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 rubrum was constructed by site-directed mutagenesis of the cooS gene to yield a CODH with ala in place of cys-531. This variant form of CODH (C531A) has 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 [Fe 4 S 4 ] clusters or for incorporation of nickel. The K m for methyl viologen is identical 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-limiting step has been introduced at the point of electron transfer from the Ni site to an associated [Fe 4 S 4 ] C cluster. Examination of indigo carmine-poised, 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 dithionite-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 C531A CODH did not involve a concurrent decrease in the molar extinction coefficient at 420 nm, indicating that the [Fe 4 S 4 ] clusters remained in the 2+ oxidation state. 61 Ni-substituted CO-pretreated C531A CODH, when poised with indigo carmine, showed no broadening of the resonances, indicating that no detectable spin density resides upon Ni. Comparisons of the EPR spectrum of the g ave = 2.16 species to Ni-C(CO) and Ni-C of Alcaligenes eutrophus [NiFe] hydrogenase are presented. On the basis of these comparisons and on the lack of 61 Ni broadening, the g ave = 2.16 resonance is interpreted as arising from a [(CO L )Fe 3+ -Ni 2+ -H - ] 4+ ( S = 1 / 2 ) system, where CO L is an activating nonsubstrate CO ligand. On the basis of the absence of spectroscopic features present in wild-type CODH, and representing coupled forms of the putative [FeNi] cluster with a [Fe 4 S 4 ], cys-531 is proposed to be directly involved in the coupling of the putative [FeNi] site with the associated [Fe 4 S 4 ] cluster.