The Heterodisulfide Reductase from Methanobacterium Thermoautotrophicum Contains Sequence Motifs Characteristic of pyridine‐Nucleotide‐Dependent Thioredoxin Reductases

Abstract

The genes hdrA, hdrB and hdrC, encoding the three subunits of the iron-sulfur flavoprotein heterodisulfide reductase, have been cloned and sequenced. HdrA (72.19 kDa) was found to contain a region of amino acid sequence highly similar to the FAD-binding domain of pyridine-nucleotidedependent disulfide oxidoreductases. Additionally, 11 0 amino acids C-terminal to the FAD-binding consensus, a short polypeptide stretch (VX,CATID) was detected which shows similarity to the region of thioredoxine reductase that contains the active-site cysteine residues (VX,CATCD). These findings suggest that HdrA harbors the site of heterodisulfide reduction and that the catalytic mechanism of the enzyme is similar to that of pyridine-nucleotide-dependent thioredoxin reductase. HdrA was additionally found to contain four copies of the sequence motif CX,CX,CX3C(P), indicating the presence of four [4Fe-4S] clusters. Two such sequence motifs were also present in HdrC (21.76 kDa), the N-terminal amino acid sequence of which showed sequence similarity to the ysubunit of the anaerobic glycerol-3-phosphate dehydrogenase of Escherichiu coli. HdrC is therefore considered to be an electron carrier protein that contains two [4Fe-4S] clusters. HdrB (33.46 kDa) did not show sequence similarity to other known proteins, but appears to possess a C-terminal hydrophobic a-helix that might function as a membrane anchor. Although hdrB and hdrC are juxtaposed, these genes are not near hdrA.

Two distinct heterodisulfide reductase‐like enzymes in the sulfate‐reducing archaeon Archaeoglobus profundus

“…marburgensis HdrCB, which are conserved in both enzymes [3,5]. A detailed spectroscopic characterization showed that the active site harbours a [4Fe)4S] cluster [7,8] …”

“…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].…”

“…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.…”

“…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.…”

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

“…HdrB contains no sequence motif characteristic for the binding of known cofactors but has two unique cysteine-rich sequence motifs (CX [31][32][33][34][35][36][37][38][39] CCX [35][36] CXXC) 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).…”