Institut für Mikrobiologie und Genetik,
Georg-August-Universität, 37077 Göttingen, Germany
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INTRODUCTION |
Methanosarcina mazei
Gö1 belongs to the methylotrophic methanogens of the order
Methanosarcinales and can grow on H2 plus CO2, methanol, methylamines, and acetate. The pathways of
methanogenesis from these substrates have been analyzed in detail in
recent years (8, 11, 29). All substrates are converted to
methyl-S-CoM (2-methylthioethanesulfonate), either by reduction of
CO2 or by demethylation of methanol and acetate. Methane is
formed from methyl-S-CoM by the catalytic activity of the methyl-S-CoM
reductase, which uses HS-CoB (7-mercaptoheptanoylthreonine phosphate)
as electron donor. The reaction leads to the production of
a heterodisulfide (CoM-S-S-CoB) from HS-CoM and HS-CoB, which
is the terminal electron acceptor of the membrane-bound electron
transport systems of M. mazei Gö1. The source of
reducing equivalents necessary for the reduction of CoM-S-S-CoB
depends on the growth substrate. If molecular hydrogen is
present, a membrane-bound F420
[(N-L-lactyl-
-L-glutamyl)-L-glutamic acid phosphodiester of 7,8 didemethyl-8-hydroxy-5-deazariboflavin-5'-phosphate]-nonreducing hydrogenase channels
electrons via b-type cytochromes to the heterodisulfide reductase which reduces the terminal electron acceptor (6, 13). This electron transport system, referred to as
H2:heterodisulfide oxidoreductase, is coupled to proton
translocation across the cytoplasmic membrane (8). When
cells are grown on methanol, part of the methyl groups are oxidized to
CO2 and reducing equivalents are transferred to coenzyme
F420. In Methanosarcina strains, reduced F420 (F420H2) is reoxidized by the
membrane-bound F420H2 dehydrogenase which is
part of the F420H2:heterodisulfide
oxidoreductase (7). Parallel to the H2-dependent
system, electrons are channeled to the heterodisulfide reductase,
resulting in the reduction of CoM-S-S-CoB and in the formation of an
electrochemical proton gradient. It was shown that the resulting
µH+ (transmembrane electrochemical gradient of
H+) is the driving force for ATP synthesis from ADP plus
Pi as catalyzed by an A1A0-type ATP
synthase (25). Since other components of the systems were
unknown at that time, only the overall electron transport reaction
could be analyzed. Recently, a new redox-active component was
isolated from the cytoplasmic membrane of strain Gö1 and
named methanophenazine (1). Furthermore, it was shown that
key enzymes of the membrane-bound electron transfer systems were able
to interact with 2-hydroxyphenazine (2-OH-phenazine), which is a
water-soluble analogue of methanophenazine (2). By using
2-OH-phenazine, the process of electron transfer from molecular
hydrogen to the heterodisulfide can be divided into two partial
reactions (6):
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(1)
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(2)
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In this report, we show that both reactions are coupled to proton
translocation and that the resulting electrochemical proton gradient is
used for ATP synthesis.
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MATERIALS AND METHODS |
Growth of cells and preparation of washed vesicles.
M.
mazei Gö1 (DSM 3647) was grown in 1-liter glass bottles or,
for mass culturing, in 20-liter carboys on 150 mM methanol in a medium
described previously (7). Washed vesicles of strain Gö1 (21) were prepared as described by Deppenmeier et
al. (7) except that the final protein concentration was 10 to 15 mg/ml.
Assay conditions.
Proton translocation was monitored by a pH
electrode (model 8103 Ross; Orion Research, Küsnacht,
Switzerland) which was inserted into a glass vessel (11 ml) from the
top through a rubber stopper. The electrode was connected with an Orion
model EA 920 pH meter and a chart recorder. After gassing with
H2 or N2, the vessel was filled with 3 ml of 40 mM potassium thiocyanate solution containing 0.5 M sucrose, 1 mg of
resazurine per liter, and 10 mM dithioerythritol, followed by the
addition of 50 to 80 µl of washed vesicles (1 to 1.4 mg of
protein/assay). The medium was continuously stirred, and the pH was
adjusted to 6.8 to 6.9. Additions were made with a microliter syringe
from the side arm. Proton uptake coupled to reaction 1 was followed by
the addition of 4.5 to 20 nmol of 2-OH-phenazine (4.5 or 20 mM stock
solution in ethanol) under an atmosphere of molecular hydrogen. To
determine the H+ transfer in the course of reaction 2, the
vessel was gassed with N2 and 240 nmol of CoM-S-S-CoB was
added. The reaction was started by the addition of 4.5 to 20 nmol of
dihydro-2-OH-phenazine. After completion of the experiments, the pH
changes were calibrated with standard solutions of HCl or NaOH.
CoB-S-S-CoM was synthesized by the method of Noll et al.
(23) and Kamlage and Blaut (15). 2-OH-phenazine
was prepared as described by Abken et al. (1) and was
reduced as described by Bäumer et al. (2).
Electron transfer reactions and ATP synthesis were investigated in
1.5-ml glass cuvettes filled with 0.6 ml of 40 mM potassium phosphate
(pH 7) containing 20 mM MgSO4, 0.5 M sucrose, 10 mM dithioerythritol, and 1 mg of resazurin per liter (buffer A) under anaerobic conditions; 10 µl of ADP (10 mM stock solution) and 10 µl
of AMP (100 mM stock solution) were added. AMP inhibits the
membrane-bound adenylate kinase of M. mazei Gö1
(8). This enzyme activity leads to an ADP disproportionation
which would otherwise interfere with the electron transfer-driven ATP
synthesis. Additions were made as indicated. 2-OH-phenazine,
dihydro-2-OH-phenazine, N,N'-dicyclohexylcarbodiimide (DCCD), and
3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) were added as ethanolic solutions. The controls received ethanol only. To determine the ATP concentration, 1- to 2-µl aliquots were withdrawn with a syringe and analyzed by the luciferin-luciferase assay (16) in combination with a biocounter M1500 (Lumac,
Landgraaf, The Netherlands). Redox reactions with 2-OH-phenazine and
dihydro-2-OH-phenazine were monitored photometrically at 475 nm (
= 2.5 mM
1 cm1) under atmospheres of
H2 and N2, respectively.
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RESULTS |
Proton translocation due to H2-dependent
heterodisulfide reduction.
Washed inverted vesicles from M. mazei Gö1 were tested for the ability to couple electron
transfer with the translocation of protons across the cytoplasmic
membrane. Therefore, concentrated vesicles were diluted with a
sucrose-thiocyanide solution under an atmosphere of molecular hydrogen
and were pulsed with 2-OH-phenazine as shown in Fig.
1A. When the electron acceptor was added,
a short period of alkalinization of the medium which is due to a rapid proton movement into the lumen of the inverted vesicles was monitored. In the second phase, a reacidification was observed until a stable pH
value was reached again. It is thought that the consumption of
2-OH-phenazine is responsible for this effect. The energy-conserving electron transport comes to an end, leading to a decay of the generated
µH+ by passive diffusion of protons from the
lumen of the inverted vesicles to the medium. After calibration of the
system, the extent of reversible alkalinization was calculated, resulting in an average ratio of 0.9 protons translocated per 2-OH-phenazine reduced (Table 1). No
alkalinization was observed when 2-OH-phenazine was replaced by ethanol
(Table 1) or when the reaction was performed under an atmosphere of
molecular nitrogen (not shown), indicating that proton transfer was
specifically coupled to the H2-dependent 2-OH-phenazine
reduction. The addition of the protonophore SF 6847 led to a complete
inhibition of measurable proton movement (Fig. 1A). In the presence of
this proton-conducting agent, the membrane becomes specifically
permeable to protons and can no longer sustain a proton potential.
H+ translocation was not affected by DCCD up to a
concentration of 250 nmol/mg of protein (Table 1), which was sufficient
to inhibit ATP synthesis (Fig. 2). Since
DCCD inhibits the catalytic activity of the
A1A0-type ATP synthase of M. mazei
Gö1 (3), this enzyme was not responsible for proton
translocation via ATP hydrolysis.
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FIG. 1.
Proton uptake by washed inverted vesicles from M. mazei Gö1. The experiments were performed as described in
Materials and Methods. The amount of translocated protons was
calculated from the difference between maximal alkalinization and the
final baseline after reacidification. The reaction was started by
pulses of 2-OH-phenazine or dihydro-2-OH-phenazine as indicated. SF
6847 was added as an ethanolic solution to a final concentration of 15 nmol/mg of protein. (A) H2-dependent reduction of
2-OH-phenazine under an atmosphere of molecular hydrogen; (B)
dihydro-2-OH-phenazine-dependent reduction of CoM-S-S-CoB under an
atmosphere of molecular nitrogen.
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FIG. 2.
Redox-driven ATP synthesis as catalyzed by washed
inverted vesicles from M. mazei Gö1. The experiments
were performed as described in Materials and Methods. The
concentrations of ADP, SF 6847, and DCCD were 0.16 mM, 25 µM, and 250 nmol/mg of protein, respectively. (A) ATP synthesis in the course of
H2-dependent-2-OH-phenazine reduction. The glass cuvette
was gassed with H2 and contained 600 µl of buffer A, 1.6 mM AMP, and 125 µM 2-OH-phenazine. The reaction was started by the
addition of washed vesicles (14 µg of protein). The following
additions were made: ADP ( ), ADP and SF 6847 ( ), ADP and DCCD
( ), ADP, SF 6847, and DCCD ( ), ADP under N2 ( ).
 , ADP omitted. (B) ATP synthesis coupled to
dihydro-2-OH-phenazine-dependent heterodisulfide reduction. Details of
the experiment and symbols are as described for panel A except that (i)
the reaction mixture contained dihydro-2-OH-phenazine (instead of
2-OH-phenazine) and 240 nmol of CoM-S-S-CoB and (ii) open circles
represent assays with ADP added and CoM-S-S-CoB omitted. The atmosphere
was N2.
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Interestingly, reaction 2 of the H2:heterodisulfide
oxidoreductase system was also able to generate a proton
gradient. As evident from Fig. 1B, the addition of
dihydro-2-OH-phenazine resulted in the transfer of protons into the
lumen of the inverted vesicles when incubated in the presence of
CoM-S-S-CoB under an atmosphere of nitrogen. A maximal stoichiometry of
0.9 H+/2e was determined (Table 1). The
addition of oxidized 2-OH-phenazine or ethanol instead of
dihydro-2-OH-phenazine prevented proton translocation (Table 1),
indicating that the latter process is strictly coupled with the
electron transport from reduced phenazine to CoM-S-S-CoB in washed
inverted vesicles of M. mazei Gö1. Again, the electron
transfer-dependent formation of a proton gradient was abolished when
the uncoupler SF 6847 was present in the reaction mixture (Fig. 1B). As
is evident, the H+/2e stoichiometries of the
partial reactions summed to 1.8, which is in the same range as the
coupling efficiency of the electron transport from H2 to
heterodisulfide (Table 1) (8). These results indicate that
strain Gö1 possesses two different proton-translocating segments
in the H2:heterodisulfide oxidoreductase system.
Coupling of electron transport and ATP synthesis.
It was
previously shown that the H2-dependent heterodisulfide
reduction was coupled to ATP synthesis (8). The question arose as to whether ATP formation could also be observed in the course
of the phenazine-dependent reactions, both of which were coupled to the
generation of an electrochemical proton gradient (Fig. 1). Under an
atmosphere of molecular hydrogen, washed vesicles catalyzed the
2-OH-phenazine reduction with an initial rate of 6.0 µmol
min1 mg of protein1 (Table 2). After 1 min,
the rate slowly decreased due to the depletion of 2-OH-phenazine. The
reaction was coupled to the phosphorylation of ADP, as indicated by a
rapid increase of the ATP concentration upon start of the reaction
(Fig. 2A). The estimated value for the first minute was 1.5 µmol of
ATP min1 mg of protein1, resulting in a
ratio of 0.25 mol of ATP formed per mol of OH-phenazine reduced. In the
presence of the ATP synthase inhibitor DCCD, the phosphorylation of ADP
was strongly inhibited (Fig. 2A) and 2-OH-phenazine reduction slowed to
4.8 U/mg of protein (Table 2). The latter effect was also observed when ADP was omitted. Addition of the uncoupler SF 6847 to H2-metabolizing vesicles in the
presence of DCCD or in the absence of ADP led to an increase of the
phenazine reduction rate of up to 95 or 97%, respectively, of the
control rate (Table 2). Furthermore, SF 6847 abolished ATP synthesis (Fig. 2A) due to the decay of µH+. No ATP was formed when ADP was omitted (Fig. 2A), indicating that the latter nucleotide was not present in washed inverted vesicles.
When the electron transfer from dihydro-2-OH-phenazine to CoM-S-S-CoB
was analyzed, the initial kinetics of ADP phosphorylation was found to
be 0.6 µmol min1 mg of protein1 (Fig. 2B)
and the initial rate of substrate conversion was 2.4 µmol
min1 mg of protein1 (Table 2), indicating
an ATP/2e stoichiometry of 0.25 within the first minute
of the reaction. ATP synthesis was abolished when either the uncoupler
SF 6847 was added or one of the substrates (dihydro-2-OH-phenazine or CoM-S-S-CoB) was omitted. In parallel to the H2-dependent
2-OH-phenazine reduction, the addition of DCCD led to a decrease of
electron transport from dihydro-2-OH-phenazine to CoM-S-S-CoB (Table 2) and to inhibition of ATP synthesis (Fig. 2B). Furthermore, in the
absence of ADP the heterodisulfide reductase activity decreased to 50%
of the control rate (ADP added [Table 2]) and ATP synthesis was
abolished (Fig. 2B). Inhibition of electron transfer under these
conditions was relieved by the addition of SF 6847, indicated by the
stimulation of electron transport activities 2.4-fold (addition of
DCCD) and 2.3-fold (ADP omitted) (Table 2) in comparison to the assays
performed with no uncoupler. Thus, the effects of SF 6847 and DCCD on
both partial electron transport reactions resemble the phenomenon of
respiratory control observed in mitochondria.
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DISCUSSION |
Several membrane-bound proteins and enzyme systems from
Methanosarcina strains have been found to be involved in the
generation of transmembrane electrochemical ion gradients. The
H2:heterodisulfide oxidoreductase, the
F420H2:heterodisulfide oxidoreductase, and the
CO:heterodisulfide oxidoreductase generate a proton motive force by
redox potential-driven H+ translocation (8, 24).
In contrast, the methyltetrahydromethanopterin:HS-CoM methyltransferase
(20) and probably the formylmethanofuran dehydrogenase (14) are reversible sodium ion pumps. It is remarkable that protons as well as sodium ions are employed by M. mazei
Gö1 as coupling ions in energy conservation.
In this report, we focus on the catalytic activity of the
H2:heterodisulfide oxidoreductase system which is composed
of a membrane-bound, F420-nonreducing hydrogenase and the
heterodisulfide reductase (29). Until recently, detailed
analysis of the system was hindered by the lack of information about
the nature of electron carriers mediating electron transfer between the
enzymes. With the identification of methanophenazine and the
elucidation of the reactivity of its water-soluble analogue
2-OH-phenazine, the overall mechanism of electron transfer became
evident (Fig. 3). It was found that the
F420-nonreducing hydrogenase (VhoGA) catalyzes the
oxidation of molecular hydrogen and transfers electrons via a
b-type cytochrome (VhoC) to 2-OH-phenazine (reaction 1). The resulting dihydro-2-OH-phenazine is oxidized by the heterodisulfide reductase and the electrons are transferred to CoM-S-S-CoB (reaction 2)
(6). Here it is shown that both partial reactions of the H2:heterodisulfide oxidoreductase are coupled to proton
translocation, exhibiting stoichiometries of 0.9 H+/2e. Thus, the ratios summed to 1.8 H+/2e, which corresponds to the efficiency of
the overall electron transport from H2 to CoM-S-S-CoB.
Keeping in mind that about 50% of the membrane structures present in
the vesicle preparations catalyzed electron transport but were unable
to establish a proton gradient (8), the
H+/2e values of the partial reactions and of
the overall reaction increase to 1.8 and 3.6, respectively.
Accordingly, the sum of the ATP/2e ratios of both
reactions would rise from 0.5 to about 1.0. In agreement with this
hypothesis is the fact that 3 to 4 H+/2e were
transferred by whole-cell preparations of M. barkeri when methane formation from methanol plus H2 was analyzed
(5). Under standard conditions, the sum of the changes of
free energy associated with reaction 1 and 2 is about 
40 kJ/mol,
which is sufficient to drive the phosphorylation of 1 mol of ATP
(G0' = 31.8 kJ/mol [28]).
However, in the environment of the cell, the G value is
probably more negative. It is to note that the methyl-CoM reductase,
which is present in abundance in the cell, catalyzes the irreversible
formation of the heterodisulfide from methyl-CoM and HS-CoB
(G0' = 45 kJ/mol). Therefore, it is likely
that the intercellular [CoM-S-S-CoB]/[HS-CoM] [HS-CoB] ratio is
very high. As a consequence, the span of available free energy would be
greater than calculated from standard conditions, indicating that the
energy-conserving reduction of CoM-S-S-CoB is driven forward by the
preceding methane-forming reaction (29).
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FIG. 3.
Tentative scheme of membrane-bound electron transfer
coupled to proton translocation in M. mazei Gö1.
Mphen, methanophenazine; MphenH2, dihydromethanophenazine;
VhoG, 40-kDa subunit of the F420-nonreducing hydrogenase;
VhoA, 60-kDa subunit of the F420-nonreducing hydrogenase;
VhoC, cytochrome b1 (Cytb1) encoded
by the third gene (vhoC) of the hydrogenase operon; HdrDE,
subunits of the heterodisulfide reductase; FeS, iron-sulfur clusters;
Ni, nickel-iron center of the F420-nonreducing
hydrogenase.
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A tentative scheme of the mechanism of proton translocation, which is
based on the structures and locations of the key enzymes of the system,
is shown in Fig. 3. The operon encoding the
F420-nonreducing hydrogenase from M. mazei
Gö1 contains three genes (8). The deduced amino acid
sequences revealed that the small (VhoG) and large (VhoA) subunits of
the protein are homologous to the corresponding polypeptides of
membrane-bound NiFe hydrogenases from several bacteria (9, 18,
22). The organisms contain a b-type cytochrome which
is connected to the core enzyme. In M. mazei, this function is fulfilled by VhoC (cytochrome b1), which is
encoded by the third gene of the hydrogenase operon. Electron
microscopic immunogold labeling experiments revealed that at least part
of the large subunit of the enzyme from Ralstonia eutropha
is exposed toward the periplasm (10). Therefore, it is
likely that the active center of the enzyme is located at the
periplasmic face of the membrane. During H2 oxidation by
R. eutropha, a electrochemical proton gradient is generated
by linking movement of electrons from the periplasmic side to the
cytoplasmic side of the membrane, with the release of protons in the
periplasm and uptake of protons from the cytoplasm in the course of
ubiquinone reduction (4, 17). The same mechanism was
suggested for p generation by fumarate respiration with
H2 in Wolinella succinogenes (12).
Since the membrane-bound hydrogenases from R. eutropha and
W. succinogenes are homologous to the corresponding enzyme
from M. mazei Gö1, the same kind of energy
conservation may be involved in the latter organism. The oxidation of
molecular hydrogen at the periplasmic site of the cytoplasmic membrane
of strain Gö1 would lead to the production of two scalar protons.
The electrons derived from the reaction are transferred to cytochrome
b1, which would accept two protons from the
cytoplasm for the reduction of methanophenazine (Fig. 3).
The mechanism of energy conservation in the course of partial reaction
2 might also be based on scalar proton transfer. The purified
heterodisulfide reductase from Methanosarcina species, which
catalyzes the dihydro-2-OH-phenazine-dependent CoM-S-S-CoB reduction,
is composed of two subunits (19, 26). HdrD harbors the
active center and contains two Fe4S4 clusters
which are involved in the reaction mechanism (26). The
subunit is predicted to be membrane-associated since membrane-spanning
helices are absent (27). HdrE is a b-type
cytochrome and contains two distinct heme groups. Hydropathy plots
revealed that the protein possesses five membrane-spanning helices
(19). Redox-driven H+ translocation was detected
in assays using washed vesicles from strain Gö1. However, the
purified heterodisulfide reductase also catalyzed the reduction of
CoM-S-S-CoB with dihydro-2-OH-phenazine as the electron donor, with
high rates indicating that the enzyme is directly involved in proton
translocation. A tentative mechanism of this process is shown in Fig.
3. We assume that in vivo electrons from dihydromethanophenazine are
transferred to the heme group of HdrE and scalar protons are released
at the outer phase of the cytoplasmic membrane. In a second step, the
electrons are channeled to FeS clusters of HdrD and in the reactive
center, CoM-S-S-CoB is reduced to HS-CoM and HS-CoB. We propose that
protons necessary for this reaction are derived from the cytoplasm and are transferred to the active site by a proton-conducting channel built
around a selected number of polar amino acid side chains as well as
bound water molecules.
This work was supported by a grant of the Deutsche
Forschungsgemeinschaft (Bonn-Bad Godesberg).
We are indebted to G. Gottschalk, Göttingen, Germany, for support
and stimulating iscussions.
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Abstract
Introduction
