Tài liệu Báo cáo khoa học: Purification and characterization of a membrane-bound enzyme complex from the sulfate-reducing archaeon Archaeoglobus fulgidus related to heterodisulfide reductase from methanogenic archaea pdf

Purification and characterization of a membrane-bound enzyme
complex from the sulfate-reducing archaeon
Archaeoglobus fulgidus
related to heterodisulfide reductase from methanogenic archaea
Gerd J. Mander
1
, Evert C. Duin
1
, Dietmar Linder
2
, Karl O. Stetter
3
and Reiner Hedderich
1
1
Max-Planck-Institut fu
¨
r terrestrische Mikrobiologie, Marburg, Germany;
2
Biochemisches Institut, Fachbereich Humanmedizin,
Justus-Liebig-Universita
¨
t Giessen, Germany;
3
Lehrstuhl fu
¨
r Mikrobiologie und Archaeenzentrum, Universita
¨
t Regensburg, Germany
Heterodisulfide reductase (Hdr) is a unique disulfide reduc-
tase that plays a key role in the energy metabolism of

methanogenic archaea. The genome of the sulfate-reducing
archaeon Archaeoglobus f ulgidus encodes s everal proteins of
unknown function with high sequence similarity to the
catalytic subunit of Hdr. Here we report on the purification
of a multisubunit membrane-bound enzyme complex from
A. fulgidus that contains a subunit related to the catalytic
subunit of Hdr. The purified enzyme is a heme/iron-sulfur
protein, as deduced by UV/Vis spectroscopy, EPR spec-
troscopy, and the primary structure. It is composed of four
different subunits encoded by a putative transcription unit
(AF499, AF501–AF503). A fifth protein (AF500) encoded
by this transcription unit could not be detected in the purified
enzyme preparation. S ubunit A F502 is clo sely r elated to the
catalytic subunit HdrD of Hdr from Methanosarcina bark-
eri. AF501 encodes a membrane-integral cytochrome, and
AF500 e ncodes a second integral m embrane protein. AF499
encodes an e xtracytoplasmic iron-sulfur protei n, and A F503
encodes an extracytoplasmic c- type cytochrome with th ree
heme c-binding motifs. All of the subunits show high
sequence similarity to proteins encoded by the dsr lo cu s of
Allochromatium vinosum and to subunits of the Hmc
complex from Desulfovibrio v ulgaris. The heme groups of the
enzyme are rapidly reduced by redu ced 2,3-dimethyl-1,4-
naphthoquinone (DMNH
2
), which indicates that the
enzyme functions as a menaquinol–acceptor oxidoreduc-
tase. The physiological electron acceptor has not yet been
identified. Redox t itrations monitored by EPR spectroscopy
were carried out to characterize the iron-sulfur cl usters of the

enzyme. In addition to EPR signals due to [4Fe-4S]
+
clus-
ters, signals of an unusual p aramagnetic species with g values
of 2.031, 1.994, and 1.951 were obtained. The paramagnetic
species could be reduced in a one-electron transfer reaction,
but could not be further oxidized, and shows EPR properties
similar to t hose of a paramagnetic species r ecently identified
in Hdr. In Hdr this paramagnetic species is specifically
induced b y the substrates of the e nzyme a nd is thought to be
an inte rmediate of the catalytic cycle. Hence, Hdr and the
A. fulgidus enzyme not only share sequence similarity, but
may also have a similar active site and a similar catalytic
function.
Keywords: Archaeoglobus f ulgidus; heterodisulfide reductase;
Hmc complex; iron-sulfur proteins; sulfate-reducing
bacteria.
Heterodisulfide reductase (Hdr) is a key enzyme in the
energy metabolism of methanogenic archaea. In the final
step of methanogenesis, the mixed disulfide of the metha-
nogenic thiol coenzymes coenzyme M and coenzyme B is
generated in a reaction catalyzed by methyl-coenz yme M
reductase [1]. This disulfide is reduced by a unique disulfide
reductase, designated heterodisulfide reductase (Hdr). Two
types of Hdr from phylo genetically distantly related meth-
anogens have been identified a nd characterized. Neither
type of enzyme belongs to t he family of pyridine nucleotide
disulfide oxidoreductases [2].
Hdr from Methanothermobacter marburgensis is an
iron-sulfur flavoprotein composed of the subunits HdrA,

HdrB, and HdrC. The enzyme has been purified from the
soluble fraction, and none of its subunits are predicted to
form transmembran e helices. From sequence data, it has
been deduced that HdrA contains an FAD-binding motif
and four binding motifs for [4Fe-4S] clusters. HdrC
contains two additional binding motifs for [4Fe-4S]
clusters [2].
Hdr in t he two closely relate d Methanosarcina sp ecies
M. barkeri and M. thermophila is tightly membrane-
bound [3–5]. The enzyme is composed of two s ubunits,
a membrane-bound b-type cytochrome (HdrE) and a
hydrophilic subunit (HdrD) containing two binding
motifs for [4Fe-4S] clusters. Subunit HdrD of the
M. barkeri enzyme is a homologue of a hypothetical
fusion protein of the M. marburgensis HdrCB subunits
Correspondence to R. Hedderich, Max-Planck-Institut fu
¨
r terrestrische
Mikrobiologie, Karl-von-Frisch-Strabe, D-35043 Mar burg/Germany.
Fax: + 49 6421 178299, Tel.: + 49 6421 178230,
E-mail:
Abbreviations: Hme, Hdr-like menaquinol-oxidizing enzyme; Hdr,
heterodisulfide reductase; DMN, 2 ,3-dimethyl-1,4-naphthoquinone;
H-S-CoM, coenzyme M or 2-mercaptoethanesulfonate; H-S-CoB,
coenzyme B or 7-mercaptoheptanoylthreonine phosphate;
CoM-S-S-CoB, heterodisulfide of H-S-CoM and H-S-CoB; Hmc,
high-molecular-mass c-type cytochrome; Dsr, dissimilator y sulfite
reductase.
Enzyme: heterodisulfide reductase (EC 1.99.4 ).
(Received 10 October 2001, revised 12 February 2002, accepted 15

February 2002)
Eur. J. Biochem. 269, 1895–1904 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02839.x
[4]. A homologue of the M. marburge nsis HdrA subunit is
lacking in H dr from Methanosarcina species. It has
therefore been suggested that the conserved subunits
HdrD and HdrCB must harbor the catalytic site for the
reduction of the disulfide substrate. The active site of Hdr
was recently shown to contain a [4Fe-4S] cluster that is
directly involved in mediating heterodisulfide reduction
[6,7]. This extra iron-sulfur cluster has been proposed to
be co-ordinated by cysteine residues of the highly
conserved sequence motif CX
31)38
CCX
33)34
CXXC found
in subunits HdrD and HdrB. The nonconserved subunits
HdrE and HdrA are thought to interact with the physio-
logical electron donor, which differs in t he two types of
Hdr. The physiological electron donor of Hdr from
Methanosarcina species is thought to be the membrane-
soluble electron c arrier methanophenazine [8]. Hdr from
M. marburgensis forms a functional complex with the
MvhAGD hydrogenase [9]. This complex catalyzes the
reduction of CoM-S-S-CoB by H
2
.
Hdr w as originally thought to be u nique to methanogenic
archaea. However, in recent years, genes encoding pro-
teins r elated to the catalytic subunit of Hdr have been identi-

fied in a b road range of prokaryotes unable t o perform
methanogenesis [2]. No function has so far been assigned to
these Hdr-like proteins, and none has been purified and
characterized. Archaeoglobus fulgidus is one of the organ-
isms that en code the largest number of proteins related to
Hdr [10].
This extremely thermophilic sulfate-reducing archaeon
completely oxidizes organic substrates, such as lactate, t o
CO
2
[11]. Acetate is oxidized to CO
2
by a modified acetyl-
CoA pathway using typical methanogenic coenzymes
[12,13]. Some of the reducing equivalents generated in the
oxidative branch of the pathway are transferred to the
deazaflavin coenzyme F
420
, which is reoxidized by the
F
420
H
2
–menaquinone oxidoreductase. F
420
H
2
–menaqui-
none oxidoreductase is an integral membrane protein that
shows high sequence similarity to energy-conserving

NADH–quinone oxidoreductases [10,14]. It is assumed to
function as a proton or sodium ion pu mp as well. In
addition, the m embrane fraction o f A. fulgidus catalyzes the
reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by
L
-lactate, which indicates that lactate dehydrogenase direct-
ly channels the reducing equivalents generated in the
oxidation of lactate to pyruvate into the menaquinone pool
[12]. A. fulgidus has been shown t o contain a modified
menaquinone as a m embrane-soluble e lectron c arrier [15]. It
is, however, not yet known how the reduced menaquinone
pool is electrically connected to the enzymes of sulfate
reduction, namely adenosine 5 ¢-phosphosulfate reductase
and sulfite reductase.
Here we report on the isolation and characterization of a
heme-containing membrane protein from A. fulgidus related
to Hdrfrom M. barkeri. Afunction ofthis enzymeas reduced
menaquinone–acceptor oxidoreductase is discussed.
MATERIALS AND METHODS
Materials
Redox dyes were obtained from Aldrich–Sigma. DMN w as
from Sigma. Potassium trithionate was a gift from Peter
M. H. Kroneck (Universita
¨
t K onstanz). All other chemicals
were from Merck. The chromatographic materials were
from Amersham Pharmacia Biotech.
Growth of the organism
A. fulgidus (VC16, DSMZ 304) was grown in a 300-L
fermenter at 83 °C on lactate/sulfate medium as described

previously [11]. Cells were harvested after shock cooling to
4 °C w ith a continuous flow centrifuge (Z61; Padberg Lahr,
Germany) at 17 000 g; the pellet was frozen in liquid
nitrogen and stored at )80 °Cbeforeuse.
Enzyme purification
All purification steps were carried out under strictly anoxic
conditions under an atmosphere of N
2
/H
2
(95 : 5, v/v) at
18 °C. Cells were lysed by sonication and then centrifuged
at 6400 g for 1 h. The supernatant was ultracentrifuged at
150 000 g for 2 h. The pellet was resuspended in 50 m
M
Mops (pH 7.0) using a Teflon homogenizer. Protein was
solubilized from the membranes with 15 m
M
dodecyl-b-
D
-
maltoside [ 2 mg dodecyl-b-
D
-maltosideÆ(mg protein)
)1
]at
4 °C for 12 h. The unsolubiliz ed proteins and the mem-
branes were removed by u ltracentrifugation as described
above. The supernatant was applied to a Q-Sepharose
HighLoad column (2.6 · 10 cm) equilibrated with 50 m

M
Mops/KOH (pH 7.0) containing 2 m
M
dodecyl-b-
D
-malto-
side (buffer A). Protein was eluted in a s tepwise NaCl
gradient (80 mL each in buffer A): 0 m
M
, 300 m
M
,
400 m
M
,500m
M
,600m
M
,and1
M
. The majority of the
heme-containing protein(s) were eluted at 600 m
M
NaCl.
These f ractions were applied to a Superdex 200 gel-filtration
column (2.6 · 60 cm) equilibrated w ith buffer A containing
100 m
M
NaCl. Protein was eluted using the same buffer.
Heme-containing protein(s) were eluted after 120 mL (peak

maximum). These fractions were applied to a Mono Q
anion-exchange column (HR 10/10) equilibrated with
buffer A. Protein was eluted using a linear NaCl gradient
(0–1
M
, 100 mL). Heme-containing protein(s) were eluted
at 600 m
M
NaCl. The enzyme was concentrated by
ultrafiltration (Molecular/Por ultrafiltration membranes;
100-kDa cut off; Spectrum, Houston, USA) and stored in
buffer A at 4 °C und er N
2
. P rotein was judged to be >95%
pure by SDS/PAGE.
UV/Vis spectroscopy
Spectra of samples in 1-mL quartz cuvettes in a n anaerobic
chamber under N
2
/H
2
(95 : 5, v/v) were recorded using a
Zeiss Specord S10 diode array spectrophotometer connected
to a quartz photoconductor ( Hellma Mu
¨
llheim, Germany).
The oxidation or reduction of the heme groups of the
enzyme by DMN or DMNH
2
were followed spectropho-

tometrically. DMN or DMNH
2
was added to the enzyme
solution [1 mgproteinÆmL
)1
in 50 m
M
Mops/KOH (pH 7.0)]
to a final concentration of 150 l
M
, and spectra were
recorded every 5 s. DMNH
2
was prepared a s described
previously [16].
Analytical methods
Non-heme iron was quantified colorimetrically with neo-
cuproin (2,9-dimethyl-1,10-phenanthroline) and ferrozine
1896 G. J. Mander et al.(Eur. J. Biochem. 269) Ó FEBS 2002
[3-(2-pyridyl)-5,6-bis-(4-phenylsulfonate)-1,2,4-triazine] as
described by Fish [17]. Acid-labile sulfur was analyzed as
methyleneblue[18].
The protein concentration was routinely determined by
the method of Bradford (Rotinanoquant; Roth Karlsruhe,
Germany) using BSA as standard.
Heme was extracted with acetone/HCl and the pyridine
hemochrome derivate was formed as described. Reduced
minus oxidize d differe nce spectra were recorded at room
temperature [19]. The spectra obtained were compared with
the pyridine hemochrome spectra obtained with heme

extracted from hemoglobin.
EPR spectroscopy measurements
EPR spectra at X-band (9 GHz) were obtained with a
Bruker EMX spectrometer. All spectra were recorded with
a field modulation frequency of 100 kHz. The sample was
cooled by an Oxford Instruments ESR 900 flow cryostat
with an ITC4 temperature controller. Spin quantitations
were carried out under nonsaturating conditions using
10 m
M
copper perchlorate as the standard (10 m
M
CuSO
4
,
2
M
NaClO
4
,10m
M
HCl). When the EPR signals over-
lapped with o ther signals, e.g. radical signals from the redox
dyes, the signals were simulated, and the simulations were
double integrated t o obtain the spin intensity. Temperature-
dependence studies were carried out under nonsaturating
conditions where possible. For all signals, the peak ampli-
tude was measured at different temperatures. These values
were used to obtain Curie plots describing the temperature
behavior of the respective signal. EPR signals were simu-

lated using nonco mmercial programs based on formulas
described previously [20].
Redox titrations
Redox titrations were carried out at 18 °Cinananaerobic
chamber under N
2
/H
2
(95 : 5, v/v). Potentials w ere a djusted
with small amounts of freshly prepared sodium dithionite
(20 m
M
stock solution) or freshly prepared potassium
ferricyanide (20 m
M
stock solution). All redox potentials
quoted here are relative t o the standard hydrogen electr ode.
In these titrations, a selection of the following mediators
(final concentration 20 l
M
) were added individually to the
enzyme solution: 1,2-naphthoquinone (E°¢ ¼ +134 mV),
duroquinone (E ¢ ¼ +86 mV), 1,4-naphthoquinone
(E ¢ ¼+69 mV), thionine (E ¢ ¼+64 mV), methylene blue
(E ¢ ¼ +11 mV), indigodisulfonate (E ¢ ¼ )125 mV),
2-hydroxy-1,4-naphthoquinone (E ¢ ¼ )145 mV), anthra-
quinone-1,4-disulfonate (E ¢ ¼ )170 mV), phenosafranin
(E ¢ ¼ )252 mV), anthraquinone-2-sulfonate ( E°¢¼
)255 mV), safranin O (E ¢ ¼ )289 mV), and neutral red
(E ¢ ¼ )325 mV). The final concentration of Hdr-like

menaquinol-oxidizing enzyme (Hme) was 7 l
M
in 50 m
M
Mops/KOH (pH 7.0) containing 2 m
M
dodecyl-b-
D
-malto-
side. After equilibration of the desired potential, a 0.3-mL
aliquot was transferred to a calibrated EPR tube and
immediately f rozen in liquid nitrogen. The redox potential
wasmeasuredwithanAg/AgClredoxcombinationelec-
trode (Mettler Toledo Giessen, Germany). To obtain
potentials relative t o the standard hydrogen electrode, a
value of 207 mV (corresponding to the potential of Ag/
AgCl at 25 °C) was added to t he measured redox potentials.
Determination of amino-acid sequences
For determination of N-terminal amino-acid sequences,
polypeptides w ere separated by SDS/PAGE and b lotted
on to poly(vinylidene difluoride) membranes (Applied
Biosystems) as described previously [4]. Sequences were
determined using an Applied Biosystems 4774 protein/
peptide sequencer and the protocol given by the m anu-
facturer.
Amino-acid sequence analysis
For the prediction of transmembrane helices in proteins,
noncommercial programs were u sed ( />miklos/DAS/; />2.0/). For sequence comparisons, multiple sequence align-
ments were generated using the
FASTA

3server(http://
www.ebi.ac.uk/fasta3/).
RESULTS
Purification of a heme-containing enzyme complex
from the membrane fraction of
A. fulgidus
The genome of A. fulgidus encodes several membrane-
bound oxidoreductases that share sequence similarity with
subunits of Hdr from methanogenic archaea, in particular
with the membrane-bound enzyme from M. barkeri [4,10],
which is anchored in the cytoplasmic membrane via a b-
type cytochrome [3]. We used this knowledge to identify
and purify heme-containing membrane-bound enzymes
from A. fulgidus cells cultivated on lactate/sulfate medium
by following the characteristic absorption of heme proteins.
The membrane fraction was isolated, and proteins were
solubilized with the detergent dodecyl-b-
D
-maltoside. On
anion-exchange chromatography on Q-Sepharose, the
major heme-containing fraction was eluted at 600 m
M
NaCl. Approximately 70% of the heme presen t in solubi-
lized membranes was found in this fraction. A further
purification of the proteins in this heme-containing fraction
by gel fi ltration on Superdex 200 resulted again in only one
heme-containing fraction eluted after 120–150 mL. In the
final purification step, the sample was chromatographed on
a Mono Q anion-exchange column. The protein thus
purified was subjected to SDS/PAGE (Fig. 1). Samples

were either boiled for 5 min in SDS buffer or incubated in
SDS buffer at room temperature for 1 h before electro-
phoresis. The samples incubated at room temperature
yielded four major polypeptide bands with apparent
molecular masses of 53, 34, 31, and 16 kDa after SDS/
PAGE (Fig. 1, lane A1). In the boiled sample, the 34-kDa
polypeptide was only detectable at lower intensities. This
may be due to protein aggregation, which is typical of
integral membrane proteins (Fig. 1, lane B2). From the
results of SDS/PAGE, it can be deduced that the 16-kDa
protein is only present in substoichiometric amounts. In
some preparations, this protein was completely absent
(Fig. 1B).
As will be described below, the enzyme complex purified
from A. fulgidus shows similarity to Hdr and has a
menaquinol-oxidizing activity. The enzyme was therefore
preliminarily designated Hdr-like menaquinol-oxidizing
enzyme complex, abbreviated as Hme complex.
Ó FEBS 2002 Hdr-like enzyme complex from A. fulgidus (Eur. J. Biochem. 269) 1897
Identification of the genes encoding the subunits
of the Hme complex and sequence analysis
The N-terminal sequences of the four polypeptides present
in the purified enzyme preparation were determined by
Edman degradation (Table 1). Using these sequences, the
corresponding genes (AF499, AF501–503) were identified in
thegenomeofA. fulgidus [10]. The noncoding regions
between the different genes are short (less than 12 bp) or
nonexistent (the genes overlap). The sequence region
upstream o f A F499 is AT-rich and contains typical a rchaeal
promoter elements. The sequence AAAGGTTAATATA

was f ound 64 bp upstream of the start codon of AF499; this
corresponds to the BRE element and the box A element of
archaeal promoters [21,22]. The A F499–AF503 gene cluster
can therefore be predicted to form a transcription unit
(Fig. 2). This transcription unit contains one gene (AF500)
for which no corresponding protein was found in the
purified enzyme preparation. The results of the sequence
analyses of the deduced proteins are given in Table 2.
The protein encoded by AF502 has a calculated mole-
cular mass of 64.4 kDa. The protein shows about 35%
sequence identity with the proposed catalytic s ubunit H drD
from M. barkeri. The closest relative of t he protein e ncoded
by AF502 (40% sequence identity) is the dissimilatory
sulfite reductase (Dsr)K protein from the sulfur-oxidizing
phototrophic bacterium Allochromatium vinosum. The DsrK
protein is encoded by the dsr locus, which also encodes the
subunits of the siroheme sulfite reductase [23]. Another
relative of the protein encoded b y AF502 is the high-
molecula r-ma ss c-type cytochrome (Hmc)F protein of
Desulfovibrio vulgaris (20% sequence identity) [24].
A characteristic of HdrD o f M. barkeri is the presence of
two t ypical [4Fe-4S] cluster binding motifs in th e N -terminal
part of the protein. HdrD contains 10 additional cysteine
residues found in two CX
31)38
CCX
33)34
CXXC sequence
motifs at the C-terminal part of the protein [4]. Multiple
sequence alignments of HdrD, AF502, DsrK, and HmcF

clearly identified the t wo typical CXXCXXCXXXCP
binding motifs for [4Fe-4S] clusters in the N-terminal part
of these proteins. AF502, DsrK, and HmcF also contain
one of the two CX
31)38
CCX
33)34
CXXC motifs present in
HdrD. Only in AF502 does an aspartate residue replace one
of the five cysteines present in this motif [25].
The AF501 protein has a calculated molecular mass of
38 k Da. The molecular mass of this protein was estimated
by SDS/PAGE to be 34 kDa. The protein shows the highest
sequence similarity (30% identity) to the DsrM protein
from A. vinosum, encoded b y the dsr locus,andtotheb-type
cytochrome subunit of nitrate reductase from various
organisms, such as NarI of nitrate reductase of Escherichia
coli [26]. AF501 also has low sequence similarity to the
b-type cytochrome (HdrE) of Hdr. A topological analysis
suggests that AF501, like NarI, has five membrane-span-
ning helices. In t he b-type c ytochromes of nitrate reductases,
four histidine residues are conserved, two in helix b and two
Fig. 1. SDS/PAGE of the purified Hme complex. Proteins were sepa-
ratedina12%slabgel(8· 7 c m) which was subsequently stained
with Coomassie Brilliant Blue R250. The polypeptide with an apparent
molecular mass of 16 kDa, identified as a c-type cytochrome by
N-terminal sequencing, was not found in all preparations. The pre-
paration shown in (A) still contains the c-type cytochrome, while the
preparation shown in (B) lacks this polypeptide. M, Low-molecular-
mass markers ( Amersham Pharmacia Biotech). The m olecular mass es

of the marker proteins are given o n the right side. Lane A1, 15 lgof
the A. fulgidus Hme c omplex denatured fo r 30 min at room t emper-
ature in SDS sample buffer; lane B1, 10 lg Hme complex denatured
for 30 min at room temperatu re i n SDS sam ple buffer; lane B2, 10 lg
Hme complex denatured for 5 min at 100 °C in SDS sample buffer.
The polypeptide with an apparent molecular mass of 34 kDa, identi-
fied as a b-type cytochro me-like protein by N-terminal sequencing,
shows a lower intensity in the b oiled sample; it probably forms
aggregates that do not run into the gel (lane B2). This behavior is
typical of integral membrane proteins. The polypeptide with an
apparent molecular mass of 53 kDa appears as a double band in
unboiled samples (lanes A1 and B1).
Table 1. N-Terminal sequences of the polypeptides of the purified enzyme. N-Terminal sequen ces were eit her obtained by Edman degradation
(column 1) or d er ived from the genome sequence of A. fulgidus (column 2). The c orresp onding genes are given in column 3. Amino acids p re sent in
both sequences are u nd erlined, a nd am ino acids th at co uld not be determined with ce rtainty i n the Ed man d egradation are g iven in parentheses.
Sequence derived by Edman
degradation
Sequence derived from the A. fulgidus
genome sequence Identified ORF
(
M)(E)RMRE(I)IEIKAKFP MEEMPERIEIKQKFP AF502
MIGVIFGVIVFYIAV MIGVIFGVIVFYIAV AF501
(
K)TQFIESPEEV(V)EK MMSRRKFLLLTGAAAAGAILTPQISA
KTQFIESPEEVREK
AF499
MYNK-YVIPLILVFL MSEMYNKKYVIPLILVFL AF503
1898 G. J. Mander et al.(Eur. J. Biochem. 269) Ó FEBS 2002
in helix d. These histidine residues have been assigned as
b-heme axial ligands for two heme groups that are located

on different h alves o f t he membrane bilayer [26]. AF501 not
only has the same topology as NarI, but also contains the
two h istidine residues in h elix b and two in helix d. AF501 is
therefore predicted to ligate two heme groups.
The AF499 protein has a calculated molecular mass of
30.5 kDa. Sequence analysis revealed that the protein
belongs to a group of iron-sulfur proteins with 16 conserved
cysteine residues predicted to co-ordinate four [4Fe-4S]
clusters. Members of this family include DsrO from
A. vinosum, HmcB from D. vulgaris, and HybA, DmsB,
and N rfC f rom E. coli. Some members, including AF499,
have an N-terminal Ôtwin-arginineÕ signal sequence that is
characteristic of cofactor-containing proteins translocated
into the periplasm via the Tat translocase [27]. As deduced
from the N-terminal sequence of AF499, the signal peptide
is not present in the mature enzyme (Table 1).
The AF503 protein has a calculated molecular mass of
16.7 kDa. The protein contains three CxxCH sequence
motifs characteristic of proteins th at co-ordinate heme c.
The protein is therefore predicted to co-ordinate three
heme c molecules. AF503 shows the highest sequence
similarity to a protein encoded by the dsr locus of
A. vinosum, the DsrJ protein. The mature form of the
AF503 protein contains an N-terminal hydrophobic s tretch
predicted to form a transmembrane a helix, which may
anchor the protein in the membrane. This stretch may
function as a signal peptide of the Sec pathway [28].
The AF500 protein, which was not detected in the
purified enzyme, has a calculated molecular mass of
43 kDa. This protein shows highest sequence identity to

the DsrP protein from A. vinosum. It shows low sequence
similarity to the HmcC protein of D. vulgaris. Topological
analysis suggests that AF500, like DsrP and HmcC, has 10
membrane-spanning helices. These three proteins are also
related to the DmsC protein of dimethylsulfoxide reductase
[29]. The latter protein contains only eight predicted
transmembrane helices.
Catalytic properties of the Hme complex
and characterization by UV/Vis spectroscopy
To determine whether the cytochrome present in the Hme
complex is reduced by menaquinone, in vitro assays were
performed using the more hydrophilic analogue of men-
aquinone, DMN. The enzyme purified u nder anoxic condi-
tions generally contained the heme groups in the reduced
state. Any enzyme molecules that contained oxidized h eme
groups could be rapidly reduced by sodium dithionite.
Addition of DMN to the reduced enzyme resulted in rapid
oxidation of the heme present in the enzyme. The oxidized
heme groups could be rapidly reduced using DMNH
2
as
electron donor. The r ates of h eme reduction by DMNH
2
or
oxidation by D MN were too rapid to be resolved. Figure 3
shows t he dithionite-reduced minus air-oxidized absorbance
difference spectrum of an enzyme preparation containing
only minor amounts of th e 16-kDa c-type cytochrome. The
Fig. 2. Genomic o rganization of the genes e ncoding the subunits of the Hme complex from A. fulgidus. ThegenenamesannotatedbyTIGRaregiven
above the arro w representing t he genes a n d their direction of transcription. The s ize in bp is given below each gene. Betwee n the genes A F498 and

AF499 is a 385-bp-long noncoding region. The genes within the putative transcription unit from AF499 to AF503 have an intergenic region ranging
from 1 t o 11 bp or even overlap (AF500 and AF501 overlap by 3 bp). The region 81–65 bp upstream of t he start c odon of AF499 was identified as
an archaeal promoter element by seque nce analysis. The sequ ence AAAGGTTAATATA shows a high le vel of identity with th e consensus se quence
()35 to )23, AAANNN
TTATATA) ; the sequence of the so-called BRE (transcription factor B recognition element) is in italics; the sequence of the
so-called Box A is underlined. These eleme nts have been id entified as essentia l elements for archa eal transcription [21,22 ].
Table 2. Features of the subunits of the Hme complex from A. fulgidus. Data are eith er derived from the analysis of the sequence (calculated
molecular mass, predicted transmembrane helices, cofactor binding sites, sequence identities) o r obtained experimentally (apparent molecular mass,
cofactor content).
Gene AF502 AF501 AF499 AF503 AF500
Apparent/calculated
molecular mass
53/64.4 kDa 34/38 kDa 31/30.5 kDa 16/16.7 kDa – /43 kDa
Transmembrane helices None 5 None 1 10
Cofactor binding sites 2[4Fe-4S],
4 highly conserved
cysteine residues
2 heme
groups
4[4Fe-4S] 3 heme c
(CX
2
CH)
–
Highest sequence
identity with
DsrK DsrM DsrO DsrJ DsrP
Further comments Related to the
catalytic subunit
of Hdr

Cytochrome,
integral membrane
protein
Extracytoplasmic
iron-sulfur protein
Extracytoplasmic
c-type cytochrome
Integral membrane
protein
Ó FEBS 2002 Hdr-like enzyme complex from A. fulgidus (Eur. J. Biochem. 269) 1899
absorption maxima at 420 nm (c band), 530 nm (b band)
and 557 nm (a band) are characteristic of cytochrome b.
Heme was extracted from the protein with acidic acetone
and the pyridine hemochrome spectrum (reduced minus
oxidized) was recorded. T he spectrum contained maxima of
the a and b band at 553 and 521 nm, respectively. These
maxima are blue-shifted by a bout 4 nm relative t o published
values for protoheme IX [30]. In a control experiment the
pyridine hemochrome spectrum of hemoglobin was deter-
mined under identical conditions resulting in maxima
identical with published values (525 nm for the b band
and 557 nm for the a band). A similar blue shift was f ound
in heme o of cytochrome bo [30]. As pyridine hemochrome
spectra are very sensitive to substitutions on the porphyrin
ring, the results indicate that th e e xtractable heme of Hme is
not protoheme IX. Further studies are necessary to
elucidate the nature of the extractable heme present in this
enzyme.
The oxidation of the heme groups of the enzyme by
various compounds was tested to identify the physiological

electron acceptor of the enzyme. The enzyme was not
oxidized by the heterodisulfide of coenzyme M and
coenzyme B, or by the homodisulfides of these two
coenzymes. Also potassium trithionate and sodium tetra-
thionate, w hich have been identified i n dissimilatory sulfate
reducers [31], failed t o oxidize the enzyme.
Characterization of the iron-sulfur clusters
by EPR spectroscopy
The enzyme preparation was shown to contain
90–110 nmol nonheme iron and 105–115 nmol acid-labile
sulfur. I f one enzyme molecule has a mass of 150 kDa,
this corresponds to  19–21 mol acid-labile sulfurÆ(mol
enzyme)
)1
and 16–20 mol nonheme ironÆ(mol enzyme)
)1
.
Analysis of the amino-acid sequence of the enzyme leads to
the p rediction that the enzyme contains six [4Fe-4S] c lusters,
four in AF499 and two in AF502. In addition to the
conserved cysteine residues that co-ordinate these iron-
sulfur clusters, the AF502 protein contains a cysteine cluster
that is conserved in Hdr from various methanogens and in
all Hdr-like proteins. Some of these cysteines in Hdr are
thought to co-ordinate a special iron-sulfur cluster in the
active site of the enzyme that is directly i nvolved in the
reduction of disulfide substrate. The Hdr-like protein from
A. fulgidus was t herefore studied by EPR spectroscopy.
Redox titrations were monitored by EPR to characterize
the different iron-sulfur clusters presen t in the enzyme. As

expected, t he enzyme showed broad unresolved EPR signals
at redox potentials u p to )100 mV that w ere only detectable
at temperatures below 1 0 K. These signals are most
probably due to the bulk of [4Fe-4S]
+
clusters present in
the enzyme. At potentials higher than 0 mV, an unusual
paramagnetic species was detected with g values at 2.031,
1.994, and 1.951. The resonance started to develop at
potentials ‡ 0 mV and was stable at potentials up to
+350 mV. The loss and formation of the resonance was
associated with a one-electron redox process with a
midpoint potential of +90 ± 10 mV (Fig. 4). The spin
concentration of the signals in the different titrations was
generally near 0.4 spinÆ(mol enzyme)
)1
. Because of overlap
with radical signals around g ¼ 2, the signal was simulated
(Fig. 4) and double integrated to obtain the spin intensity.
Temperature studies showed that the signal is readily power
saturated at 4.5–15 K. At 15–35 K, the signal could be
measured under nonsaturating conditions. At higher tem-
peratures, the signal started to broaden a nd was broadened
beyond detection at 60 K.
The EPR signal observed has EPR characteristics very
similar to a unique signal described for Hdr from metha-
nogens. The two paramagnetic species have similar g values,
show the same temperature behavior, and are only detect-
able in the o xidized enzyme. The g value at 2.016 present in
Hdr [6] is shifted in the A. fulgidus enzyme to 2.031. The

midpoint potential of the paramagnetic species found in the
A. fulgidus enzyme is shifted to higher redox potentials. In
Hdr, this paramagnetic species is only observed in titrations
carried out in the presence of one of the substrates of the
enzyme. The physiological electron acceptor of the A. ful-
gidus enzyme is still unknown. Therefore, no substrate could
be added to the titration mixture.
DISCUSSION
A large number of protein sequences related to the catalytic
subunit of Hdr from methanogenic archaea have been
deposited in the databases. None of these putative pro teins
has b een c haracterized and no f unction has been assigned to
any of them [2]. In this study, we chose the sulfate-reducing
archaeon A. fulgidus for the isolation of one of the Hdr-like
proteins encoded by the genome of this organism. In cells
cultivated on lactate/sulfate medium, the enzyme turned o ut
to be a major membrane protein and contained m ost of the
heme present in t he membran e fraction. Hdr from M. bark-
eri is composed of only two subunits, a b-type cytochrome
and the hydrophilic catalytic subunit [7]; the subunit
structure of the Hme complex isolated from A. fulgidus is
considerably more complex. The a nalysis o f the sequence o f
the gene cluster encoding the enzyme predicts the presence
of five subunits, but only four were detected in the purified
enzyme preparation. The integral m embrane subunit A F500
Fig. 3. Room temperature reduced–oxidized difference spectrum of the
purified Hme complex. Hme [1 mg proteinÆml
)1
in 50 m
M

Tris/HCl
(pH 7 .6)] was reduced with so dium dithionite and su bsequently oxi-
dized by air. The oxidized spectrum was subt racted from the red uced
spectrum. When the enzyme was oxidized by DMN, the same differ-
ence spectrum was observed (not shown). The arrow indicates the
absorption maximum of the aband at 557 nm.
1900 G. J. Mander et al.(Eur. J. Biochem. 269) Ó FEBS 2002
could not be detected after SDS/PAGE in gels stained with
either Coomassie or silver (data not shown), which suggests
that this subunit does no t copurify with the other subunits
of the enzyme complex.
From the primary structure, the b-type cytochrome-like
protein AF501 and subunit AF500 are clearly predicted to
be integral membrane proteins. The iron-sulfur protein
AF499 contains a characteristic twin-arginine leader pep-
tide. This strongly suggests t hat this p rotein is located at t he
extracytoplasmic side of the membrane [27]. The c-type
cytochrome AF503 contains a typical Sec-dependent hydro-
phobic leader peptide [28] that is not cleaved off by a l eader
peptidase as i t was still present in the purified protein.
Therefore, this protein can also be predicted t o be located
on the extracytoplasmic side of the membrane and to have
an N-terminal membrane anchor. The AF502 protein,
which is related to the catalytic subunit of Hdr, is a
hydrophilic iron-sulfur protein. The protein does not
contain a leader sequence and therefore may be attached
to the integral membrane subunits on the cytoplasmic side.
It cannot, however, be excluded that this protein binds to
the AF499 protein in the cytoplasm and that this protein
complex then is translocated across the cytoplasmic mem-

brane via the TAT translocase. Such a mechanism has been
found for periplasmic oxidoreductases [27].
The characterization of the A. fulgidus protein complex
by EPR spectroscopy identified an unusual paramagnetic
species with EPR characteristics and redox properties
similar to those of the unusual paramagnetic species that
has recently been described for Hdr from M. marburgensis
and M. barkeri. In Hdr, this paramagnetic species, desig-
nated CoM-Hdr, is formed on reaction o f the oxidized
enzyme with coenzyme M (H-S-CoM) in the absence of
coenzyme B (H-S-CoB).
This paramagnetic species can be reduced in a one-
electron step with a midpoint potential of )185 mV
(M. marburgensis enzyme) or )142 mV (M. barkeri
enzyme), but cannot be further oxidized. A broadening of
the EPR sign al in the
57
Fe-enriched enzyme indicates that it
is at least partially iron-based. The g values (g
xyz
¼ 2.013,
1.991, 1.938 for the M. marburgensis enzyme and
g
xyz
¼ 2.011, 1.993, 1.944 for the M. barkeri enzyme) and
the midpoint potential argue against a conventional [2Fe-
2S]
+
,[3Fe-4S]
+

, [4Fe-4S]
+
,or[4Fe-4S]
3+
cluster. CoM-
Hdr reacts with H-S-CoB to produce an EPR-silent form.
This indicates that only a half reaction is catalyzed when
only H -S-CoM is pr esent and that a reaction intermediate of
the catalytic cycle is trapped [6]. Variable-temperature
magnetic circular dichroism spectroscop y studies of CoM-
Hdr have provided compelling evidence f or the p resence of a
novel type of [4Fe-4S]
3+
cluster at t he active site of Hdr [6,7].
When oxidized Hdr is incubated with H-S-CoB, an EPR
signal with similar g values is obtained, but the midpoint
potential is shifted t o higher values ()30 mV for Hdr from
M. marburgensis and >0 mV for Hdr from M. barkeri).
From these data it has been concluded that H-S-CoB also
reacts with the active site of the enzyme. As this reaction is
only observed at nonphysiological redox potentials, it has
been proposed that this species could not be an intermediate
of the catalytic cycle, but rather is the product of a side
reaction that occurs at these high redox potentials. Similar
results have been obtained with other thiols, such as
dithiothreitol, which are not substrates of the enzyme [6].
In contrast with Hdr, the paramagnetic species in the
enzyme co mple x f rom A. fulgidus co uld a lready be observed
when the enzyme was poised at redox potentials higher than
0 mV. No substrate was added in these experiments. It

cannot, however, b e excluded that the purified enzyme
contains an unidentified tightly bound substrate. It also has
to be considered that the formation of the paramagnetic
species is an intrin sic property of the enzyme. In this case, the
signal could, for example, b e g enerated by the co-ordination
of a redox-active cysteine residue of the enzyme to a metal
cluster. The midpoint-potential of the paramagnetic species
in the A. fulgidus enzyme was determined to be +90 mV.
Fig. 4. EPR-monitored r edox titration of the A. fulgidus Hme c omplex.
Hme ( 7 l
M
)in50m
M
Mops/KOH (pH 7.0) was used. Titrations were
carried out as described in Materials and methods. (A) Data points
correspondtotheamplitudeofthetroughcenteredatg ¼ 1.951; as in
the low po tential range, the radical signal of the dyes overlap in the
g ¼ 2.0 region. The maximal spin concentration was 0.4 per enzyme
molecule as determined by double integration of t he simulated EPR
signal. (B) EPR spectrum obtained at +176 mV (solid line) and the
EPR simulation (dashed line). EPR conditions: temperature, 20 K;
microwave power, 2.007 mW; microwave frequency, 9458 MHz;
modulation amplitude, 0.6 mT. Simu lation parameters: g
123
¼ 2.031,
1.994, and 1.951; W
123
¼ 1.25, 1.2, and 1.15 mT.
Ó FEBS 2002 Hdr-like enzyme complex from A. fulgidus (Eur. J. Biochem. 269) 1901
This value is more positive than the standard redox

potentials of the APS/sulfite couple ()60 mV) and the
sulfite/sulfide couple ()116 mV), which are thought to be
the final electron a cceptors (see b elow). It therefore has to be
considered that the signal in the A. fulgidus enzyme is
generated nonspecifically at high redox potentials. The
reaction of the e nzyme with its physiological substrate may
result in a shift of the midpoint potential of this species to
lower values, as has been observed w ith Hdr [6].
The sequence analysis of the A. fulgidus enzyme clearly
shows that t he AF502 protein is related to the catalytic
subunit HdrD of H dr from M. barkeri. I n particular, AF502
and HdrD share a common cysteine motif that in Hdr is
thought to co-ordinate the special [4Fe-4S] cluster in the
active site. I n the four Hdr sequences currently available, this
motif (CX
31)38
CCX
33)34
CXXC) is present in two copies in
each sequence. The Hdr-like p roteins contain either one or
two copies of this sequence motif. The three closely related
proteins AF502, DsrK, and HmcF contain only one copy,
and this may be sufficient for metal-cluster binding. Only in
the A F502 protein does an aspartate residue replace one of
the five cysteine residues. Aspartate can in principle also
function as a ligand of an iron-sulfur cluster [25].
Enzymes related to the Hme complex from A. fulgidus
described in this work are also encoded by the genomes
of the sulfate-reducing bacterium D. vulgaris and the
phototrophic sulfur bacterium A. vinosum. Anaerobic sul-

fate-reducing bacteria such as D. vulgaris contain a high-
molecular-m ass cytochrome c with 16 covalently bound
hemes [32]. This multiheme cytochrome has been purified
and extensively characterized. In D. vulgaris, this protein is
encoded by a large operon, called the hmc operon [24]. The
operon consists of eight genes: two encode regulatory
proteins and six encode the s tructural proteins o f the enzyme
complex (hmcA to hmcF). hm cA encodes a high-molecular-
mass c-type cytochrome, hmcB encodes a periplasmic i ron-
sulfur protein, hmcE encodes a b-type cytochrome, hmcD
encodes a small hydrophilic protein with a single hydro-
phobic, potentially membrane-spanning sequence, hmcC
encodes an integral membrane p rotein, a nd hmcF encodes
an iron-sulfur protein related to the catalytic subunit of Hdr.
A comparison of the Hmc complex from D. vulgaris with
the Hme complex from A. fulgidus shows that the two
complexes h ave a set o f s equence-related subunits, with only
two major differences: a homologue of the H mcD p rotein is
not encoded by the operon from A. fulgidus, and the high-
molecular-mass c ytochrome c of D. vulgaris is replaced b y a
low-molecular-mass cytochrome c with only three heme -
binding motifs in A. fulgidus.
The D. vulgaris Hmc complex has not yet been purified,
but expression of the hmc operon has been monitored in an
immunoassay using HmcA-specific or HmcF-specific anti-
sera. T he le vel of expression is highest in cells cultivated on
H
2
/sulfate medium, and expression is about fourfold lower
in cells cultivated on lactate/sulfate or pyruvate/sulfate

medium [33]. In addition, a mutant strain in which most of
the hmc operon is deleted has been constructed. This
deletion mutant grows normally when lactate or pyruvate
serve as e lectron donors for sulfate r eductio n. T he mutant is
still able to grow on H
2
/sulfate, although at a growth rate
lower t han that of the wild-type. The mutant is a lso d eficien t
in low-redox-potential niche establishment [34]. From these
various observations, it has been concluded that the Hmc
complex is involved in t he electron transfer from H
2
,which
is activated by a periplasmic hydrogenase, to an electron
acceptor on the cytoplasmic side o f the membrane. As
growth of the hmc deletion mutant on H
2
/sulfate is not
completely abolished, the organism may be able to synthe-
size an alternative enzyme complex with a function similar
to that of Hmc.
Proteins with the highest sequence similarity to the five
subunits of the A. fulgidus enzyme complex were found to
be encoded by the dsr locus of A. vinosum [23]. dsrA and
dsrB encode the a and b subunit of the dissimilatory sulfite
reductase of this organism. These two g enes are o rganized in
a cluster with genes encoding proteins highly related to the
AF499–AF503 proteins (Table 2) [23]. Polar insertion
mutations immediately downstream of dsrA,andindsrB,
dsrH,anddsrM, lead to an inability of the cells to oxidize

intracellular sulfur to sulfite under photolithoautotrophic
conditions. The ability of the mutant cells to oxidize sulfide
to sulfur, thiosulfate to tetrathionate, or sulfite to sulfate
under photolithoautotrophic conditions is unaltered. Two
models suggesting a function of the dsr gene products in the
oxidation of sulfur to sulfite have been presented [23]. In
these m odels, t he pr oducts of the dsrO to dsrN genes a re not
yet included. These genes have o nly recently b een identified
(EMBL accession number U84760).
A. fulgidus strain VC16 completely oxidizes organic
substrates, s uch a s l actate, to C O
2
. T he reducing equivalents
thus generated are transferred to the menaquinone pool by
different oxidoreductases of the oxidative branch of t he
pathway. It is, however, not yet k nown how the reduced
menaquinone pool is electrically connected to th e enzymes
of sulfate reduction, namely APS reductase and sulfite
reductase. APS reductase from A. fulgidus is an iron-sulfur
flavoprotein composed of two subunits [35]. The enzyme
has been isolated from the soluble fraction, and t he primary
structure does not indicate any transmembrane helices. The
enzyme is closely related to APS reductase from sulfate-
reducing b acteria a nd from chemotrophic and phototrophic
sulfur-oxidizing bacteria [36]. Sulfite reductase from A. ful-
gidus has also been characterized [37]. I n c ommon with
other Dsrs, the enzyme h as an a
2
b
2

structure and contains
siroheme, nonheme iron, and acid-labile sulfur [36]. An
additional protein with an apparent molecular mass of
11 k Da is associated with sulfite reductase from D. vulgaris
[38] and De sulfovibrio desulfuricans [39,40]. The function of
this so calle d c subunit is not yet known. In most of the
organisms t hat h ave been studied, t he enzyme has been
isolated from the soluble fraction. Sulfite reductase from
D. desulfuricans was found to be partially membrane-
associated after gentle disruption of t he cells [39,40].
On the basis of our results and comparisons with
published results for other organisms, we propose that the
Hme complex of A. fulgidus functions as a menaquinol
oxidoreductase. The sequence analysis of the enzyme
indicates that it is composed of two modules that may have
distinct functions. The first module is related to Hdr from
M. barkeri [3]. It is composed of the b-type cytochrome-like
protein AF501 and the AF502 protein, which has sequence
similarity to the catalytic subunit of Hdr. We propose that
this module of the enzyme complex mediates the electron
transfer from menaquinol to an unidentified electron
acceptor on the cytoplasmic side o f the membrane. This is
supported by the findin g that the heme g roups of the
1902 G. J. Mander et al.(Eur. J. Biochem. 269) Ó FEBS 2002
purified A. fulgidus enzyme were rapidly reduced by
DMNH
2
or were rapidly oxidized by DMN. Furthermore,
it has b een shown that the membrane fr action of A. fulgidus
catalyzes the reduction of the heme groups present in the

membrane fraction by F
420
H
2
at high ra tes [14 ].
The Hme com plex contains th ree a dditional s ubunits: th e
integral membrane protein AF500, the extracytoplasmic
iron-sulfur protein AF499, and the extracytoplasmic c-type
cytochrome AF503. AF500 shows low sequence similarity
to the subunit DmsC of dimethylsulfoxide reductase, which
functions as a menaquinol oxidase [41]. Likewise, AF500
may function as a second menaquinol-oxidizing site of the
Hme complex, and, together with the iron-sulfur protein
AF499 and the c-type cytochrome AF503, may f orm a
second module of the enzyme complex. We propose that
this modu le catalyzes the electron transfer from menaquinol
to the c-type cytochrome. The c-type cytochrome may
function as the electron donor of alternative electron
acceptors or oxidoreductases. One possible candidate is
oxygen. It has been shown that sulfate-reducing bacteria
first consume oxygen i n their environment and then start to
reduce sulfate. I n Desulfovibrio species, the highest oxygen-
uptake activity is found in the periplasmic fraction, with H
2
as electron donor. C ytochrome c
3
was found to play a
major role in oxygen reduction [42,43].
It has been proposed that the Hmc complex from
D. vulgaris mediates the electron t ransfer from a periplasmic

hydrogenase to t he cytoplasmic side where reduction of
sulfate occurs [24,44]. A. fulgidus is also able to grow with H
2
as electron donor for sulfate reduction [11]. The genome of
A. fulgidus, however, does not encode a s oluble periplasmic
hydrogenase as found in sulfate-reducing bacteria. Instead,
thegenomeofA. fulgidus encodes an extracytoplasmic
hydrogenase (AF1379 to AF1381), which is predicted to
contain a b-type cytochrome subunit (AF1379) as a
membrane anchor [10]. This hydrogenase is therefore
predicted to catalyze the hydrogen-dependent reduction of
menaquinone, as do oth er hydrogenases of this type [45].
Methanogenic archaea belonging to the family Meth-
anosarcinales contain two different energy-conserving elec-
tron-transport chains t hat catalyze the reduction of the
heterodisulfide. W hen the organism grows on methanol,
reduced coenzyme F
420
is generated during methanol
oxidation to CO
2
[46]. The organism contains a mem-
brane-bound electron-transport chain which mediates the
reduction of the heterodisulfide by F
420
H
2
. I t is c omposed of
F
420

H
2
–methanophenazine oxidoreductase, methanophen-
azine, and Hdr [47]. When the organism g rows on H
2
/CO
2
,
H
2
serves as the electron donor for the reduction of th e
heterodisulfide. In this case, the electron-transport chain is
composed of a m ethanophenazine-reducing hydrogenase,
methanophenazine, and Hdr [47]. The reduced thiols thus
generated then function as the electron donor for the
reduction of the methyl group to methane in a nonenergy-
conserving reaction [1].
On the basis of the above, we propose that, in A. fulgidus,
two similar electron-transport chains operate in which
menaquinone and not methanophenazine is the membrane-
soluble electron carrier. Menaquinone is either reduced by
the F
420
H
2
–MQ o xidoreductase or by the h ydrogenase. T he
Hme complex described in this work then reoxidizes
menaquinol and transfers the e lectrons either to an electron
acceptor on the extracytoplasmic side of the membrane or
to an acceptor in the cytoplasm. The latter electron

acceptor, which is still unknown, is thought to function in
its reduced form as electr on donor of the e nzymes of sulfate
reduction.
ACKNOWLEDGEMENTS
This work was supported by the Max-Planck-Gesellschaft,theDeutsch e
Forschungsgemeinsc haft,theFonds der Chemischen Industrie, and by a
fellowship f rom the Humboldt Stiftung to E. D. We thank P eter M. H.
Kroneck for the gift of potassium trithionate. We thank Karen A.
Brune for editing the manuscript.
REFERENCES
1. T hauer, R.K. (1998) Biochemistry of methanogenesis: a tribute to
Marjory Stephenson. Microbiology 144, 2377–2406.
2. Hedderich, R., Klimmek, O., Kro
¨
ger, A., Dirmeier, R., Keller, M.
& Stetter, K.O. (1998) Anaerobic respiration w ith elemental sulfur
and with disulfides. FEMS Microbiol. Rev. 22, 3 53–381.
3. Heiden, S., Hedderich, R., Setzke, E. & Thauer, R .K. (1994)
Purification of a two-subunit cytochrome-b-containing het-
erodisulfide reductase from methanol-grown Methanosarcina
barkeri. Eur. J. Biochem. 221, 855–861.
4. K u
¨
nkel, A ., Vaupel, M., Heim, S., Thauer, R.K. & Hedderich, R.
(1997) Heterodisulfide reductase from methanol-grown cells of
Methanosarcina barkeri is not a flavoenzyme. Eur. J. Biochem. 244,
226–234.
5. Simianu, M., Murakami, E., Brewer, J.M. & Ragsdale, S.W.
(1998) Purification and properties of the heme- and iron-sulfur-
containing heterodisulfide reductase from Methanosarcina ther-

mophila. Biochemistry 37, 10027–10039.
6. Madadi-Kahkesh, S., Duin, E.C., Heim, S., Albracht, S.P.J.,
Johnson, M.K. & Hedderich, R. (2001) A paramagnetic species
with unique EPR characteristics in the active s ite of heterodisulfide
reductase from methano genic archaea. Eur. J. Biochem. 268,
2566–2577.
7. Duin, E.C., Madadi-Kakesh, S., Hedderich, R., Clay, M.D. &
Johnson, M.K. (2002) H eterodisulfide reductase from Metha-
nothermobacter marburgensis contains an active-site [4Fe-4S]
cluster that is directly involved in mediating heterodisulfi de
reduction. FE BS Lett. 512, 263–268.
8. Abken,H.J.,Tietze,M.,Brodersen,J.,Baumer,S.,Beifuss,U.&
Deppenmeier, U. (1998) Isolation and characterization of
methanophenazine and function of phenazines in membran e-
bound electron transport of Methanosarcina mazei Go
¨
1. J. Bac-
teriol. 180, 2027–2032.
9. Setzke, E., Hedderich, R., Heiden, S. & Thauer, R.K. (1994)
H
2
:heterodisulfide oxidoreductase complex from Methanobacter-
ium thermoautotrophicum: composition and properties. Eur. J.
Biochem. 220, 139–148.
10. K lenk, H.P., Clayton, R.A., T omb, J.F., White, O., Nel son, K.E.,
Ketchum, K.A., Dodson, R.J., Gwinn, M., Hickey, E.K.,
Peterson, J.D., et al. (1997) The complete genome s eq uence of t he
hyperthermophilic, sulphate-reducing archaeon Archaeoglobus
fulgidus. Nature 390, 364–370.
11. Stetter, K.O., Lauerer, G., Thomm, M. & Neuner, A. (1987)

Isolation of extremely thermophilic sul fate reducers: evidence for a
novel branch of archaebacteria. Science 236, 822–824.
12. Mo
¨
ller-Zinkhan, D., Bo
¨
rner,G.&Thauer,R.K.(1989)Function
of methanofuran, tetrahydromet hanopterin, and co enzyme F
420
in
Archaeoglobus fulgidus. Arch. Microbiol. 152, 362–368.
13. Mo
¨
ller-Zinkhan, D. & Thauer, R.K. (1990) Anaerobic lactate
oxidation to 3 CO
2
by Archaeoglobus fulgidus via the carbon
monoxide dehydrogenase pathway: demonstration of the acetyl-
CoA carbon-carbon cleavage reaction in cell extracts. Arch.
Microbiol. 153, 215–218.
Ó FEBS 2002 Hdr-like enzyme complex from A. fulgidus (Eur. J. Biochem. 269) 1903
14. Kun ow, J., Linder, D., S tetter, K.O. & Thauer, R.K. (1994)
F
420
H
2
:quinone oxidoreductase from Archaeoglobus fulgidus.
Characterization of a membrane-bound multisubu nit complex
containing FAD and iron-sulfur clusters. Eur. J. Biochem. 223,
503–511.

15. Tindall, B.J., Stetter, K.O. & Collins, M.D. (1989) A novel fully
saturated menaquinone from the thermophilic sulfate-reducing
archaebacterium Archaeoglobus fulgidus. J. Gen. Microbiol. 13 5,
693–696.
16. Unden, G. & Kro
¨
ger, A. (1986) Reconstitution of a functional
electron-transfer chain from purified formate dehydrogenase and
fumarate reductase complexes. Methods Enzymol. 126, 387–399.
17. Fish, W.W. (1988) Rapid colorimetric micromethod for the
quantitation of comp lexed iron in bio logical samples. Methods in
Enzymology (Riordan,J.F.&Vallee,B.L.,eds),pp.357–364.
Academic Press Inc, New York.
18. Cline, J.D . (196 9) Spectrophotometric determination o f hydrogen
sulfide in natural waters. Limnol. Oceanogr. 14, 454–458.
19. Berry, E.A. & Trumpower, B.L. (1987) Simultaneous determina-
tion of h emes a, b,andc from pyridine he mochrome spectra. Anal.
Biochem. 161, 1–15.
20. Beinert, H. & A lbracht, S.P. (198 2) New insights, ideas and
unanswered questions concerning iron-sulfur clusters in mito-
chondria. Biochim. Biophys. Acta 683, 245–277.
21. Soppa, J. (1999) Normalized nucleotide frequencies allow the
definition of archaeal promoter elements for different archaeal
groups and reveal base-specific TFB contacts upstream of the
TATA box. Mol. Microbiol. 31, 1589–1601.
22. Brown, J.W., Daniels, C.J. & Reeve, J.N. (1989) Ge ne structure,
organization, and expression in archaebacteria. Crit.Rev.Micro-
biol. 16, 287–338.
23. Pott, A.S. & D ahl, C. (1998) Sirohaem s ulfite reductase and o ther
proteins encoded by g e nes at th e dsr locus of Chrom atium vinosum

are involved in the oxidation of intracellular sulfur. M icrobiology
144, 1881–1894.
24. Rossi, M., Pollock, W.B., Reij, M.W., Keon, R.G., Fu, R. &
Voordouw, G. (1993) The hmc operon of Desulfovibrio vulgaris
subsp. vulgaris Hildenboro ugh encodes a potential transm em-
brane redox protein compl ex. J. Bacteriol. 175, 4699–4711.
25. Gorst, C.M., Zhou, Z.H., Ma, K ., Teng, Q., Howard, J .B.,
Adams, M.W. & La Mar, G .N. ( 1995) Participation of the
disulfide bridge in the redox cycle of the ferredoxin from the
hyperthermophile Pyrococcus furiosus:
1
H nuclear magnetic
resonance time resolution of t he four redox states at amb ient
temperature. Biochemistry 34, 8788–8795.
26. Berks, B.C., P age, M.D., Richardson, D .J., Reilly, A., Cavill, A.,
Outen, F. & F erguson, S.J. (1995) Sequence ana lysis of subunits of
the m embrane-bou nd nitrate reductase from a denitrifying bac-
terium: the integral membrane subunit provides a prototype for
the dihaem e lectron-carrying arm of a redox loop. Mol. Microbiol.
15, 319–331.
27. Berks, B.C., Sargent, F. & Palmer, T. (2000) The Tat protein
export pathwa y. Mol. Microbiol. 35, 260–274.
28. Fekkes, P. & Driessen A rnold, J.M. (1999) Protein t argeting to the
bacterial cytoplasmic membrane. Microbiol. Mol. Biol. Rev. 63,
161–173.
29. Bilous, P.T., Cole, S.T., Anderson, W.F. & Weiner, J.H. (1988)
Nucleotide sequence of the dmsABC operon enco ding the a nae-
robic d imethylsulph oxide reductase of E scherich ia coli. Mol.
Microbiol. 2, 785–795.
30. Puustinen, A. & Wikstro

¨
m, M. (1991) The heme groups of cyto-
chrome o from Escherichia coli. Proc. Natl Acad. Sci. USA 88,
6122–6126.
31. Sass, H., Steuber, J., Kroder, M., Kroneck, P.M.H. &
Cypionka, H. (1992) Formation of thionates by freshwater and
marine strains of sulfate-reducing bacteria. Arch. M icrobiol. 158 ,
418–421.
32. Pollock, W.B., Loutfi, M., Bruschi, M., Rapp-Giles, B.J., Wall,
J.D. & Voordouw, G. ( 1991) Cloning, seque ncing, and expression
of the gene e ncoding the high-molec ular-weight cytochrome c
from Desulfovibrio v ulgaris Hildenborough. J . Bacteriol. 173,220–
228.
33. Keon, R.G. & Voordouw, G. (1996) Identification of the Hmcf
and Topology of th e Hmcb Subunit of the Hmc Complex of
Desulfovibrio vulgaris. Anaerobe 2, 231–238.
34. Dolla, A., P ohorelic, B.K., Voordouw, J.K. & Voordouw, G.
(2000) Deletion of the hmc op eron o f Desulfovibrio v ulgaris su bsp.
vulgaris Hildenborough hampers hydrogen metabolism and low-
redox-potential niche establishm ent. Arch. Microbiol. 174 ,143–
151.
35. Speich, N., Dahl, C., Heisig, P., Klein, A., Lottspeich, F.,
Stetter, K.O. & Tru
¨
per, H.G. (1994) Adenylylsulphate r eductase
from the sulphate-reducing archaeon Archaeoglobus f ulgi dus:
cloning and characterization of the genes and c omparison of t he
enzyme with other i ron-sulphu r flavoproteins. Microbiology 140,
1273–1284.
36. Hipp,W.M.,Pott,A.S.,Thumschmitz,N.,Faath,I.,Dahl,C.&

Tru
¨
per, H.G. (1997) Towards the phylogeny of APS reductases
and sirohaem sulfite reductases in sulfate-reducing and sulfur-
oxidizing prokaryotes. Microbiology 143, 2891–2902.
37. Dahl,C.,Kredich,M.K.,Deutzmann,R.&Tru
¨
per, H.G. (1 993 )
Dissimilatory sulphite reductase from Archaeoglobus fulgidus:
physico-chemical properties of the enzyme and cloning, sequ en-
cing and a nalysis of the reductase genes. J. Gen. Microbiol. 139,
1817–1828.
38. Pierik, A.J., Duyvis, M.G., Van Helvoort, J.M.L.M., Wolbert,
R.B.G. & Hagen, W.R. ( 1992) The third subunit of desulfoviridin-
type dissimilatory sulfite reductases. Eur. J. Bi oche m. 205,111–
115.
39. Steuber, J ., C ypionka, H . & Kroneck, P.M.H. (1994) Mech anism
of dissimilatory sulfite reduction by Desulfovibrio desulfuricans:
purification of a membrane-bound sulfit e reductase and coupling
with cytochro me c-3 and hydrogenase. Arch. M icrobiol. 16 2,255–
260.
40. Steuber, J., Arendsen, A.F., Hagen, W.R. & Kroneck, P.M. (1995)
Molecular properties of the dissimilatory sulfite reductase from
Desulfovibrio desulfuricans (Essex)andcomparisonwiththe
enzyme from Desulfovibrio vulgaris (Hildenborough). Eur. J.
Biochem. 233, 873–879.
41. Sambasivarao, D. & We iner, J .H. ( 1991) Dim ethyl sulfoxide
reductase of Escherichia coli: an investigation of function and
assembly by use of in vivo complementation . J. Bacteriol. 17 3,
5935–5943.

42.Baumgarten,A.,Redenius,I.,Kranzoch,J.&Cypionka,H.
(2001) Periplasmic o xygen r eduction by Desulfovibrio species.
Arch. Microbiol. 176, 306–309.
43. Cypionka, H. (2000) Oxygen respiration by Desulfovibrio species.
Annu. Rev. Microbiol. 54, 827–848.
44. Fauque,G.,Peck,H.D.Jr,Moura,J.J.,Huynh,B.H.,Berlier,Y.,
DerVartanian, D.V., Teixeira, M ., Przybyla, A.E., Lespinat, P.A.
& Moura, I. (1988) The three cl asses of hydrogenases from sulfate-
reducing bacteria of the genus Desulfovibrio. FEMS Microbiol.
Rev. 54, 299–344.
45. Gross,R.,Simon,J.,Theis,F.&Kro
¨
ger, A. (199 8) Tw o mem-
brane anchors of Wolinella succinogenes hydrogenase and their
function in fumarate and p olysulfide respiration. Ar ch. M icrobiol.
170, 50–58.
46. Keltjens, J.T. & Vogels, G.D. ( 1993) Conversion of methanol a nd
methylamines to methane and carbon dioxide. M ethanogenesis
(Ferry, J.G., ed.), pp. 253–303. Chapman & Hall, New York &
London.
47. De ppenmeier, U., Lienard, T. & Gottsc halk, G. (1999) Novel
reactions involved in energy conservation by methanogenic ar-
chaea. FEBS Lett. 457 , 291–297.
1904 G. J. Mander et al.(Eur. J. Biochem. 269) Ó FEBS 2002