Purification of a two‐subunit cytochrome‐b‐containing heterodisulfide reductase from methanol‐grown Methanosarcina barkeri

Abstract

Heterodisulfide reductase catalyzes the terminal step in the energy-conserving electron-transport chain in methanogenic Archaea. The heterodisulfide reductase activity of the membrane fraction of methanol-grown Methanosarcina barkeri was solubilized by Chaps. Chromatography on Q-Sepharose and Superdex-200 yielded a high-molecular-mass fraction (> 700 kDa) which was dissociated by dodecyl P-D-maltoside. After chromatography on Q-Sepharose, an active heterodisulfide reductase preparation was obtained which was composed of only two different subunits of apparent molecular masses 46 kDa and 23 kDa. For each 69 kDa, the enzyme contained 0.6 rnol cytochrome b, 0.2 rnol FAD, 20 rnol non-heme iron and 20 mol acid-labile sulfur. The 23-kDa subunit possessed heme-derived peroxidase activity, showing that this polypeptide is the cytochrome b. The purified enzyme contained the cytochrome b in the reduced form. Upon addition of the heterodisulfide of coenzyme M and N-7-mercaptoheptanoylthreonine phosphate the cytochrome was instantaneously oxidized, indicating that the cytochrome b served as electron donor for heterodisulfide reduction.An energy-conserving step in the metabolism of all methanogenic Archaea (Ferry, 1993), which grow on H, and CO, (4H,+CO,-CH,+ 2H,O), is the reduction of the heterodisulfide of coenzyme M (H-S-CoM) and N-7-mercaptoheptanoylthreonine phosphate (H-S-HTP) with molecular hydrogen, which is coupled with electrogenic proton translocation (Blaut et al., 3992;Weiss and Thauer, 1993). H, + COM-S-S-HTP + H-S-CoMfH-S-HTPAGO' = -40 k.J/mol (CoM-S-S-HTP, the heterodisulfide of H-S-CoM and H-S-HTP). The reaction is catalyzed by a membrane-associated H, :heterodisulfide oxidoreductase complex which has been purified and characterized from HJCO, grown Methanobacterium thermoautotrophicum (Setzke et al., 1994) and from acetate-grown Methanosarcina barkeri (Heiden et al., 1993). The purified H, : heterodisulfide oxidoreductase complex from M. thermoautotrophicum is composed of six different subunits of apparent molecular masses 80, 51, 41, 36, 21 and 17 kDa and contains in each 250 kDa, 0.6 mol nickel, 0.9 mol FAD and approximately 26 mol non-heme iron and 22 mol acid-labile sulfur. The complex can be dissociated by Chaps into a hydrogenase and a heterodisulfide reductase subcomplex, each composed of three different subunits. The heterodisulfide reductase subcomplex, which contains the FAD and approximately 18 rnol non-heme iron and acid-labile sulfur, is composed of the 80-, 36-and 21-kDa subunits (Hedderich et al., 1990), and the hydrogenase subcomplex, which contains the nickel and approximately 10 mol nonheme iron and acid-labile sulfur, is composed of the 51-, 41-and 17-kDa subunits (Hedderich et al., 1990;Setzke et al., 1994). From DNA sequence data it can be concluded that the 80-kDa polypeptide is the catalytic subunit of the heterodisulfide reductase subcomplex (unpublished results).The purified H, :heterodisulfide oxidoreductase complex from M. barkeri is composed of nine different polypeptides of a...

Analysis of the vhoGAC and vhtGAC Operons from Methanosarcina mazei Strain Gö1, Both Encoding a Membrane‐bound Hydrogenase and a Cytochrome b

“…The same effect was described for membrane preparations of acetate-grown M s barkeri by Kemner et al (1987). Additionally, a membrane-bound multienzyme complex composed of a Fa,,-nonreactive hydrogenase, a cytochrome b, and the heterodisulfide reductase was isolated (Heiden et al, 1993(Heiden et al, , 1994 reported to accept electrons from reduced F4," (Kamlage and Blaut, 1992). Cytochrome reduction by H, was reported to be comparatively slow.…”

“…The 27-kDa cytochrome was subjected to N-terminal sequencing and the sequence MKSNNNPKAMVVERY was obtained. The N-terminus of the 25-kDa cytochrome was determined to be EMAYFSGLS-ALR(L)(G)FVQIM(I), which shows 95 % similarity to the cytochrome b subunit of the heterodisulfide reductase from Ms barkeri (Heiden et al, 1994).…”

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

“…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.…”

“…The second type of Hdr, designated as HdrDE, is found in Methanosarcina species [4,6]. This enzyme is tightly membrane bound.…”

“…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.…”