The role of nickel in CO oxidation and electron flow was investigated in carbon monoxide dehydrogenase from Rhodospirillum rubrum. The Fe-S centers of oxidized, nickel-containing (holo) CO dehydrogenase were completely reduced within 1 min of exposure to CO. The Fe-S centers of oxidized, nickel-deficient (apo) CO dehydrogenase were not reduced during a 35-min incubation in the presence of CO. Apo-CO dehydrogenase Fe-S centers were reduced by dithionite. The Fe-S centers of cyanide-inhibited, holo-CO dehydrogenase were not reduced in the presence of CO but were reduced by dithionite. Treatment of apo-CO dehydrogenase with cobalt(II), zinc(II), and iron(II) resulted in association of these metal ions (0.70, 1.2, and 0.86 mol of M2+/mol, respectively) with the protein but no increase in specific activity. Purified holo-CO dehydrogenase contained 1.1 mol of nickel/mol of protein and could not be further activated upon addition of NiCl2, suggesting the presence of one catalytic nickel site on the enzyme. The M2+-treated enzymes could not be further activated by addition of NiCl2 as opposed to the untreated apoenzyme, whose activity was stimulated 50-100-fold to the level of holoenzyme upon addition of NiCl2. When placed under CO, the Fe-S centers of the cobalt-treated enzyme became reduced over a 35-min time course, as opposed to the zinc- and iron-treated enzymes, which remained oxidized. We conclude that nickel, or an appropriate nickel analogue in the nickel site, mediates electron flow from CO to the Fe-S centers of CO dehydrogenase.

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...

An inactive, Ni-deficient form ofcarbon monoxide (CO) dehydrogenase [carbon-monoxide:(acceptor) oxidoreductase; EC 1.2.99.2], designated apo-CO dehydrogenase, accumulated in Rhodospirillum rubrum when cells were grown in the absence of Ni and treated with CO. In vivo, both CO dehydrogenase activity and hydrogenase activity increased several hundred fold upon addition of 2 ILM NiCl2. Apo-CO dehydrogenase was purified to homogeneity and differed from holo-CO dehydrogenase only in its activity and Ni content, containing <0.2 mol of Ni per mol of protein, and a specific activity of 35 jtmol of CO per min per mg. Optimal in vitro activation of purified apo-CO dehydrogenase resulted in an enzyme with a specific activity of 2640 ,umol of CO per min per mg. No additional enzymes or low molecular weight cofactors were required for activation. Apo-CO dehydrogenase was not activated by MgCI2, MnCI2, CuC12, ZnC12, CoCI2, or Na2MoO4. 63Ni was incorporated into apo-CO dehydrogenase during activation. The electron paramagnetic resonance (EPR) spectra of dithionite-reduced apo-and holo-enzyme were identical, indicating that, in the reduced state, the Fe-S centers observed by EPR are unchanged in the apo-enzyme.The CO dehydrogenase [carbon-monoxide:(acceptor) oxidoreductase, EC 1.2.99.2] from the photosynthetic bacterium Rhodospirillum rubrum is a Ni, Zn, and Fe-S protein (1). Ni is presumed to function as the CO-binding site in the protein and to catalyze the oxidation of CO to CO2. The Ni present in the CO dehydrogenase from Clostridium thermoaceticum has been shown to bind CO, implicating its involvement at the active site of this enzyme (2). Other CO dehydrogenases are also known to contain Ni, and Ni is also found in other classes of enzymes, such as methylcoenzyme M reductases, hydrogenases, and ureases. The role of Ni in enzymes was summarized by Hausinger (3).Much of the research with Ni proteins has been directed toward understanding their roles in cellular metabolism, the nature of the Ni binding site within the protein, and the role of Ni during enzyme catalysis. Studies of the role of a metal in an enzyme are greatly aided by the existence of a metal-free form of the protein, particularly if enzymatic activity is restored upon replacement of the metal (4-10).No description of the activation of a Ni-deficient apoprotein by Ni has been reported. The purified hydrogenase of Nocardia opaca lb exhibited an activation upon incubation with Ni; however, other metals and salts could substitute as well or better than Ni, and the authors concluded that this added Ni was not involved in enzyme catalysis (11). Hartzell and Wolfe (12) have demonstrated the reconstitution of methylcoenzyme M reductase from its subunits and the Ni-containing tetrapyrole F430.In this paper we present the purification and properties of the inactive Ni-deficient apo-CO dehydrogenase from R. rubrum and describe the in vitro activation of this protein by Ni.

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The carbon monoxide dehydrogenase from the photosynthetic bacterium Rhodospirillum rubrum was purified over 600-fold by DEAE-cellulose chromatography, heat treatment, hydroxylapatite chromatography, and preparative scale gel electrophoresis. In vitro, this enzyme catalyzed a two-electron oxidation of CO to form CO2 as the product. The reaction was dependent on the addition of an electron acceptor. The enzyme was oxygen labile, heat stable, and resistant to tryptic and chymotryptic digestion. Optimum in vitro activity occurred at pH 10.0. A sensitive, hemoglobin-based assay for measuring dissolved CO levels is presented. The in vitro Km for CO was determined to be 110 ,uM. CO, through an unknown mechanism, stimulated hydrogen evolution in whole cells, suggesting the presence of a reversible hydrogenase in R. rubrum which is CO insensitive in vivo.