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J Biol Inorg Chem. 2005 Dec;10(8):903-12. Epub 2005 Nov 8.

New insights into the mechanism of nickel insertion into carbon monoxide dehydrogenase: analysis of Rhodospirillum rubrum carbon monoxide dehydrogenase variants with substituted ligands to the [Fe3S4] portion of the active-site C-cluster.

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Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.


Carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum catalyzes the oxidation of CO to CO2. A unique [NiFe4S4] cluster, known as the C-cluster, constitutes the active site of the enzyme. When grown in Ni-deficient medium R. rubrum accumulates a Ni-deficient apo form of CODH that is readily activated by Ni. It has been previously shown that activation of apo-CODH by Ni is a two-step process involving the rapid formation of an inactive apo-CODH*Ni complex prior to conversion to the active holo-CODH. We have generated CODH variants with substitutions in cysteine residues involved in the coordination of the [Fe3S4] portion of the C-cluster. Analysis of the variants suggests that the cysteine residues at positions 338, 451, and 481 are important for CO oxidation activity catalyzed by CODH but not for Ni binding to the C-cluster. C451S CODH is the only new variant that retains residual CO oxidation activity. Comparison of the kinetics and pH dependence of Ni activation of the apo forms of wild-type, C451S, and C531A CODH allowed us to develop a model for Ni insertion into the C-cluster of CODH in which Ni reversibly binds to the C-cluster and subsequently coordinates Cys531 in the rate-determining step.

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