Heterodisulfide Reductase from Methanol‐Grown Cells of Methanosarcina Barkeri is not a Flavoenzyme

Abstract

Heterodisulfide reductase from methanol-grown cells of Methanosarcina barkeri (MbHdrDE) is a membrane-bound enzyme composed of a 46-kDa subunit MbHdrD and a 23-kDa subunit MbHdrE. The enzyme has been shown to contain 0.6 mol heme and 20 mol Fe/S per mol heterodimer. In addition, substoichiometric amounts of FAD, thought to be an essential component of the active enzyme, were detected. We have now obtained preparations of active heterodisulfide reductase in high yields completely devoid of a flavin. Cloning and sequencing of the genes encoding MbHdrD and MbHdrE, which were found to form a transcription unit hdrED, revealed that both subunits also lack an FAD-binding motif. MbHdr thus differs from heterodisulifde reductase from Methanobacterium thermoautotrophicum (MtHdr), which is a flavo iron-sulfur protein composed of the subunits MtHdrA (80 kDa), MtHdrB (36 kDa) and MtHdrC (21 kDa), the subunit HdrA harboring the flavin-binding site. Sequence comparisons revealed that the N-terminal third of MbHdrD, which contained two sequence motifs for [4Fe-4S] clusters, is similar to MtHdrC and that the C-terminal two thirds of MbHdrD are similar to MtHdrB. Thus, MbHdrD and MtHdrBC are structurally equivalent subunits. MbHdrE shows sequence similarity to b-type cytochromes, in agreement with the finding that this subunit contains a heme. These and other results indicate that MbHdrD harbors the active site of heterodisulfide reduction and that a flavin is not involved in catalysis. Since MbHdrD contains only iron-sulfur clusters, a mechanism of disulfide reduction involving one electron rather than two electron-transfer reactions has to be considered such as operative in ferredoxin :thioredoxin reductases from chloroplasts and cyanobacteria.

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

“…For heterologous production of HdrB, E. coli transformants were grown in 1 L of medium at 37 °C on a magnetic stirrer (1000 rpm). The optimized expression medium contained (per liter) 15 g of yeast extract, 20 g of Bacto casamino acids, 2 g of Na 2 HPO 4 ·2H 2 O, 1 g of KH 2 -PO 4 , and 8 g of NaCl. After autoclaving, filter-sterilized components were added: kanamycin (100 mg/L), chloramphenicol (50 mg/L), tetracycline (15 mg/L), glucose (2.5 g/L), cysteine·HCl (177 mg/L), and FeCl 2 ·4H 2 O (10 mg/L).…”

“…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).…”

“…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.…”

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

“…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.…”

“…Subunit HdrD of the Methanosarcina enzyme can be regarded as a hypothetical fusion protein of subunits HdrC and HdrB of Mt. marburgensis Hdr [5].The catalytic centre must be located on Ms. barkeri HdrD and Mt. marburgensis HdrCB, which are conserved in both enzymes [3,5].…”