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 [Fe4S4] 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 [Fe4S4](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 [Fe4S4] clusters remained in the 2+ oxidation state. Ni-61-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,,, = 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 Ni-61 broadening, the g(ave) = 2.16 resonance is interpreted as arising from a [(COL)Fe3+-Ni2+-H-](4+) (S = 1/2) system, where COL 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 [Fe4S4], cys-531 is proposed to be directly involved in the coupling of the putative [FeNi] site with the associated [Fe4S4] cluster.