AF499 encodes an extracytoplasmic iron-sulfur protein, and AF503 encodes an extracytoplasmic c-type cytochrome with three heme c-binding motifs.. All of the subunits show high sequence s
Trang 1Purification and characterization of a membrane-bound enzyme
related to heterodisulfide reductase from methanogenic archaea Gerd J Mander1, Evert C Duin1, Dietmar Linder2, Karl O Stetter3and Reiner Hedderich1
1
Max-Planck-Institut fu¨r terrestrische Mikrobiologie, Marburg, Germany;2Biochemisches Institut, Fachbereich Humanmedizin, Justus-Liebig-Universita¨t Giessen, Germany;3Lehrstuhl 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 fulgidus encodes several 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 fulgidusthat 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 Subunit AF502 is closely related to the
catalytic subunit HdrD of Hdr from Methanosarcina
bark-eri AF501 encodes a membrane-integral cytochrome, and
AF500 encodes a second integral membrane protein AF499
encodes an extracytoplasmic iron-sulfur protein, and AF503
encodes an extracytoplasmic c-type cytochrome with three
heme c-binding motifs All of the subunits show high
sequence similarity to proteins encoded by the dsr locus of
Allochromatium vinosum and to subunits of the Hmc
complex from Desulfovibrio vulgaris The heme groups of the enzyme are rapidly reduced by reduced 2,3-dimethyl-1,4-naphthoquinone (DMNH2), which indicates that the enzyme functions as a menaquinol–acceptor oxidoreduc-tase The physiological electron acceptor has not yet been identified Redox titrations monitored by EPR spectroscopy were carried out to characterize the iron-sulfur clusters of the enzyme In addition to EPR signals due to [4Fe-4S]+ clus-ters, signals of an unusual paramagnetic 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 those of a paramagnetic species recently identified
in Hdr In Hdr this paramagnetic species is specifically induced by the substrates of the enzyme and is thought to be
an intermediate of the catalytic cycle Hence, Hdr and the
A fulgidusenzyme not only share sequence similarity, but may also have a similar active site and a similar catalytic function
Keywords: Archaeoglobus fulgidus; 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-coenzyme M
reductase [1] This disulfide is reduced by a unique disulfide
reductase, designated heterodisulfide reductase (Hdr) Two types of Hdr from phylogenetically distantly related meth-anogens have been identified and characterized Neither type of enzyme belongs to the 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 transmembrane 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 the two closely related Methanosarcina species
M barkeri and M thermophila is tightly membrane-bound [3–5] The enzyme is composed of two subunits,
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 Marburg/Germany.
Fax: + 49 6421 178299, Tel.: + 49 6421 178230,
E-mail: hedderic@mailer.uni-marburg.de
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, dissimilatory sulfite
reductase.
Enzyme: heterodisulfide reductase (EC 1.99.4.-).
(Received 10 October 2001, revised 12 February 2002, accepted 15
February 2002)
Trang 2[4] A homologue of the M marburgensis HdrA subunit is
lacking in Hdr 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 CX31)38CCX33)34CXXC 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 the two types of
Hdr The physiological electron donor of Hdr from
Methanosarcina species is thought to be the
membrane-soluble electron carrier 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 H2
Hdr was originally thought to be unique to methanogenic
archaea However, in recent years, genes encoding
pro-teins related to the catalytic subunit of Hdr have been
identi-fied in a broad range of prokaryotes unable to 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 encode the largest number of proteins related to
Hdr [10]
This extremely thermophilic sulfate-reducing archaeon
completely oxidizes organic substrates, such as lactate, to
CO2[11] Acetate is oxidized to CO2by 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 F420, which is reoxidized by the
F420H2–menaquinone oxidoreductase F420H2
–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 pump as well In
addition, the membrane fraction of 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 to contain a modified
menaquinone as a membrane-soluble electron carrier [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 Hdr from M barkeri A function of this enzyme as reduced
menaquinone–acceptor oxidoreductase is discussed
M A T E R I A L S A N D M E T H O D S
Materials
Redox dyes were obtained from Aldrich–Sigma DMN was
from Sigma Potassium trithionate was a gift from Peter
M H Kroneck (Universita¨t Konstanz) 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 with a continuous flow centrifuge (Z61; Padberg Lahr, Germany) at 17 000 g; the pellet was frozen in liquid nitrogen and stored at)80 °C before use
Enzyme purification All purification steps were carried out under strictly anoxic conditions under an atmosphere of N2/H2 (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 mM Mops (pH 7.0) using a Teflon homogenizer Protein was solubilized from the membranes with 15 mMdodecyl-b-D -maltoside [2 mg dodecyl-b-D-maltosideÆ(mg protein))1] at
4°C for 12 h The unsolubilized proteins and the mem-branes were removed by ultracentrifugation as described above The supernatant was applied to a Q-Sepharose HighLoad column (2.6· 10 cm) equilibrated with 50 mM Mops/KOH (pH 7.0) containing 2 mMdodecyl-b-D -malto-side (buffer A) Protein was eluted in a stepwise NaCl gradient (80 mL each in buffer A): 0 mM, 300 mM,
400 mM, 500 mM, 600 mM, and 1M The majority of the heme-containing protein(s) were eluted at 600 mM NaCl These fractions were applied to a Superdex 200 gel-filtration column (2.6· 60 cm) equilibrated with buffer A containing
100 mM 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 (0–1M, 100 mL) Heme-containing protein(s) were eluted
at 600 mM 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 under N2 Protein was judged to be >95% pure by SDS/PAGE
UV/Vis spectroscopy Spectra of samples in 1-mL quartz cuvettes in an anaerobic chamber under N2/H2(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 DMNH2were followed spectropho-tometrically DMN or DMNH2was added to the enzyme solution [1 mg proteinÆmL)1in 50 mMMops/KOH (pH 7.0)]
to a final concentration of 150 lM, and spectra were recorded every 5 s DMNH2 was prepared as described previously [16]
Analytical methods Non-heme iron was quantified colorimetrically with neo-cuproin (2,9-dimethyl-1,10-phenanthroline) and ferrozine
Trang 3[3-(2-pyridyl)-5,6-bis-(4-phenylsulfonate)-1,2,4-triazine] as
described by Fish [17] Acid-labile sulfur was analyzed as
methylene blue [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 oxidized difference 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 mMcopper perchlorate as the standard (10 mMCuSO4,
2M NaClO4, 10 mM HCl) When the EPR signals
over-lapped with other signals, e.g radical signals from the redox
dyes, the signals were simulated, and the simulations were
double integrated to 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 noncommercial programs based on formulas
described previously [20]
Redox titrations
Redox titrations were carried out at 18°C in an anaerobic
chamber under N2/H2(95 : 5, v/v) Potentials were adjusted
with small amounts of freshly prepared sodium dithionite
(20 mM stock solution) or freshly prepared potassium
ferricyanide (20 mM stock solution) All redox potentials
quoted here are relative to the standard hydrogen electrode
In these titrations, a selection of the following mediators
(final concentration 20 lM) 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 lM in 50 mM
Mops/KOH (pH 7.0) containing 2 mMdodecyl-b-D
-malto-side After equilibration of the desired potential, a 0.3-mL
aliquot was transferred to a calibrated EPR tube and
immediately frozen in liquid nitrogen The redox potential
was measured with an Ag/AgCl redox combination
elec-trode (Mettler Toledo Giessen, Germany) To obtain
potentials relative to the standard hydrogen electrode, a
value of 207 mV (corresponding to the potential of Ag/
AgCl at 25°C) was added to the measured redox potentials
Determination of amino-acid sequences For determination of N-terminal amino-acid sequences, polypeptides were separated by SDS/PAGE and blotted
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 manu-facturer
Amino-acid sequence analysis For the prediction of transmembrane helices in proteins, noncommercial programs were used (http://www.sbc.su.se/
miklos/DAS/; http://www.cbs.dtu.dk/services/TMHMM-2.0/) For sequence comparisons, multiple sequence align-ments were generated using the FASTA3 server (http:// www.ebi.ac.uk/fasta3/)
R E S U L T S
Purification of a heme-containing enzyme complex from the membrane fraction ofA 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 mM NaCl Approximately 70% of the heme present in solubi-lized membranes was found in this fraction A further purification of the proteins in this heme-containing fraction
by gel filtration 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
Trang 4Identification 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
the genome of A fulgidus [10] The noncoding regions between the different genes are short (less than 12 bp) or nonexistent (the genes overlap) The sequence region upstream of AF499 is AT-rich and contains typical archaeal promoter elements The sequence AAAGGTTAATATA was found 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 AF499–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 subunit HdrD from M barkeri The closest relative of the protein encoded
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 by AF502 is the high-molecular-mass c-type cytochrome (Hmc)F protein of Desulfovibrio vulgaris(20% sequence identity) [24]
A characteristic of HdrD of M barkeri is the presence of two typical [4Fe-4S] cluster binding motifs in the N-terminal part of the protein HdrD contains 10 additional cysteine residues found in two CX31)38CCX33)34CXXC sequence motifs at the C-terminal part of the protein [4] Multiple sequence alignments of HdrD, AF502, DsrK, and HmcF clearly identified the two 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 CX31)38CCX33)34CXXC 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 kDa 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 by the dsr locus, and to the b-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 the b-type cytochromes 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-rated in a 12% slab gel (8 · 7 cm) 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 molecular Low-molecular-masses
of the marker proteins are given on the right side Lane A1, 15 lg of
the A fulgidus Hme complex denatured for 30 min at room
temper-ature in SDS sample buffer; lane B1, 10 lg Hme complex dentemper-atured
for 30 min at room temperature in SDS sample 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 cytochrome-like protein by N-terminal sequencing,
shows a lower intensity in the boiled 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 sequences were either obtained by Edman degradation (column 1) or derived from the genome sequence of A fulgidus (column 2) The corresponding genes are given in column 3 Amino acids present in both sequences are underlined, and amino acids that could not be determined with certainty in the Edman degradation are given in parentheses Sequence derived by Edman
degradation
Sequence derived from the A fulgidus
KTQFIESPEEVREK
AF499
Trang 5in helix d These histidine residues have been assigned as
b-heme axial ligands for two heme groups that are located
on different halves of the membrane bilayer [26] AF501 not
only has the same topology as NarI, but also contains the
two histidine residues in helix 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 NrfC from 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 that 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 stretch
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 under anoxic condi-tions generally contained the heme groups in the reduced state Any enzyme molecules that contained oxidized heme 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 DMNH2 as electron donor The rates of heme reduction by DMNH2or oxidation by DMN were too rapid to be resolved Figure 3 shows the dithionite-reduced minus air-oxidized absorbance difference spectrum of an enzyme preparation containing only minor amounts of the 16-kDa c-type cytochrome The
Fig 2 Genomic organization of the genes encoding the subunits of the Hme complex from A fulgidus The gene names annotated by TIGR are given above the arrow representing the genes and their direction of transcription The size in bp is given below each gene Between the genes AF498 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 to 11 bp or even overlap (AF500 and AF501 overlap by 3 bp) The region 81–65 bp upstream of the start codon of AF499 was identified as
an archaeal promoter element by sequence analysis The sequence AAAGGTTAATATA shows a high level of identity with the consensus sequence ( )35 to )23, AAANNNTTATATA); 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 elements have been identified as essential elements for archaeal transcription [21,22].
Table 2 Features of the subunits of the Hme complex from A fulgidus Data are either derived from the analysis of the sequence (calculated molecular mass, predicted transmembrane helices, cofactor binding sites, sequence identities) or obtained experimentally (apparent molecular mass, cofactor content).
Apparent/calculated
molecular mass
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
Further comments Related to the
catalytic subunit
of Hdr
Cytochrome, integral membrane protein
Extracytoplasmic iron-sulfur protein
Extracytoplasmic c-type cytochrome
Integral membrane protein
Trang 6absorption 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 The spectrum contained maxima of
the a and b band at 553 and 521 nm, respectively These
maxima are blue-shifted by about 4 nm relative to 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 found
in heme o of cytochrome bo [30] As pyridine hemochrome
spectra are very sensitive to substitutions on the porphyrin
ring, the results indicate that the extractable 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, which have been identified in dissimilatory sulfate
reducers [31], failed to 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 If 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 prediction that the enzyme contains six [4Fe-4S] clusters,
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 involved in the reduction of disulfide substrate The Hdr-like protein from
A fulgiduswas therefore studied by EPR spectroscopy Redox titrations were monitored by EPR to characterize the different iron-sulfur clusters present in the enzyme As expected, the enzyme showed broad unresolved EPR signals
at redox potentials up to)100 mV that were only detectable
at temperatures below 10 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 g¼ 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 and 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 oxidized 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 fulgidusenzyme 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-gidusenzyme is still unknown Therefore, no substrate could
be added to the titration mixture
D I S C U S S I O N
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 proteins has been characterized and no function 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 out
to be a major membrane protein and contained most of the heme present in the membrane fraction Hdr from M bark-eriis 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 analysis of the sequence of the gene cluster encoding the enzyme predicts the presence
of five subunits, but only four were detected in the purified enzyme preparation The integral membrane subunit AF500
Fig 3 Room temperature reduced–oxidized difference spectrum of the
purified Hme complex Hme [1 mg proteinÆml)1in 50 m M Tris/HCl
(pH 7.6)] was reduced with sodium dithionite and subsequently
oxi-dized by air The oxioxi-dized spectrum was subtracted from the reduced
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 a band at 557 nm.
Trang 7could not be detected after SDS/PAGE in gels stained with
either Coomassie or silver (data not shown), which suggests
that this subunit does not 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 that this protein is located at the
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 leader
peptidase as it was still present in the purified protein Therefore, this protein can also be predicted to 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 of 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 signal in the57Fe-enriched enzyme indicates that it
is at least partially iron-based The g values (gxyz¼ 2.013, 1.991, 1.938 for the M marburgensis enzyme and
gxyz¼ 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 present and that a reaction intermediate of the catalytic cycle is trapped [6] Variable-temperature magnetic circular dichroism spectroscopy studies of CoM-Hdr have provided compelling evidence for the presence of a novel type of [4Fe-4S]3+cluster at the 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 to 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 complex from A fulgidus could already be observed when the enzyme was poised at redox potentials higher than
0 mV No substrate was added in these experiments It cannot, however, be 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 intrinsic property of the enzyme In this case, the signal could, for example, be generated 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 redox titration of the A fulgidus Hme complex.
Hme (7 l M ) in 50 m M Mops/KOH (pH 7.0) was used Titrations were
carried out as described in Materials and methods (A) Data points
correspond to the amplitude of the trough centered at g ¼ 1.951; as in
the low potential 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 the 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 Simulation parameters: g 123 ¼ 2.031,
1.994, and 1.951; W 123 ¼ 1.25, 1.2, and 1.15 mT.
Trang 8This 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 acceptors (see below) 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 enzyme with its physiological substrate may
result in a shift of the midpoint potential of this species to
lower values, as has been observed with Hdr [6]
The sequence analysis of the A fulgidus enzyme clearly
shows that the AF502 protein is related to the catalytic
subunit HdrD of Hdr from M barkeri In 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 In the four Hdr sequences currently available, this
motif (CX31)38CCX33)34CXXC) is present in two copies in
each sequence The Hdr-like proteins 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 AF502 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-mass 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 structural proteins of the enzyme
complex (hmcA to hmcF) hmcA encodes a
high-molecular-mass c-type cytochrome, hmcB encodes a periplasmic
iron-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 protein, and 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 have a set of sequence-related subunits, with only
two major differences: a homologue of the HmcD protein is
not encoded by the operon from A fulgidus, and the
high-molecular-mass cytochrome c of D vulgaris is replaced by 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 The level of expression is highest in cells cultivated on
H2/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 electron donors for sulfate reduction The mutant is
still able to grow on H2/sulfate, although at a growth rate
lower than that of the wild-type The mutant is also deficient
in low-redox-potential niche establishment [34] From these
various observations, it has been concluded that the Hmc
complex is involved in the electron transfer from H2, which
is activated by a periplasmic hydrogenase, to an electron acceptor on the cytoplasmic side of the membrane As growth of the hmc deletion mutant on H2/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 dsrBencode the a and b subunit of the dissimilatory sulfite reductase of this organism These two genes are organized in
a cluster with genes encoding proteins highly related to the AF499–AF503 proteins (Table 2) [23] Polar insertion mutations immediately downstream of dsrA, and in dsrB, dsrH, and dsrM, 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 models, the products of the dsrO to dsrN genes are not yet included These genes have only recently been identified (EMBL accession number U84760)
A fulgidus strain VC16 completely oxidizes organic substrates, such as lactate, to CO2 The reducing equivalents thus generated are transferred to the menaquinone pool by different oxidoreductases of the oxidative branch of the pathway It is, however, not yet known how the reduced menaquinone pool is electrically connected to the 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 the primary structure does not indicate any transmembrane helices The enzyme is closely related to APS reductase from sulfate-reducing bacteria and from chemotrophic and phototrophic sulfur-oxidizing bacteria [36] Sulfite reductase from A ful-gidus has also been characterized [37] In common with other Dsrs, the enzyme has an a2b2structure and contains siroheme, nonheme iron, and acid-labile sulfur [36] An additional protein with an apparent molecular mass of
11 kDa is associated with sulfite reductase from D vulgaris [38] and Desulfovibrio desulfuricans [39,40] The function of this so called c subunit is not yet known In most of the organisms that have been studied, the enzyme has been isolated from the soluble fraction Sulfite reductase from
D desulfuricans was found to be partially membrane-associated after gentle disruption of the 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 of the membrane This is supported by the finding that the heme groups of the
Trang 9purified A fulgidus enzyme were rapidly reduced by
DMNH2or were rapidly oxidized by DMN Furthermore,
it has been shown that the membrane fraction of A fulgidus
catalyzes the reduction of the heme groups present in the
membrane fraction by F420H2at high rates [14]
The Hme complex contains three additional subunits: the
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 form a
second module of the enzyme complex We propose that
this module 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 in their environment and then start to
reduce sulfate In Desulfovibrio species, the highest
oxygen-uptake activity is found in the periplasmic fraction, with H2
as electron donor Cytochrome c3 was found to play a
major role in oxygen reduction [42,43]
It has been proposed that the Hmc complex from
D vulgarismediates the electron transfer from a periplasmic
hydrogenase to the cytoplasmic side where reduction of
sulfate occurs [24,44] A fulgidus is also able to grow with H2
as electron donor for sulfate reduction [11] The genome of
A fulgidus,however, does not encode a soluble periplasmic
hydrogenase as found in sulfate-reducing bacteria Instead,
the genome of A 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 other hydrogenases of this type [45]
Methanogenic archaea belonging to the family
Meth-anosarcinalescontain two different energy-conserving
elec-tron-transport chains that catalyze the reduction of the
heterodisulfide When the organism grows on methanol,
reduced coenzyme F420 is generated during methanol
oxidation to CO2 [46] The organism contains a
mem-brane-bound electron-transport chain which mediates the
reduction of the heterodisulfide by F420H2 It is composed of
F420H2–methanophenazine oxidoreductase,
methanophen-azine, and Hdr [47] When the organism grows on H2/CO2,
H2 serves as the electron donor for the reduction of the
heterodisulfide In this case, the electron-transport chain is
composed of a methanophenazine-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 F420H2–MQ oxidoreductase or by the hydrogenase The
Hme complex described in this work then reoxidizes
menaquinol and transfers the electrons 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 electron donor of the enzymes of sulfate reduction
A C K N O W L E D G E M E N T S
This work was supported by the Max-Planck-Gesellschaft, the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and by a fellowship from the Humboldt Stiftung to E D We thank Peter M H Kroneck for the gift of potassium trithionate We thank Karen A Brune for editing the manuscript.
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