Characterization of the Intramolecular Electron Transfer
Pathway from 2-Hydroxyphenazine to the Heterodisulfide Reductase from
Methanosarcina thermophila*
Eisuke
Murakami
,
Uwe
Deppenmeier§, and
Stephen W.
Ragsdale
¶
From the
Department of Biochemistry, Beadle Center,
University of Nebraska, Lincoln, Nebraska 68588-0664 and
§ Institut Für Mikrobiologie, Der
Georg-August-Universität Göttingen, Grisebachstra
e 8, Göttingen D-37077, Germany
Received for publication, June 2, 2000, and in revised form, October 13, 2000
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ABSTRACT |
Heterodisulfide reductase (HDR) is a component of
the energy-conserving electron transfer system in methanogens. HDR
catalyzes the two-electron reduction of coenzyme B-S-S-coenzyme M
(CoB-S-S-CoM), the heterodisulfide product of the methyl-CoM
reductase reaction, to free thiols, HS-CoB and HS-CoM. HDR from
Methanosarcina thermophila contains two b-hemes and two
[Fe4S4] clusters. The physiological electron
donor for HDR appears to be methanophenazine (MPhen), a membrane-bound
cofactor, which can be replaced by a water-soluble analog,
2-hydroxyphenazine (HPhen). This report describes the electron transfer
pathway from reduced HPhen (HPhenH2) to CoB-S-S-CoM. Steady-state kinetic studies indicate a ping-pong mechanism for heterodisulfide reduction by HPhenH2 with the following
values: kcat = 74 s
1 at 25 °C,
Km (HPhenH2) = 92 µM, Km (CoB-S-S-CoM) = 144 µM. Rapid freeze-quench EPR and stopped-flow kinetic
studies and inhibition experiments using CO and diphenylene iodonium
indicate that only the low spin heme and the high potential FeS cluster are involved in CoB-S-S-CoM reduction by HPhenH2. Fe-S
cluster disruption by mersalyl acid inhibits heme reduction by
HPhenH2, suggesting that a 4Fe cluster is the initial
electron acceptor from HPhenH2. We propose the following
electron transfer pathway: HPhenH2 to the high potential
4Fe cluster, to the low potential heme, and finally, to
CoB-S-S-CoM.
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INTRODUCTION |
Heterodisulfide reductase
(HDR)1 plays important roles
in methane production by methanogenic archaea. In the last step of
methanogenesis, the methyl group of methylated coenzyme M
(2-mercaptoethane sulfonic acid, HS-CoM) is reduced to methane by
methyl-S-CoM reductase. Coenzyme B
(7-mercaptoheptanoyl-threonine phosphate, HS-CoB) is the electron
donor, and the product of the reaction is the heterodisulfide, CoB-S-S-CoM (reaction 1) (1). The cofactors, HS-CoM and HS-CoB, are
regenerated by reduction of the disulfide bond of CoB-S-S-CoM in a
reaction catalyzed by HDR (reaction 2). CoB-S-S-CoM reduction by
H2 or reduced coenzyme F420 drives proton
translocation across the cytoplasmic membrane, which is coupled to ATP
synthesis by an A1A0-type ATP synthase
(2-4).
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(Eq. 1)
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(Eq. 2)
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The ultimate electron donor to HDR can be the CO
dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex when bacteria grow on acetate. In this case, the electron transfer pathway involves ferredoxin and possibly an iron-sulfur flavoprotein (5, 6). When
bacteria grow on methanol, the ultimate donor is reduced coenzyme
F420H2, generated by F420
dehydrogenase. The F420 dehydrogenases from
Methanosarcina mazei (7), Methanolobus tindarius
(8), and Archaeoglobus fulgidus (9) have been isolated and
shown to contain Fe-S clusters and FAD. The direct electron donor for HDR appears to be a membrane-bound cofactor, methanophenazine (MPhen)
(10). This cofactor has been isolated from membranes of M. mazei strain Gö1 (11) and Methanosarcina
thermophila.2 It has a
25-carbon isoprenoid chain attached to position 2 of phenazine via an
ether bond, which makes it insoluble in aqueous solution (11). The
2-hydroxyphenazine (HPhen) derivative is a suitable water-soluble
substitute for MPhen that can accept electrons from
F420H2 and can donate electrons to the purified HDR from M. thermophila (10). Electron transfer from
F420H2 to HPhen results in the translocation of
two protons per two electrons transferred (12). Another two protons
(per two electrons) are translocated during reduction of CoB-S-S-CoM by
HPhen (13).
HDR from M. thermophila consists of two subunits. A 53-kDa
subunit contains two distinct [Fe4S4]
clusters with midpoint potentials of
100 and
400 mV (6). A 27-kDa
membrane-associated subunit contains two b-type hemes, one that
is low spin and is hexacoordinate and another that is high spin and is
five-coordinate. The midpoint potentials of the low and high spin hemes
are
180 and
23 mV, respectively (6).
We have used steady-state and pre-steady-state kinetics to answer some
key questions about the HDR mechanism. Which of the metal centers in
HDR is the initial electron acceptor from reduced HPhen
(HPhenH2)? The midpoint potentials of some of the metal centers are outside the range of the HPhen/HPhenH2 and
CoB-S-S-CoM/CoB-SH, CoM-SH couples; therefore, are all of the metal
clusters involved in the electron transfer reaction? What is the
intramolecular electron transfer pathway? Based on our results, we
propose that the physiological electron transfer pathway from
methanophenazine to the heterodisulfide is: MPhenH2
[Fe4S4]high
hemelow
CoB-S-S-CoM.
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EXPERIMENTAL PROCEDURES |
Materials--
HPhen was synthesized as described (11). Other
chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo).
Cell Growth and Enzyme Preparation--
M.
thermophila TM1 was cultured on acetate at 50 °C and pH
6.8 in a 5-liter New Brunswick fermentor equipped with a pH auxostat (14) as described (15). HDR was purified as described previously (6),
except for enzyme concentration steps. As mentioned by Thauer et
al. (16), higher activity was recovered when membranes with
molecular/pore molecular mass cut-off of 50 kDa (Spectrum) were used.
Enzyme Assays--
HDR activity was measured by monitoring the
oxidation of reduced methyl viologen at 604 nm (
= 13.9 mM
1 cm
1) and 55 °C (6, 17,
18). One unit of HDR activity corresponds to 1 µmol of CoB-S-S-CoM
reduced per minute. HDR activity was also assayed using
2-hydroxyphenazine as the electron donor. A solution containing HPhen
(final concentration of 200 µM) in Buffer A (50 mM Tris, pH 7.6, and 10% glycerol) was reduced by bubbling with 100% hydrogen gas for 20 min, adding partially purified
hydrogenase from M. thermophila, and incubating at 55 °C
for 2 h. For steady-state kinetic experiments, varying amounts of
the HPhenH2 stock solution were added to Buffer A. Then,
HDR was added and the reaction was started by adding CoB-S-S-CoM. The
oxidation of HPhenH2 was monitored either at 25 °C or at
55 °C by following an increase in absorbance at 365 nm. The
difference (oxidized minus reduced, 
) extinction coefficient at
365 nm was measured to be 3.58 mM
1
cm
1. One unit is defined as 1 µmol of CoB-S-S-CoM
reduced min
1 mg
1. The data were fit to eq.
3 for a ping-pong reaction, where v0 is initial
velocity and A and B are HPhenH2 and
CoB-S-S-CoM, respectively, as follows.
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(Eq. 3)
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Protein concentrations were determined by the Bio-Rad protein
assay (19) using bovine serum albumin as a standard. UV-visible spectra
were collected on a Cary-14 spectrophotometer modified by On-Line
Instrument Systems, Inc. (Bogart, GA). EPR spectra were recorded on a
Bruker ESP300E spectrometer equipped with a temperature controller
(Oxford ITC4) and automatic frequency counter (Model 5340A,
Hewlett-Packard Co.).
Stopped-flow Experiments--
HDR was oxidized by adding thionin
(E'0 = +60 mV) until the blue color
disappeared. Excess thionin was removed by passing the solution through
a Sephadex G-25 column (Amersham Pharmacia Biotech). Oxidized HDR (6 µM before mixing) and varied concentrations of
HPhenH2 (10, 20, 50, 100, 150, and 200 µM
before mixing) were rapidly mixed at 25 °C in a 1:1 ratio. Heme
reduction was followed at 423 nm in a rapid-scanning stopped flow
instrument (On-Line Instrument Systems, Inc.).
Detergent Exchange--
Purified HDR was diluted 4-fold with 25 mM Tris, pH 7.6, 10% glycerol, 0.6% Triton X-100, 2 mM dithiothreitol, and loaded on a DEAE-Sephacel (Sigma)
column pre-equilibrated with the same buffer. After washing the column
with 25 mM Tris, pH 7.6, 10% glycerol, 2 mM
dodecylmaltoside, 2 mM dithiothreitol for at least 5 column
volumes, HDR was eluted with 0.4 M KCl-containing
dodecylmaltoside buffer. The enzyme was concentrated for freeze-quench
EPR experiments.
Freeze-quench EPR--
Thionin-oxidized HDR (60 µM) was rapidly mixed with 200 µM
HPhenH2 at room temperature using a chemical/freeze-quench
apparatus (Update Instrument, Inc., Madison, WI). The HDR-containing
and HPhenH2-containing syringes (2 ml) were connected by a
mixer, and the reaction time was controlled using aging hoses of
different lengths. The solutions were mixed using four sequential ram
displacements of 1.3 mm (83 µl per shot) and ram velocity of 8 cm/s.
The reaction was quenched by rapidly freezing the mixture in a funnel
attached to an EPR tube that was filled with low temperature
(
120 °C) isopentane. The frozen snow containing the quenched
reaction mixture was then packed tightly in EPR tubes. The 0-ms time
point was obtained by mixing the oxidized HDR with buffer.
Mersalyl Acid Treatment--
Oxidized HDR (above) was treated
with 1 mM (final concentration) mersalyl acid for 30 min.
Then, excess mersalyl acid was removed by centrifuging the
solution through a Sephadex G-25 column (Amersham Pharmacia Biotech).
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RESULTS |
Steady State--
Steady-state kinetic experiments were performed
at 55 °C at varying concentrations of HPhenH2 and
CoB-S-S-CoM to determine the overall mechanism of the HDR reaction. The
data were fitted to the Michaelis-Menten equation (Fig.
1), which yielded the
Km values for HPhenH2 and CoB-S-S-CoM of
92 ± 22 and 144 ± 33 µM, respectively. The
specific activity was 52 µmol min
1 mg
1
(kcat of 70 s
1, assuming a dimeric
unit of 80 kDa). In a ternary-complex mechanism, the
V/K value for one substrate increases with the
concentration of the other substrate. The
Vmax/Km values for
CoB-S-S-CoM do not increase with HPhenH2 concentration
(Fig. 1, inset), indicating that the reaction follows a
ping-pong mechanism. At higher concentrations of HPhenH2,
the V/K value decreases, indicating some degree
of substrate inhibition.

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Fig. 1.
Steady-state kinetics of the reduction of
CoB-S-S-CoM by HPhenH2. The rate of
HPhenH2 oxidation was followed at 365 nm
( 365 = 3.58 mM 1
cm 1). The concentration of CoB-S-S-CoM was varied at
fixed HPhenH2 concentrations of 200 (closed
circles), 100 (open circles), 50 (closed
triangles), and 20 µM (open triangles).
The data were globally fit to the equation for a two-substrate
ping-pong mechanism (see "Experimental Procedures") to yield the
following Michaelis parameters: Vmax = 54 ± 7 units/mg, Km for phenazine = 93 ± 28 µM, and Km for CoB-S-S-CoM = 296 ± 65 µM. Inset: plot of
Vmax/Km for CoB-S-S-CoM at
different concentrations of HPhenH2.
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Pre-steady-state Kinetics--
The electron transfer pathway from
HPhenH2 to HDR was studied by rapid-scanning stopped-flow
kinetics at 25 °C. The enzyme used in the stopped-flow experiments
was highly active, with a turnover number at 25 °C of 72 s
1. HPhenH2 was rapidly mixed with oxidized
HDR, and heme reduction was monitored at 423 nm. Two phases of equal
amplitude were evident, and the data fit well to a biexponential
equation, corresponding to reduction of the two b-type hemes of HDR
(Fig. 2). Only the first phase of this
reaction appears to be kinetically relevant (87 s
1),
because the rate constant for the second phase (9.4 s
1)
is significantly slower than the turnover number (72 s
1)
for the enzyme. Results described below indicate that it is the low
potential heme that is reduced at catalytically competent rates.
Furthermore, only the first rate constant is dependent on
HPhenH2 concentration; the heme reduced in the second phase was HPhen-independent. These results suggest that one heme (the high
potential heme, see below) is not involved in CoB-S-S-CoM reduction by
HPhenH2.

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Fig. 2.
Stopped-flow kinetics of the reduction of HDR
by HPhenH2. Thionin-oxidized HDR (6 µM
before mixing) was rapidly mixed with HPhenH2 (10, 20, 50, 100, 150, and 200 µM before mixing). Reduction of the
hemes was followed at 423 nm. Two phases with equal amplitude were
observed, a fast phase with kobs = 87 s 1 ( ) and a slow phase with a rate constant of 9.4 s 1 ( ).
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We had hoped to independently monitor reduction of the heme and the
Fe-S cluster; however, the heme absorbance dominated the spectra of
HDR. When the HPhenH2 concentration is similar to that of
HDR and is well below its Km value, there are two clearly distinguishable phases: a rapid increase in absorbance (kobs1 = 34 s
1) followed by a
slower decay (kobs2 = 2.3 s
1)
(Fig. 3). The difference spectrum
generated by subtracting the spectrum collected at 300 ms from that at
70 ms (Fig. 3B) matches that of the difference spectrum of
the reduced minus oxidized HDR. The amplitudes of the two phases are
equal and correspond to 0.25 heme. The electron acceptor is likely to
be either an FeS cluster or some other redox site on the protein,
possibly a redox-active disulfide. If the acceptor is an FeS cluster,
the results would be most consistent with reoxidation of the heme group
by the high-potential Fe-S cluster. Given the slow rate of heme
reoxidation, however, this event is unlikely to be involved in
catalysis.

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Fig. 3.
Stopped flow kinetics of the HDR reaction at
low concentrations of HPhenH2. A, oxidized
HDR (3 µM) was mixed with HPhenH2 (5 µM), and the reaction was followed at 420 nm.
B, spectra were collected at 0, 70, and 300 ms after mixing
by rapid-scanning stopped-flow, and the difference spectrum was
generated by subtracting the 300-ms spectrum from the 70-ms
spectrum.
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Freeze-quench EPR--
Because we are unable to follow the
reduction of FeS clusters by stopped-flow due to dominating absorbance
of hemes at 400-450 nm, rapid freeze-quench EPR studies were
performed. The FeS cluster is fully reduced within 35 ms (Fig.
4A). The rate constant for Fe-S reduction by HPhen is 73 ± 31 s
1 at 20 °C
(Fig. 4B). These results clearly show that the FeS cluster is reduced at catalytically competent rates. Given the standard error
in the freeze-quench measurement, we cannot conclude whether the
cluster is reduced before, after, or simultaneously with the low
potential heme. It is the high potential cluster that undergoes reduction, because HPhenH2 reduces only the high-potential
FeS cluster; the low potential cluster remains oxidized (see
below).

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Fig. 4.
Rapid freeze-quench EPR studies of the
reduction of HDR (60 µM) by
HPhenH2 (200 µM).
The enzyme and substrate were kept in separate syringes at 20 °C
before mixing. A, reduction of the high potential FeS
cluster. The spectra shown from the top to the bottom were collected 0, 7.8, 35, 115, and 215 ms after rapidly mixing HDR with HPhen at
20 °C. B, signal intensity (g = 1.95-1.90) was plotted and fitted in a single-exponential
curve. C, reduction of the high potential heme. The spectra
shown from the top to the bottom were collected 0, 7.8, 215, and 415 ms
after mixing. D, signal intensity at g = 6.2 was plotted and fitted in a single-exponential curve. The
kcat for the HDR used in this experiment was 48 s 1 (at 25 °C). EPR conditions were: temperature, 10 K;
power, 40 milliwatts; gain, 20,000; modulation frequency, 100 kHz;
modulation amplitude, 10 G; microwave frequency, 9.4767 GHz.
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The EPR signal of the low potential heme is observed at very low
intensity (6) and could not be detected in the freeze-quench experiments; however, we were able to follow the rate at which HPhenH2 reduces the high potential high spin heme (Fig.
4C). The high spin heme signal (g = 6.2)
remained more than 60% oxidized 415 ms after reaction with
HPhenH2. The rate constant for reduction of the high spin
heme is 1.3 ± 0.1 s
1 (Fig. 4D). Thus,
the freeze-quench EPR and stopped-flow results show that reduction of
the high potential heme by HPhenH2 occurs about 10-fold
slower than kcat, indicating that this heme is
not involved in electron transfer from HPhenH2 to
CoB-S-S-CoM. This is consistent with stopped-flow and inhibition
studies described below.
Effects of CO on the HDR Reaction--
We showed earlier that CO
binds tightly to one of the hemes (Kd = 0.8 µM) (6). However, even at 1 mM concentration, CO does not affect the rate of reduction of CoB-S-S-CoM when
HPhenH2 or methyl viologen is the electron donor (Fig.
5). Because only the high spin high
potential heme binds CO (6), these results indicate that only the low
potential heme is required for CoB-S-S-CoM reduction.

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Fig. 5.
Effect of CO on HDR activity.
Top, reduction of CoB-S-S-CoM with methyl viologen as the
electron donor in the absence (solid trace) and the presence
(dashed trace) of CO. Bottom, reduction of
CoB-S-S-CoM with HPhenH2 as electron donor in the absence
(solid trace) and the presence (dashed trace) of
CO.
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When dithionite is added to HDR, both hemes are reduced [Fig.
6A]. When excess CoB-S-S-CoM is then
added, the UV-visible spectrum shows a broad Soret peak around 420 nm
(Fig. 6B, solid line). This spectrum can be fit to the sum
of two hemes with 66% in the oxidized and 34% in the reduced state
(dashed line). Therefore, the remaining reduced heme is not
involved in substrate reduction. To determine which of the two hemes is
involved in catalysis, we added CO to the CoB-S-S-CoM treated enzyme.
The Soret peak for the reduced heme shifts to 420 nm (dotted
line), which corresponds to the CO-bound form of HDR (6) and the
spectrum of the oxidized heme was unchanged. This result clearly shows
that, when CoB-S-S-CoM is added, the high potential heme remains
reduced and the low potential hexacoordinate heme undergoes
oxidation.

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Fig. 6.
Visible spectra of various states of
HDR. A, spectra of the thionin-oxidized enzyme
(solid line), and after reduction with excess dithionite
(dashed line). B, spectra after addition of
excess CoB-S-S-CoM (1 mM) to the reduced enzyme
(solid line), and after incubating the enzyme with CO
(dotted line). The dashed spectrum is a simulated
spectrum assuming 66% of the heme is oxidized and 34% is
reduced.
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These combined results strongly indicate that the high potential heme
is not involved in CoB-S-S-CoM reduction and that electrons from the
reduced low potential heme can reduce CoB-S-S-CoM to the dithiol
products at kinetically relevant rates.
Effects of Mersalyl Acid--
Mersalyl acid is known to disrupt
FeS clusters (20). Addition of mersalyl acid to HDR only slightly
affects the heme spectra. This indicates that mercury treatment does
not alter the heme environment. This is expected, because mercury only
affects hemes with sulfur ligands, not histidine-ligated hemes like
those in HDR (6). The difference spectrum between the native and the mersalyl acid-treated HDR showed a broad band around 400-500 nm that
is characteristic of FeS clusters (Fig.
7). There is a small peak at 420 nm above
the broad absorption band, which is likely to be from the heme.
However, this would constitute less than 10% alteration of the heme.
Using a typical extinction coefficient for ferredoxin, which is 16 mM
1 cm
1 per cluster (21), these
results indicate that the mersalyl acid disrupted 1.8 clusters per
dimeric unit. HDR contains two [4Fe-4S] clusters (6).

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Fig. 7.
Effect of mersalyl acid on the optical
spectrum of HDR. Oxidized HDR (solid line) was treated
with mersalyl acid (dashed line). Inset:
difference spectrum of HDR oxidized minus mersalyl-treated HDR.
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HPhenH2 was unable to reduce the hemes of the mersalyl
acid-treated oxidized enzyme, whereas dithionite reduced both hemes. The mersalyl acid-treated enzyme was also unable to catalyze the reduction of CoB-S-S-CoM, when either HPhenH2 or methyl
viologen was used as the electron donor. Assuming that mersalyl acid
only affects the FeS cluster, these results indicate that an
iron-sulfur cluster is the initial acceptor of electrons from
HPhenH2. Another possibility is that disruption of the FeS
cluster damages the HPhen binding site, which would prevent heme
reduction. The redox potentials of the two clusters are
100 and
400
mV (6). Because the midpoint redox potential for
HPhen/HPhenH2 is ~
250 mV, it seems likely that the high
potential cluster is the electron acceptor from HPhenH2.
Because the results described above indicated that the high potential
heme is not involved in CoB-S-S-CoM reduction, we hypothesize that the
electron pathway from HPhenH2 to the heme is:
HPhenH2
[Fe4S4]H
[heme b]L, where the H and L subscripts designate the
high and low potential centers. However, this is not the most
thermodynamic electron transfer pathway, because the midpoint potential
of the low potential heme is 80 mV more negative than that of the high
potential cluster.
Effects of Diphenylene Iodonium--
Diphenylene iodonium (DPI) is
a lipophilic reagent that inhibits a variety of flavoproteins such as
NAD(P)H-dependent dehydrogenases and oxidases (22-24). The
inhibitor is thought to interact with flavins and low potential
b-type cytochromes in these enzymes. DPI also inhibits the
reduction of CoB-S-S-CoM by factor F420H2 dehydrogenase in the membrane-bound electron transport chain of M. mazei Gö1 (25). To elucidate the intramolecular
electron transfer pathway among the centers of HDR, we studied
inhibition of the purified enzyme by DPI.
Inhibition of the HDR Reaction by DPI--
When
HPhenH2 is the electron donor, DPI is a strong inhibitor of
CoB-S-S-CoM reduction (Fig.
8A). DPI inhibits
heterodisulfide reduction in a competitive manner with respect to
HPhenH2 with a Ki value below 1 µM (Fig. 9). Surprisingly,
it increases the rate of methyl viologen oxidation by CoB-S-S-CoM (Fig.
8B). When CoB-S-S-CoM is absent, methyl viologen oxidation
is not observed. These results suggest that the mechanism of
CoB-S-S-CoM reduction is different with the two electron donors. One
possibility is that DPI, in competing with the HPhen binding site,
blocks electron transfer to the high potential Fe-S cluster. However,
methyl viologen, which can reduce CoB-S-S-CoM in the presence of DPI,
has a different binding site than HPhen; it may transfer electrons
directly to the heme.

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Fig. 8.
Effect of DPI on HDR activity.
Top, reduction of CoB-S-S-CoM with methyl viologen as the
electron donor in the absence (solid trace) and the presence
(dashed trace) of DPI. Bottom, reduction of
CoB-S-S-CoM with HPhenH2 as electron donor in the absence
(solid trace) and the presence (dashed trace) of
DPI.
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Fig. 9.
Competitive inhibition of the HDR reaction by
DPI. Steady-state kinetic studies were performed at 25 °C in
the presence of DPI at the following concentrations: 0 ( ), 1 ( ),
2 ( ), and 5 ( ) µM. The data points were fit
globally to the equation describing competitive inhibition. The derived
kinetic parameters were: Km for HPhenH2,
31 µM; Ki for DPI, 0.7 µM; Vmax, 38 µmol
min 1 mg 1.
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UV-visible Spectra of DPI-treated HDR--
The effects of DPI on
the oxidation states of the metal centers of HDR are summarized in
Table I. When HPhenH2 is used
as the electron donor, both of the hemes are reduced (Fig.
10). Adding DPI to the
HPhenH2-reduced enzyme causes the Soret band at 425 nm to
shift to 410 nm, corresponding to the oxidized form of HDR. This
indicates that both hemes are oxidized by DPI. Dithionite reduces both
hemes in the absence of DPI. However, when dithionite is then added to
HPhenH2-reduced and DPI-oxidized HDR, a composite spectrum
is obtained with peaks at 425 and 410 nm. The spectrum fits a mixture
of 50% oxidized and 50% reduced heme. These results indicate that,
although dithionite has a low enough potential to reduce both hemes,
reduction of one of the two hemes is inhibited by prior treatment with
DPI. CO shifts the spectrum of the reduced heme in the DPI-treated
enzyme indicating that, after treatment of HDR with DPI, only the high
potential 5-coordinate heme can be reduced. Reduction of the low
potential hexacoordinate heme is inhibited. This constitutes further
evidence that the low potential, but not the high potential, heme is
involved in electron transfer from HPhenH2 to the
heterodisulfide.

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Fig. 10.
Effects of DPI on the visible spectra of
HDR. A, spectra of the thionin-oxidized HDR
(solid line), after reduction with HPhenH2
(dashed line), and after addition of DPI to
HPhenH2-reduced enzyme (dotted line).
B, spectra of HDR after addition of dithionite to
DPI-oxidized enzyme (solid line), and after incubating the
enzyme with CO (dashed line).
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EPR Spectra of DPI-treated Enzyme--
EPR spectroscopy was used
to evaluate the effects of DPI on the metal centers in HDR (Fig.
11). Oxidized HDR displays an EPR spectrum with g values at 6.2, 5.8, and 2.0 that derives
from the high spin (spin = 5/2) heme (6) (Fig. 11A).
The low spin heme displays a "large gmax"
EPR spectrum with very low intensity and values of
gmax that are >3. When the oxidized enzyme is
reduced with HPhenH2 (Fig. 11B), the high spin
heme spectrum disappears and a new EPR signal with g values
at 2.06, 1.95, and 1.90 appears, which is from a singly reduced
[4Fe-4S] cluster (6). Unlike dithionite (Fig. 11C),
HPhenH2 is not a strong enough electron donor to reduce the
low potential cluster. When DPI is added to HPhenH2-reduced
HDR (Fig. 11D), the EPR spectrum of the cluster disappears
as the g = 6 signal from the high spin heme reappears. These results combined with the UV-visible spectroscopic results described above indicate that the high potential FeS cluster and the
two hemes undergo oxidation by DPI. When the thionin-oxidized enzyme is
treated with dithionite (Fig. 11C), all the metal centers of
HDR are reduced; the characteristic complex spectrum of the doubly
reduced protein is observed with g values at 2.03, 1.97, 1.92, and 1.88 (6). The complicated signals result from dipolar coupling between the two clusters. Addition of DPI to the
dithionite-reduced enzyme led to the oxidation of one cluster (Fig.
11E). Presumably, DPI does not oxidize the high potential
cluster.

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Fig. 11.
Effects of DPI on EPR spectra of HDR.
A, thionin-oxidized HDR. B, oxidized HDR was
treated with HPhenH2. C, oxidized HDR was
treated with dithionite. D, HPhenH2-reduced HDR
was treated with DPI. E, dithionite-reduced HDR was treated
with DPI. EPR conditions were the same as in Fig. 4.
|
|
 |
DISCUSSION |
Electron transfer from HPhenH2 through the redox
centers of HDR to CoB-S-S-CoM drives the translocation of two protons
across the cytoplasmic membrane per two electrons transferred (13). Reduction of HDR by HPhenH2 has been studied here by
kinetic and spectroscopic methods. Stopped-flow and freeze-quench EPR
experiments indicate that only one of the two hemes and one of the two
clusters undergoes reduction by HPhenH2 with rate constants
exceeding kcat. Our CO-binding and DPI
inhibition experiments indicate that the low potential heme is involved
in the electron transfer pathway and that the high potential heme is
not involved in catalysis. This is consistent with the redox demands of
the reaction; i.e. the midpoint potential for the
CoB-S-S-CoM/(RSH)2 couple is approximately
200 mV,
whereas that of the high spin high potential heme is
23 mV (6).
Why would HDR retain an unnecessary high potential heme throughout
evolution? One possibility is that this heme is involved in stabilizing
the protein or in generating the proton gradient associated with the
HDR reaction. There is a high potential c-type cytochrome in
the membranes of M. mazei with unknown function. It is
attractive to consider an electron transfer chain leading from the
oxidized heme to cytochrome c that would be coupled to proton translocation. Such a pathway would make the HDR reaction analogous to cytochrome bc1.
Which redox center is the direct electron acceptor from
HPhenH2? HPhenH2 reduces the FeS cluster and
the low potential heme at similar rates. Thus, the rapid kinetics
experiments cannot distinguish between the following electron transfer
pathways: HPhenH2
FeS
hemeL,
HPhenH2
hemeL
FeS, or a simultaneous reduction of hemeL and FeS by HPhenH2.
Inhibition experiments provide further information about the possible
electron transfer pathways. HPhenH2 does not reduce either
heme group of mersalyl-treated HDR. Because the heme spectra are not
appreciably altered by the treatment and mercury is known to disrupt
FeS clusters, we assume that inhibition of heme reduction is due to a
direct effect of Hg2+ on the cluster. Therefore, it appears
that electrons are transferred directly from HPhenH2 to an
iron-sulfur cluster, indicating that the electron transfer pathway is
HPhenH2
FeS
hemeL.
There are two 4Fe-4S clusters in HDR (6). Which of the two clusters is
the initial electron acceptor? DPI oxidizes only the low potential Fe-S
cluster, leaving the high potential cluster reduced; however, this form
of HDR can still reduce CoB-S-S-CoM. Thus, apparently, the low
potential Fe-S cluster does not undergo redox cycling during the
catalytic mechanism. Therefore, we propose that the electron transport
pathway is HPhenH2
high potential [Fe4S4]
low potential [heme
b]L. If the low potential cluster is not involved in the
relay of electrons from HPhenH2 to CoB-S-S-CoM, why would
it be present in HDR? One possibility, described below, is that the
oxidized low potential cluster is involved in a catalytic step and not
in redox chemistry. Another is that HDR can accept electrons from
several sources and that, under some conditions, a redox partner with a
more negative midpoint potential than MPhen could donate electrons to
the low potential cluster, which would in turn donate electrons to the
high potential cluster. The electron transport pathway from CODH and
CO, with a midpoint potential below
500 mV, has not been elucidated.
The experiments described in this report were performed in aqueous
solution with the water-soluble HPhen analog of MPhen. It is important
to determine whether the membrane-bound enzyme uses the same electron
transfer pathway. If the physiologically relevant order of electron
flow is indeed HPhenH2
[Fe4S4]H
[heme
b]L, the high potential cluster is expected to be located near the subunit interface. This is because the large subunit containing the clusters is cytoplasmic and the heme is in the membrane-associated subunit (16). This would be similar to several quinone-coupled enzymes whose FeS clusters are in close contact with a
quinone-binding site in membrane, such as quinol:fumarate reductase
(26), Me2SO reductase (27), and NADH:ubiquinone oxidoreductase (28).
How do electrons passing through one-electron redox centers accomplish
the two-electron reduction of CoB-S-S-CoM? Because the two classes of
HDRs are heme iron-sulfur or flavin iron-sulfur proteins, Thauer
et al. (16) proposed that the iron-sulfur subunits harbor
the active site of heterodisulfide reduction and that the mechanism of
disulfide reduction could resemble that of the ferredoxin:thioredoxin reductases from chloroplasts and cyanobacteria. In the plant
thioredoxin reductase mechanism, a sulfur radical, which is formed as
an intermediate, appears to be stabilized by binding to a
[Fe4S4] cluster (29, 30). Evidence presented
here indicates that the low potential cluster of HDR is not involved in
electron transfer reactions. Because the midpoint potential of this
cluster (
400 mV) is much lower than that of HPhen (~
250 mV) or
CoB-S-S-CoM (~
220 mV), this cluster may play a catalytic role,
analogous to that of the cluster in thioredoxin reductase. This
putative role is to stabilize a radical anion formed by one electron
reduction of the disulfide substrate. Further studies are required to
test this hypothesis.
 |
FOOTNOTES |
*
This work was supported by Department of Energy Grant
ER20053 (to S. W. R.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Tel.:
402-472-2943; Fax: 402-472-8912; E-mail: sragsdale1@unl.edu.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M004809200
2
U. Deppenmeier, E. Murakami, and S. W. Ragsdale, manuscript in preparation.
 |
ABBREVIATIONS |
The abbreviations used are:
HDR, heterodisulfide reductase;
HS-CoM, coenzyme M or 2-mercaptoethane
sulfonic acid;
HS-CoB, coenzyme B or 7-mercaptoheptanoyl-threonine
phosphate;
MPhen, methanophenazine;
HPhen, 2-hydroxyphenazine;
DPI, diphenylene iodonium.
 |
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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.