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.

Heterodisulfide reductase (Hdr) from methanogenic archaea is an iron–sulfur protein that catalyses the reversible reduction of the heterodisulfide (CoM‐S‐S‐CoB) of the methanogenic thiol coenzymes, coenzyme M (H‐S‐CoM) and coenzyme B (H‐S‐CoB). In EPR spectroscopic studies with the enzyme from Methanothermobacter marburgensis, we have identified a unique paramagnetic species that is formed upon reaction of the oxidized enzyme with H‐S‐CoM in the absence of H‐S‐CoB. This paramagnetic species can be reduced in a one‐electron step with a midpoint‐potential of −185 mV but not further oxidized. A broadening of the EPR signal in the 57Fe‐enriched enzyme indicates that it is at least partially iron based. The g values (gxyz= 2.013, 1.991 and 1.938) and the midpoint potential argue against a conventional [2Fe−2S]+, [3Fe−4S]+, [4Fe−4S]+ or [4Fe−4S]3+ cluster. This species reacts with H‐S‐CoB to form an EPR silent form. Hence, we propose that only a half reaction is catalysed in the presence of H‐S‐CoM and that a reaction intermediate is trapped. This reaction intermediate is thought to be a [4Fe−4S]3+ cluster that is coordinated by one of the cysteines of a nearby active‐site disulfide or by the sulfur of H‐S‐CoM. A paramagnetic species with similar EPR properties was also identified in Hdr from Methanosarcina barkeri.

We aimed to validate an analytical approach based on proteomics on gastric cancer specimens for the identification of new putative diagnostic or prognostic markers. Primary screening was performed on gastrectomy specimens obtained from ten consecutive patients with gastric cancer. Gastric epithelial cells were obtained with an epithelial cell enrichment technique, homogenized and then separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The differential protein expression pattern was verified stepwise by Western blotting and immunohistochemistry on samples from 28 and 46 cancer patients, respectively. The putative clinical applicability and prognostic use were tested by an enzyme-linked immunoabsorbent assay on serum samples obtained from 149 cancer patients. One hundred-ninety-one differentially expressed protein spots were found by 2-D PAGE and identified by mass spectrometry, including cathepsin B, which was over-expressed in six (60%) patients. Western blotting confirmed that the active form of cathepsin B is over-expressed, while immunohistochemistry showed strong cytoplasmic staining in cancer tissues of 45 (98%) patients. The serum level of cathepsin B was increased in patients with gastric cancer compared to healthy controls (P = 0.0026) and correlated with T-category and the presence of distant metastases (P < 0.05). Serum levels above 129 pmol x L(-1) were associated with a reduced survival rate (P = 0.0297). Proteome analysis is a valuable tool for the identification of prognostic markers in gastric cancer: Increased cathepsin B serum levels are associated with advanced tumor stages and progressive disease, which enables the classification of some gastric cancer patients into a subgroup that should undergo aggressive therapy.

Most methanogenic Archaea contain an unusual cytoplasmic fumarate reductase which catalyzes the reduction of fumarate with coenzyme M (CoM‐S‐H) and coenzyme B (CoB‐S‐H) as electron donors forming succinate and CoM‐S‐S‐CoB as products. We report here on the purification and characterization of this thiol :fumarate reductase (Tfr) from Methanobacterium thermoautotrophicum (strain Marburg). The purified enzyme, which was composed of two different subunits with apparent molecular masses of 58 kDa (TfrA) and 50 kDa (TfrB), was found to catalyze the following reactions : (a) the reduction of fumarate with CoM‐S‐H and CoB‐S‐H (150 U/mg); (b) the reduction of fumarate with reduced benzyl viologen (620 U/mg); (c) the oxidation of CoM‐S‐H and CoB‐S‐H to CoM‐S‐S‐CoB with methylene blue (95 U/mg); and (d) the reduction of CoM‐S‐S‐CoB with reduced benzyl viologen (250 U/mg). The flavoprotein contained 12 mol non‐heme iron and approximately the same amount of acid‐labile sulfur/mol heterodimer. The genes encoding TfrA and TfrB were cloned and sequenced. Sequence comparisons with fumarate reductases and succinate dehydrogenases from Bacteria and Eucarya and with heterodisulfide reductases from M. thermoautotrophicum and Methanosarcina barkeri revealed that TfrA harbors FAD‐binding motifs and the catalytic site for fumarate reduction and that TfrB harbors one [2Fe‐2S] cluster and two [4Fe‐4S] clusters and the catalytic site for CoM‐S‐H and CoB‐S‐H oxidation.