Regulation of carbon monoxide dehydrogenase and hydrogenase in Rhodospirillum rubrum: effects of CO and oxygen on synthesis and activity

Abstract

Exposure of the photosynthetic bacterium Rhodospirillum rubrum to carbon monoxide led to increased carbon monoxide dehydrogenase and hydrogenase activities due to de novo protein synthesis of both enzymes. Two-dimensional gels of [35S]methionine-pulse-labeled cells showed that induction of CO dehydrogenase synthesis was rapidly initiated (<5 min upon exposure to CO) and was inhibited by oxygen. Both CO dehydrogenase and the CO-induced hydrogenase were inactivated by oxygen in vivo and in vitro. In contrast to CO dehydrogenase, the CO-induced hydrogenase was 95% inactivated by heating at 70°C for 5 min. Unlike other hydrogenases, this CO-induced hydrogenase was inhibited only 60% by a 100% CO gas phase.A diverse set of bacteria possess the ability to oxidize CO to CO2 (for reviews, see references 10 through 12, 14, 15, 18, 26, and 30). In acetogenic and methanogenic bacteria, CO is oxidized by multisubunit nickel-containing CO dehydrogenase complexes (8,9,21). The expression of these enzymes is not affected by the presence or absence of CO, and these enzymes appear to be involved in acetate metabolism, with CO oxidation being a secondary reaction (15,29,30).Aerobic carboxydotrophic bacteria oxidize CO by using carbon monoxide oxidase, an oxygen-stable iron-sulfur enzyme containing flavin and molybdopterin (16)(17)(18). In these bacteria CO oxidase is induced by the presence of CO, and hydrogenase is induced in cells grown with CO or with CO2 and H2 (18).Some photosynthetic bacteria tolerate CO (13), and Rhodocyclus (formerly Rhodopseudomonas) gelatinosus has been shown by Uffen and co-workers to utilize CO as its sole carbon and energy source during anaerobic growth in the dark (25). The membrane-bound CO-oxidizing system of R. gelatinosus is inducible by CO and produces CO2 and H2 as products of the oxidation of CO (27,28). CO-dependent evolution of H2 from extracts of Rhodospirillum rubrum S1 has been noted (26). We have previously reported COdependent formation of H2 and CO2 by R. rubrum cells grown in the light on ammonium-malate medium (6) and that exposure of light-grown R. rubrum cultures to CO led to significantly increased levels of CO dehydrogenase (4).In this paper these initial observations are extended to demonstrate that CO induces two enzymatic activities, CO dehydrogenase and a CO-insensitive hydrogenase, which appear to function together to accomplish the oxidation of CO to CO2 and H2. These activities are inactivated by 02 both in vivo and in vitro, and the synthesis of CO dehydrogenase is shown to be inhibited by oxygen. Chromatophore suspensions. Membrane preparations (chromatophores) were derived from cells which had been treated with CO for 24 h before harvest. Cells were broken and the chromatophores were collected by centrifugation as previously described (4) and stored in liquid nitrogen.Enzyme assays. CO dehydrogenase activity was assayed by using a CO-dependent methyl viologen reduction assay as previously described (6). Cells were lysed by grinding before the assay for CO dehydrogenas...

Redox-dependent activation of CO dehydrogenase from Rhodospirillum rubrum

“…The activation process of CODH was monitored by reduction of MV and [Fe-S] clusters of CODH in the presence of CO. The determination of CO concentration in the system was performed by a hemoglobinbinding assay (1,4). Spectra were recorded on a Shimadzu model 1605 dual-beam spectrophotometer.…”

“…Wild-type strain (UR2) R. rubrum was cultured as described (1,6,14). CODH was purified as described for the wild-type enzyme (15).…”

“…C arbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum is a nickel-iron-sulfur enzyme that carries out the reversible oxidation of CO to CO 2 (1)(2)(3)(4). Kinetic analyses have demonstrated that Ni is required for CODH activity (3,5,6).…”

Effect of Sodium Sulfide on Ni-Containing Carbon Monoxide Dehydrogenases

“…[E] tot in µM were converted from mg/mL values by assuming molecular masses of 62,000 da for each β subunit of CODH Rr (12) and 155,000 da for each αβ dimer of CODH Mt (13). diluted 100:1 with buffer A, and aliquots were added to CODH Rr as above, resulting in solutions with 0, 0.5, 1.0 and 2.5 µM of sulfide.…”

Thermosinus carboxydivorans gen. nov., sp. nov., a new anaerobic, thermophilic, carbon-monoxide-oxidizing, hydrogenogenic bacterium from a hot pool of Yellowstone National Park

“…The ability to grow anaerobically on CO with the production of H 2 as the only reduced product was first observed in the mesophilic purple, non-sulfur bacteria Rhodocyclus gelatinosus and Rhodospirillum rubrum (Uffen, 1976;Bonam et al, 1989). Carboxydothermus hydrogenoformans was the first anaerobic, thermophilic, non-photosynthetic bacterium found that performed this process (Svetlichny et al, 1991).…”