Rhodospirillum rubrum CO-Dehydrogenase. Part 1. Spectroscopic Studies of CODH Variant C531A Indicate the Presence of a Binuclear [FeNi] Cluster

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• 作者：Christopher R. Staples ; Jongyun Heo ; Nathan J. Spangler ; Robert L. Kerby ; Gary P. Roberts ; and Paul W. Ludden
• 刊名：Journal of the American Chemical Society
• 出版年：1999
• 出版时间：December 8, 1999
• 年：1999
• 卷：121
• 期：48
• 页码：11034 - 11044
• 全文大小：170K
• 年卷期：v.121,no.48(December 8, 1999)
• ISSN：1520-5126

文摘

A variant of the carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum wasconstructed by site-directed mutagenesis of the cooS gene to yield a CODH with ala in place of cys-531. Thisvariant form of CODH (C531A) has a metal content identical to that of wild-type CODH but has an extremelyslow turnover rate. Cys-531 is not essential for construction of the [Fe₄S₄] clusters or for incorporation ofnickel. The K_m for methyl viologen is identical to that of wild-type CODH, but the K_m for CO is approximately30% that of wild-type CODH. The data suggest that in C531A CODH a rate-limiting step has been introducedat the point of electron transfer from the Ni site to an associated [Fe₄S₄]_C cluster. Examination of indigocarmine-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 torepresent as much as 0.90 ± 0.10 spins/molecule, yet production of the species from fully oxidized C531ACODH 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. ⁶¹Ni-substituted CO-pretreated C531A CODH, when poisedwith indigo carmine, showed no broadening of the resonances, indicating that no detectable spin density residesupon Ni. Comparisons of the EPR spectrum of the g_ave = 2.16 species to Ni-C(CO) and Ni-C of Alcaligeneseutrophus [NiFe] hydrogenase are presented. On the basis of these comparisons and on the lack of ⁶¹Nibroadening, the g_ave = 2.16 resonance is interpreted as arising from a [(CO_L)Fe³⁺-Ni²⁺-H^-]⁴⁺ (S = ¹/₂) system,where CO_L is an activating nonsubstrate CO ligand. On the basis of the absence of spectroscopic featurespresent 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.