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J Biol Chem, Vol. 273, Issue 7, 4059-4064, February 13, 1998

Substitution of Valine for Histidine 265 in Carbon Monoxide Dehydrogenase from Rhodospirillum rubrum Affects Activity and Spectroscopic States

Nathan J. Spangler, Monica R. Meyers^§, Karin L. Gierke, Robert L. Kerby^§, Gary P. Roberts^§, and Paul W. Ludden

From the Departments of  Biochemistry and ^§ Bacteriology, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706

In carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum, histidine 265 was replaced with valine by site-directed mutagenesis of the cooS gene. The altered form of CODH (H265V) had a low nickel content and a dramatically reduced level of catalytic activity. Although treatment with NiCl₂ and CoCl₂ increased the activity of H265V CODH by severalfold, activity levels remained more than 1000-fold lower than that of wild-type CODH. Histidine 265 was not essential for the formation and stability of the Fe₄S₄ clusters. The K_m and K_D for CO as well as the K_D for cyanide were relatively unchanged as a result of the amino acid substitution in CODH. The time-dependent reduction of the [Fe₄S₄]²⁺ clusters by CO occurred on a time scale of hours, suggesting that, as a consequence of the mutation, a rate-limiting step had been introduced prior to the transfer of electrons from CO to the cubanes in centers B and C. EPR spectra of H265V CODH lacked the g_av = 1.86 and g_av = 1.87 signals characteristic of reduced forms of the active site (center C) of wild-type CODH. This indicates that the electronic properties of center C have been modified possibly by the disruption or alteration of the ligand-mediated interaction between the nickel site and Fe₄S₄ chromophore.

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