplasmic and
membrane fractions
Sphaeroplasts were prepared by a modified version of a procedure developed originally for
Escherichia coli [11]. Optimum experimental conditions were sought in an attempt to ensure sufficient release of periplasmic proteins from the
sphaeroplasts and at the same time to keep the
latter (or most of them) still intact. A typical
operation is described as follows. The pellet of the
washed cells (130 mg protein), reddish in colour,
was suspended in 10 ml of 1 M sucrose, 5 mM
EDTA and 0.2 M Tris-HCI, pH 8.0. Then 8.5 mg
of iysozyme was added and a mild osmotic shock
administered by dilution with an equal volume of
distilled water. The suspension was incubated at
26°C for 25 min, and centrifuged at 12000 x g for
20 min at 4°C to yield a clear pink-orange supernatant (periplasm) and a pellet (sphaeroplasts) of
a rather paler colour than the original whole cells.
The periplasmic fraction was dialysed against 25
mM Tris-HCl, pH 7.5, at 4°C for 2 h ' a n d then
concentrated to about 2 ml by removal of water
with polyethyleneglycol. The pellet of sphaeroplasts was lysed by resuspension in 20 mi of 25
mM Tris-HCI, pH 7.5, then adding 50 mg DNase
and 20 #1 of 0.1 M MgCI z to reduce the viscosity
of the resulting suspension, and centrifuged at
150000 x g for 1 h to obtain a straw-coloured
supernatant (cytoplasm) and a pellet (membrane)
which was subsequently resuspended in 25 mM
Tris-HCl, pH 7.5.
3.3. Assay of enzyme activities
Activity of thiosulphate : c y t o c h r o m e c
oxidoreductase i.e., the thiosulphate-oxidising
multi-enzyme system (for terminology, see [2]) was
measured by following the reduction of horse heart
cytochrome c essentially as described previously
[1]. The reaction mixture (1 ml) contained (ttmol):
Na2S203, 2; Tris-HCi, pH 7.5, 90; cytochrome c,
0.07; and enzyme solution, 2 mg. The same procedure was employed to assay the activity of
sulphite : cytochrome ¢ oxidoreductase except that
0.03 /zmol cytochrome and 0.05 mg enzyme protein were used. Units were ~mol cytochrome c
reduced min 1 for both enzymes. Sulphite:cytochrome c oxidoreductase was used as an indicator
of the thiosulphate-oxidising multi-enzyme system, since the enzyme has been shown to be
closely associated with cytochrome c551 and enzyme B [121.
Malate dehydrogenase activity [13] was measured in a reaction mixture (1 ml) that contained
(/.tmol): phosphate buffer, pH 7.4, 25; NADH,
0.3; oxaloacetate, 0.5; and enzyme solution, 0.07
mg. Activity was expressed as /~mol N A D H
oxidised min-1. All enzyme assays were done at
ambient temperature (about 30°C) in a l-cm
light-path cuvette using a Beckman DU7 spectrophotometer.
3. 4. Measurement of c-type ~ytochrome and protein
Dithionite reduced minus ferricyanide oxidised
difference spectra of cytochromes were recorded
on a Shimadzu UV-240 spectrophotometer. The
content of c-type cytochrome was calculated from
the absorbance difference at 551-535 nm by using
a millimolar extinction coefficient of 19 cm -1.
Units were expressed as nmol cytochrome c. Protein was determined by the Lowry method using
bovine serum albumin as standard [21].
3.5. Assay of the effect of pH on the inhibition by
azide of thiosulphate oxidation by whole cells
The assay was essentially as described before
[14,15] in a Clark oxygen electrode (Rank Brothers,
Bottisham, U.K.) at 32°C. The reaction mixture
contained: cells, 0.5 or 0.75 mg protein; NazS20~,
1 mM; phosphate, 0.1 M (pH 6 or 6.4), or TrisHCI, 0.1 M (pH 7.2 or 8.2) in a final volume of 2
315
ml. Ki values for azide inhibition at different pH
values were calculated graphically from the common equation for uncompetitive inhibition [14]:
1
-v =
Km
I
vm s +
far. Since only about 7~ of the total activity of
malate dehydrogenase was detected in the periplasm, and few periplasmic components (thiosulphate or sulphite : cytochrome c oxidoreductase
and c-type cytochrome) were found in the cytoplasm, it is concluded that the original sphaeroplast preparation was largely intact. The simplest
interpretation of the result is that the thiosulphate-oxidising multi-enzyme system of T.
versutus is located in the periplasm.
Results from several repeat experiments with
the same treatment all indicated a periplasmic
location of the multi-enzyme system, though proportions of thiosuiphate or sulphite : cytochrome c
oxidoreductases released into the periplasm varied
from 95~ to 70%, and corresponding release of
malate dehydrogenase from 5% to 30%. These
variations seemed to relate to the factors such as
growth phase of cells harvested (i.e., length of
cultivation) and the conditions used in the preparation of sphaeroplasts (e.g., length of incubation with lysozyme).
1
(2)
+ vm
where V was the specific activity of oxidation of
thiosulphate, S the concentration of thiosulphate
and I the concentration of azide. In practice,
because the concentration of thiosulphate used
was much higher than its K m [7], the term
Km/V~S was omitted from the calculation.
3.6. Chemicals
Lysozyme (egg white), cytochrome c (horse
heart), bovine serum albumin desoxyribonuclease,
valinomycin and N A D H were obtained from
Sigma.
4. RESULTS
4.1. Cellular location of the thiosulphate-oxidising
multi-enzyme system
Three fractions, namely periplasm, membrane
and cytoplasm, were resolved after sequential lysis
of the cells with a modified EDTA-iysozyme treatment, osmotic shock and subsequent differential
centrifugations. Analysis of the resultant fractions
showed (Table 1) that a very high proportion of
the total activities of thiosulphate or sulphite:cytochrome c oxidoreductases and 80% of the c-type
cytochrome were present in the periplasm, whereas
the cytoplasm contained most malate dehydrogenase activity, indicating this to be a cytoplasmic
protein as in other bacterial systems studied so
4.2. The location of the cytochrome oxidase aa 3
reaction
Inhibition by azide ion of the oxidation of
thiosulphate by the organism was used to locate
the terminal oxidase (Fig. 1). The results showed
that the apparent inhibition constants (Ki) for
azide were dependent on the pH of the reaction
medium: the values of K i were low in acidic
media and high in neutral alkaline media. By
analogy with other cytochrome oxidases it is coneluded that 0 2 reduction takes place on the inner
face of the cell membrane, since azide with a pK
of 4.6, is expected to penetrate cells more readily
Table 1
Cellular distribution of thiosulphate or sulphite:cytochrome c oxidoreductases, malate dehydrogenas¢, c-type cytochrome and
protein
Fraction
Protein
(mg)
(%) °
Periplasm
38.0
36
Membrane
18.6
17
Cytoplasm
50.0
47
a Sum of the 3 fractions as 100%.
Sulphite : cytc
oxidoreductase
Thiosuiphate
: cytc Malate
oxidoreductase dehydrogenase
c-type cytochrome
(U)
(%)
(U)
(7o)
(U)
(~)
(U)
(%)
8132
0
345
96
91
0
5
95
0
5
32.6
0
415.0
7.3
0
92.6
102.0
20.5
3.5
81
16
3
4
316
9
/
t
iI
K~
/
(mM
l
tI
2
i
/
t
t
I
.0
I
/
J
o"
i
6
i
7
pH
L
.
g
Fig. 1. Effect of pH of the reaction medium on the K, for
azide inhibition of thiosulphate oxidation by whole cells of T.
versutus. The experimental conditions were as described in
M A T E R I A L S A N D METHODS. Apparent K i values were
determined with (zx) and without ( O ) inclusion of 20 ng
valinomycin and l0 m M KCI.
at low external pH than at high external pH, and
it is generally accepted that cytochrome oxidases
are inhibited by azide at a site very close to that at
which 0 2 is reduced.
Fig. 1 also reveals that the values for K, were
largely decreased in the presence of valinomycin
and K ÷. giving further evidence for the conclusion, because the ionophore and cation facilitate
the azide accumulation in cells by a mechanism of
co-transport with the permeant cation.
5. DISCUSSION
The results described in this paper clearly show
that the thiosulphate-oxidising multi-enzyme system of T. versutus is located in the periplasmic
space. This is consistent with the electron acceptors for the system being c-type cytochrome which,
by analogy with their counterparts in mitochondria
and other well-studied bacterial systems, is
expected to be on the external surface of the
membrane. Apparently the previous observation
of a cytoplasmic location of the multi-enzyme
system was premature and can be attributed to
insufficient breakdown of the cell wall under the
experimental conditions used previously (e.g.,
using phosphate rather than Tris buffer [5]). In
addition, cells used previously were collected from
thiosulphate-limited chemostat culture, which
might have strengthened the interaction between
the multi-enzyme system and the membrane, resulting in the retention of the activity of thiosulphate:cytochrome c oxidoreductase by the
sphaeroplasts. In this sense it is more likely that
the multi-enzyme system is attached to the periplasmic side of the respiratory membrane.
These results, together with the data for redox
potentials of the membrane and soluble cytochromes [6,16], allow us to draw a new scheme (Fig. 2),
modified from the previous proposal [7], for the
topographical organisation of various components
of the electron transport chain and the electron
and proton movements associated with the thiosulphate oxidation in T. versutus. Thus, reaction
(1) takes place in the periplasmic space with a
release of 10 protons and a concomitant reduction
of 2 oxygen molecules in the cytoplasm by the
electrons released from the reaction, hence establishing a transmembrane proton gradient without
the necessity of pumping protons by the cytochrome oxidase aa 3, which would be required if
the thiosulphate-oxidising multi-enzyme system
were situated on the inside of the membrane. The
proton motive properties of the system for oxidation of thiosulphate are attributable to a redox
arm mechanism similar to that found in hydroxylamine oxidation in Nitrosomonas europaea [17],
methanol oxidation in Methylophilus methylotrophus [18] and Paracoccus denitrificans [19],
and dissimilatory sulphate reduction by hydrogen
in Desulfovibrio oulgaris [20]. Such a mechanism
implies that proton release could directly contribute to the generation of a proton motive force.
Two further soluble T. oersutus c-type cytochromes [2,16] namely c550 (acidic) and csso (basic),
are not shown in Fig. 2. Both of these are probably also located in the periplasmic space, since it
was apparent that virtually all of the soluble c-type
cytochromes were periplasmic proteins (Table 1).
They might also be involved in the dehydrogenation of thiosulphate (e.g., as electron mediators)
but their exact role is still obscure.
The scheme (Fig. 2) proposed here makes more
likely the concept that thiosulphate oxidation (Eqn.
317
Eh ( v o l t s )
periplasm membrune
c~opIo.sr.
-o. 15
0
-s-sog~
!
5H20
0.15
2SO 2 O3
rJ
This work was supported by the Science Funds of
the Chinese Academy of Sciences and by Fudan
University, Shanghai, People's Republic of China.
1OH+
Css~
]
.... : ....
0.45
Fig. 2. Scheme for the probable mechanism of electron and
proton movements associated with thiosulphate oxidation in T.
versutus. For details see text [7]. The cytochromes involved and
their E m values are indicated. Membrane-bound cytochrome
c552 is assumed to be anchored to the outer surface of the
membrane. Cytochrome oxidase (aa3) presumably has no
function as a proton pump. Enzyme A is the component for
the initial binding of thiosulphate [4].
1) is a n i n t e g r a t e d process a n d that free interm e d i a t e s d o n o t o c c u r [3,7], as a n y i n t e r m e d i a t e s
(e.g., sulphite) l i b e r a t e d in the p e r i p l a s m i c space
m i g h t easily diffuse o u t of the cells, r e s u l t i n g in a n
i n c o m p l e t e o x i d a t i o n of t h i o s u l p h a t e . T h e perip l a s m i c l o c a t i o n for t h i o s u l p h a t e o x i d a t i o n also
has the a d v a n t a g e s that t h i o s u l p h a t e - i m p o r t a n d
s u l p h a t e - e x p o r t systems are u n n e c e s s a r y . These
t r a n s p o r t a t i o n processes m i g h t otherwise i n v o l v e
the c o n s u m p t i o n of extra e n e r g y a n d require
specific p o r t s o n the m e m b r a n e . A c c o r d i n g to the
scheme, however, the p r e v i o u s s p e c u l a t i o n [7,16]
of a n e l e c t r o n flow c o u p l i n g directly to cytoc h r o m e b562 via the n e g a t i v e E m centres of cytoc h r o m e s c551 a n d c55z5, in the e n e r g y - c o n s u m i n g
r e d u c t i o n of N A D ( P ) ~" d u r i n g the o x i d a t i o n of
t h i o s u l p h a t e , seems i n v a l i d b e c a u s e it is u n l i k e l y
that the b - t y p e c y t o c h r o m e w o u l d be o n the perip l a s m i c surface of the m e m b r a n e . At present, a
f u n c t i o n for the n e g a t i v e E m c e n t r e s of cytoc h r o m e c551 a n d csszs c a n n o t be inferred.
ACKNOWLEDGEMENTS
I a m greatly i n d e b t e d to Prof. D.P. Kelly for
m a n y i n v a l u a b l e d i s c u s s i o n s a n d critical r e a d i n g
of the m a n u s c r i p t . T h e technical assistance of J a n e
Z h a n g a n d H a o W a n g is g r a t e f u l l y a c k n o w l e d g e d .
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