Showing papers by "Reiner Hedderich published in 2007"

A cysteine-rich CCG domain contains a novel [4Fe-4S] cluster binding motif as deduced from studies with subunit B of heterodisulfide reductase from Methanothermobacter marburgensis.

Abstract: Heterodisulfide reductase (HDR1) (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 the family of pyridine nucleotide disulfide oxidoreductases (7). HDR from M. marburgensis is an iron–sulfur flavoprotein composed of three subunits: HdrA, HdrB, and HdrC. The primary sequence of HdrA indicates that it contains the FAD binding site and four canonical binding motifs for [4Fe-4S] clusters. HdrB contains no sequence motif characteristic for the binding of known cofactors but has two unique cysteine-rich sequence motifs (CX31–39CCX35–36CXXC) of unknown function, designated as the CCG domain in the Pfam protein families database (accession number PF02754) (8). The ferredoxin-like subunit HdrC contains two canonical binding motifs for [4Fe-4S] clusters (9). HDR from Methanosarcina species lacks a homologue of the M. marburgensis HdrA subunit, whereas subunits HdrC and HdrB are conserved in the putative fusion protein HdrD (Figure 1). Subunits HdrC and HdrB are also conserved in subunit TfrB of thiol:fumarate reductase (TFR) (Figure 1), an anabolic enzyme of methanogens that catalyzes the reduction of fumarate to succinate with CoM-SH plus CoB-SH as electron donors (10). HDR with highly conserved HdrB and HdrC subunits is also encoded by the genomes of uncultivated anaerobic methanotrophic archaea in which the enzyme is thought to catalyze formation of CoM-S-S-CoB from CoM-SH and CoB-SH during anaerobic methane oxidation (11). Figure 1 Schematic alignment of heterodisulfide reductase from Methanothermobacter marburgensis (Mt Hdr), heterodisulfide reductase from Methanosarcina barkeri (Mb Hdr), and thiol: fumarate reductase from Methanothermobacter marburgensis (Mt Tfr). Homologous subunits ... Studies focused on elucidation of the catalytic mechanism of HDR performed with both the enzyme from M. marburgensis and M. barkeri led to identification of a mechanistic-based paramagnetic intermediate generated upon half-reaction of the oxidized enzyme with CoM-SH in the absence of CoB-SH (12, 14). The S = 1/2 species, designated as CoM-HDR, is observed at temperatures below 50 K with principal g values of 2.013, 1.991, and 1.938 (M. marburgensis HDR). The resonance is lost on reduction (Em = −185 mV versus NHE at pH 7.6) and on reaction with CoB-SH. Hence, it was attributed to the product of the oxidative half-reaction that occurs in the absence of CoB-SH in which case it is likely to correspond to a trapped intermediate in the catalytic cycle. Signal broadening in the 57Fe-enriched enzyme indicated that the intermediate is iron based. The combination of variable-temperature magnetic circular dichroism (VT-MCD) spectroscopy and EPR spectroscopy with 33S-labeled CoM-SH led to the proposal that the CoM-HDR reaction intermediate is a novel substrate-bound [4Fe-4S]3+ cluster with two thiolate ligands at a unique Fe site (13, 14). 57Fe-pulsed ENDOR at two very different frequencies, 9 and 94 GHz, provided direct evidence for a [4Fe-4S] cluster with unusually large 57Fe isotropic hyperfine coupling values, which reveals the unusual nature of the cluster (15). From these data it was concluded that HDR uses an active-site iron–sulfur cluster to mediate disulfide reduction in two one-electron steps via site-specific cluster chemistry (6, 13). The sequence of HDR is not related to that of ferredoxin: thioredoxin reductase (FTR), the only other known enzyme which uses an active-site [4Fe-4S] cluster to mediate disulfide reduction (16, 17). An assignment of the active-site cluster to a specific subunit has not yet been achieved. It has been suggested that cysteine residues present in the two CCG domains of M. marburgensis HdrB, M. barkeri HdrD and M. marburgensis TfrB (Figure 1), could provide the ligands to this cluster (4, 10). A database search indicates that the CCG domain is also conserved in a large number of proteins from non-methanogens in the archaeal and bacterial domain. This protein family currently has 1871 members. In most of these proteins the CCG domain is present in two copies, but in some proteins the N-terminal CCG domain is degenerated and conserved cysteine residues are replaced by other amino acid residues. The role of a CCG domain has not been defined for any of these proteins (6). In previous studies HdrB was produced in Bacillus subtilis to address the function of its CCG domains (12). M. marburgensis HdrB rather than M. barkeri HdrD was selected because it contains no additional canonical iron–sulfur cluster binding motifs (Figure 1). Heterologous production of HdrB resulted in a protein containing a [2Fe-2S] cluster as deduced by UV/vis absorption, MCD, and resonance Raman spectroscopies. The cluster was labile and irreversibly lost upon reduction. Since a [2Fe-2S] cluster was not observed in native HdrABC it was considered as an artifact of the heterologous expression system (12). Here we reinvestigated the heterologous production of HdrB using E. coli as the expression host. We present evidence that after an in vitro reconstitution step a [4Fe-4S] cluster with spectroscopic properties reminiscent of CoM-HDR was generated. The cluster ligands are located in the C-terminal CCG domain as shown by site-directed mutagenesis. Via X-ray absorption spectroscopy a Zn site was identified in HdrB, proposed to be coordinated by cysteine residues of the N-terminal CCG domain. This work provides the first assignment of a CCG domain as an iron–sulfur cluster binding site and establishes the basis for future studies on the widespread CCG domain proteins.