Rhodospirillum rubrum CO-dehydrogenase. Part 1. Spectroscopic studies of CODH variant C531A indicate the presence of a binuclear [FeNi] cluster

A variant of the carbon monoxide dehydrogenase (CODH) from Rhodospirillum r ubrum was constructed by site-directed mutagenesis of the cooS gene to yiel d a CODH with ala in place of cys-531. This variant form of CODH (C531A) ha s a metal content identical to that of wild-type CODH but has an extremely slow turnover rate. Cys-531 is not essential for construction of the [Fe4S4 ] clusters or for incorporation of nickel. The K-m for methyl viologen is i dentical to that of wild-type CODH, but the K-m for CO is approximately 30% that of wild-type CODH. The data suggest that in C531A CODH a rate-limitin g step has been introduced at the point of electron transfer from the Ni si te to an associated [Fe4S4](C) cluster. Examination of indigo carmine-poise d, CO-pretreated C531A CODH revealed the presence of a paramagnetic species (g = 2.33, 2.10, 2.03; g(ave) = 2.16), which was also observed in dithioni te-treated samples. This species was shown to represent as much as 0.90 +/- 0.10 spins/molecule, yet production of the species from fully oxidized C53 1A CODH did not involve a concurrent decrease in the molar extinction coeff icient at 420 nm, indicating that the [Fe4S4] clusters remained in the 2+ o xidation state. Ni-61-substituted CO-pretreated C531A CODH, when poised wit h indigo carmine, showed no broadening of the resonances, indicating that n o detectable spin density resides upon Ni. Comparisons of the EPR spectrum of the g,,, = 2.16 species to Ni-C(CO) and Ni-C of Alcaligenes eutrophus [N iFe] hydrogenase are presented. On the basis of these comparisons and on th e lack of Ni-61 broadening, the g(ave) = 2.16 resonance is interpreted as a rising from a [(COL)Fe3+-Ni2+-H-](4+) (S = 1/2) system, where COL is an act ivating nonsubstrate CO ligand. On the basis of the absence of spectroscopi c features present in wild-type CODH, and representing coupled forms of the putative [FeNi] cluster with a [Fe4S4], cys-531 is proposed to be directly involved in the coupling of the putative [FeNi] site with the associated [ Fe4S4] cluster.