Rhodospirillium 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{sub 4}S{sub 4}] clusters or for incorporation of nickel. The K{sub m} for methyl viologen is identical to that of wild-type CODH, but the K{sub 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{sub 4}S{sub 4}]{sub 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{sub 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{sub 4}S{sub 4}] clusters remained in the 2+ oxidation state. {sup 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{sub 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 {sup 61}Ni broadening, the g{sub ave} = 2.16 resonance is interpreted as arising from a [(CO{sub L})Fe{sup 3+}-Ni{sup 2+}-H{sup {minus}}]{sup 4+} (S = 1/2) system, where CO{sub 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{sub 4}S{sub 4}], cys-531 is proposed to be directly involved in the coupling of the putative [FeNi] site with the associated [Fe{sub 4}S{sub 4}] cluster.
- Research Organization:
- Univ. of Wisconsin, Madison, WI (US)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- FG02-87ER13691
- OSTI ID:
- 20014097
- Journal Information:
- Journal of the American Chemical Society, Vol. 121, Issue 48; Other Information: PBD: 8 Dec 1999; ISSN 0002-7863
- Country of Publication:
- United States
- Language:
- English
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