F(420)-non-reducing hydrogenase (Mvh) from Methanothermobacter marburgensis is a [NiFe] hydrogenase composed of the three subunits MvhA, MvhG, and MvhD. Subunits MvhA and MvhG form the basic hydrogenase module conserved in all [NiFe] hydrogenases, whereas the 17-kDa MvhD subunit is unique to Mvh. The function of this extra subunit is completely unknown. In this work, the physiological function of this hydrogenase, and in particular the role of the MvhD subunit, is addressed. In cells of Mt. marburgensis from Ni(2+)-limited chemostat cultures the amount of Mvh decreased about 70-fold. However, the amounts of mvh transcripts did not decrease in these cells as shown by competitive RT-PCR, arguing against a regulation at the level of transcription. In cells grown in the presence of non-limiting amounts of Ni(2+), Mvh was found in two chromatographically distinct forms-a free form and in a complex with heterodisulfide reductase. In cells from Ni(2+)-limited chemostat cultures, Mvh was only found in a complex with heterodisulfide reductase. The EPR spectrum of the purified enzyme reduced with sodium dithionite was dominated by a signal with g(zyx)=2.006, 1.936 and 1.912. The signal could be observed at temperatures up to 80 K without broadening, indicative of a [2Fe-2S] cluster. Subunit MvhD contains five cysteine residues that are conserved in MvhD homologues of other organisms. Four of these conserved cysteine residues can be assumed to coordinate the [2Fe-2S] cluster that was detected by EPR spectroscopy. The MvhG subunit contains 12 cysteine residues, which are known to ligate three [4Fe-4S] clusters. Data base searches revealed that in some organisms, including the Methanosarcina species and Archaeoglobus fulgidus, a homologue of mvhD is fused to the 3' end of an hdrA homologue, which encodes a subunit of heterodisulfide reductase. These data allow the conclusion that the only function of Mvh is to provide reducing equivalents for heterodisulfide reductase and that the MvhD subunit is an electron transfer protein that forms the contact site to heterodisulfide reductase.

Heterodisulfide reductase (Hdr) is a unique disulfide reductase 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. 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 spectroscopy, 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 barkeri. 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 oxidoreductase. 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]+ clusters, 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. fulgidus enzyme not only share sequence similarity, but may also have a similar active site and a similar catalytic function.

Heterodisulfide reductase (HDR) of methanogenic archaea with its active-site [4Fe-4S] cluster catalyzes the reversible reduction of the heterodisulfide (CoM-S-S-CoB) of the methanogenic coenzyme M (CoM-SH) and coenzyme B (CoB-SH). CoM-HDR, a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH, is a novel type of [4Fe-4S] 3+ cluster with CoM-SH as a ligand. Subunit HdrB of the Methanothermobacter marburgensis HdrABC holoenzyme contains two cysteine-rich sequence motifs (CX 31-39 CCX 35-36 CXXC), designated as CCG domain in the Pfam database and conserved in many proteins. Here we present experimental evidence that the C-terminal CCG domain of HdrB binds this unusual [4Fe-4S] cluster. HdrB was produced in Escherichia coli, and an iron-sulfur cluster was subsequently inserted by in vitro reconstitution. In the oxidized state the cluster without the substrate exhibited a rhombic EPR signal (g zyx = 2.015, 1.995, and 1.950) reminiscent of the CoM-HDR signal. 57 Fe ENDOR spectroscopy revealed that this paramagnetic species is a [4Fe-4S] cluster with 57 Fe hyperfine couplings very similar to that of CoM-HDR. CoM-33 SH resulted in a broadening of the EPR signal, and upon addition of CoM-SH the midpoint potential of the cluster was shifted to values observed for CoM-HDR, both indicating binding of CoM-SH to the cluster. Site-directed mutagenesis of all 12 cysteine residues in HdrB identified four cysteines of the C-terminal CCG domain as cluster ligands. Combined with the previous detection of CoM-HDR-like EPR signals in other CCG domain-containing proteins our data indicate a general role of the C-terminal CCG domain in coordination of this novel cluster. In addition, Zn K-edge X-ray absorption spectroscopy identified an isolated Zn site with † This work was supported by the Max Planck Society, the Deutsche Forschungsgemeinschaft, and the Fonds der Chemischen SUPPORTING INFORMATION AVAILABLESequences of oligonucleotides used in polymerase chain reactions (Table S1) and primer combinations and restriction sites used for mutations in hdrB from Methanothermobacter marburgensis (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2013 January 13. Heterodisulfide reductase (HDR 1 ) (EC 1.8.98.1) is a unique disulfide reductase with a key function in the energy metabolism of methane-producing archaea. The enzyme catalyzes the reversible reduction of the mixed disulfide (CoM-S-S-CoB) of the two methanogenic thiol coenzymes, designated coenzyme M (CoM-SH) and coenzyme B (CoB-SH). This disulfide is generated in the final step of methanogenesis (1, 2).Two types of HDRs from phylogenetically distantly related methanogens, represented by the enzymes from Methanothermobacter marburgensis (3) and from Methanosarcina barkeri and Methanosarcina thermophila (4, 5), have been identified and characterized (1,6). Neither type of enzyme belongs to t...

Heterodisulfide reductase (Hdr) is a unique disulfide reductase that plays a key role in the energy metabolism of methanogenic archaea. Two types of Hdr have been identified and characterized from distantly related methanogens. Here we show that the sulfate-reducing archaeon Archaeoglobus profundus cultivated on H 2 /sulfate forms enzymes related to both types of Hdr. From the membrane fraction of A. profundus, a two-subunit enzyme (HmeCD) composed of a b-type cytochrome and a hydrophilic iron-sulfur protein was isolated. The amino-terminal sequences of these subunits revealed high sequence identities to subunits HmeC and HmeD of the Hme complex from A. fulgidus. HmeC and HmeD in turn are closely related to subunits HdrE and HdrD of Hdr from Methanosarcina spp. From the soluble fraction of A. profundus a six-subunit enzyme complex (Mvh:Hdl) containing Ni, iron-sulfur clusters and FAD was isolated. Via amino-terminal sequencing, the encoding genes were identified in the genome of the closely related species A. fulgidus in which these genes are clustered. They encode a three-subunit [NiFe] hydrogenase with high sequence identity to the F 420 -nonreducing hydrogenase from Methanothermobacter spp. while the remaining three polypeptides are related to the three-subunit heterodisulfide reductase from Methanothermobacter spp. The oxidized enzyme exhibited an unusual EPR spectrum with g xyz ¼ 2.014, 1.939 and 1.895 similar to that observed for oxidized Hme and Hdr. Upon reduction with H 2 this signal was no longer detectable.Keywords: Archaeoglobus; heterodisulfide reductase; Hmc complex; iron-sulfur proteins; sulfate-reducing bacteria.Heterodisulfide reductase (Hdr) is a unique disulfide reductase, which has a key function in the energy metabolism of methanogenic archaea. The enzyme catalyses the reversible reduction of the mixed disulfide (CoM-S-S-CoB) of the two methanogenic thiol-coenzymes, called coenzyme M (CoM-SH) and coenzyme B (CoB-SH). This disulfide is generated in the final step of methanogenesis [1]. Two types of Hdr have been identified and characterized from distantly related methanogens [2][3][4][5][6].One type of Hdr, which was purified and characterized from Methanothermobacter marburgensis, is a soluble ironsulfur flavoprotein composed of the three subunits HdrA, HdrB and HdrC [2,3]. For clarity this enzyme will be called HdrABC throughout this paper. From sequence data it has been deduced that HdrA contains an FAD-binding motif and four binding motifs for [4Fe)4S] clusters. HdrC was shown to contain two binding motifs for [4Fe)4S] clusters while in subunit HdrB no characteristic binding motif of any known cofactor could be identified. However, this subunit contains 10 highly conserved cysteine residues present in two Cx 31)38 CCx 33)34 Cx 2 C motifs.The second type of Hdr, designated as HdrDE, is found in Methanosarcina species [4,6]. This enzyme is tightly membrane bound. It is composed of two subunits, a membrane anchoring b-type cytochrome (HdrE) and a hydrophilic iron-sulfur protein (HdrD). The ...