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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.

Room temperature near-infrared magnetic circular dichroism and low-temperature electron paramagnetic resonance measurements have been used to characterize the ligands of the heme iron in mitochondrial cytochromes c, c1, and b and in cytochrome f of the photosynthetic electron transport chain. The MCD data show that methionine is the sixth ligand of the heme of oxidized yeast cytochrome c1; the identify of this residue is inferred to be the single conserved methionine identified from a partial alignment of the available cytochrome c1 amino acid sequences. A different residue, which is most likely lysine, is the sixth heme ligand in oxidized spinach cytochrome f. The data for oxidized yeast cytochrome b are consistent with bis-histidine coordination of both hemes although the possibility that one of the hemes is ligated by histidine and lysine cannot be rigorously excluded. The neutral and alkaline forms of oxidized yeast cytochrome c have spectroscopic properties very similar to those of the horse heart proteins, and thus, by analogy, the sixth ligands are methionine and lysine, respectively.

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Dinitrogenase reductase from Klebsiella pneumoniae strain UN1041 has a histidine residue substituted for arginine at position 101. The mutant dinitrogenase reductase was purified and characterized in order to determine the importance of arginine-101 in the interaction between dinitrogenase and dinitrogenase reductase during electron transfer. Purified dinitrogenase reductase from UN1041 is a dimer of 67 kDa, contains a functional 4Fe-4S cluster, undergoes a MgATP-dependent conformational change, and is competent for ATP hydrolysis uncoupled from substrate reduction in the presence of dinitrogenase. However, the mutant protein is unable to support the reduction of protons or acetylene by dinitrogenase. A 100-fold molar excess of Kp2 from UN1041 does not inhibit electron transfer from wild-type dinitrogenase reductase to dinitrogenase. It is concluded that the interaction of dinitrogenase reductase with dinitrogenase during reductant-independent ATP hydrolysis is different than the interaction between the two proteins during electron transfer; the substitution of histidine for arginine at position 101 disrupts only the latter interaction. The same conclusions are reached using wild-type dinitrogenase reductase which has been ADP-ribosylated at arginine-101.