The Role of Arginine-127 at the Proximal NO-Binding Site in Determining the Electronic Structure and Function of 5-Coordinate NO-Heme in Cytochrome c′ of Rhodobacter sphaeroides
The Role of Arginine-127 at the Proximal NO-Binding Site in Determining the Electronic Structure and Function of 5-Coordinate NO-Heme in Cytochrome c′ of Rhodobacter sphaeroides
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DOI：
10.1021/bi900833f
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发表时间：
2009-09-29
期刊：
影响因子：
2.9
通讯作者：
Scholes, Charles P.
中科院分区：
文献类型：
--
作者：
Lee, Byunghoon;Usov, Oleg M.;Scholes, Charles P.
Cytochrome c' is a heme protein from a denitrifying variant of Rhodobacter sphaeroides which may serve to store and transport metabolic NO while protecting against NO toxicity. Its heme site bears resemblance through its 5-coordinate NO-binding capability to the regulatory site in soluble guanylate cyclase. A conserved arginine (Arg-127) abuts the 5-coordinate NO-heme binding site, and the alanine mutant R127A provided insight into the role of the Arg-127 in establishing the electronic structure of the heme-NO complex and in modifying the heme-centered redox potential and NO-binding affinity. By comparison to R127A, the wild-type Arg-127 was determined to Increase the heme redox potential, diminish the NO-binding affinity, perturb and diminish the (NO)-N-14 hyperfine coupling determined by ENDOR (electron nuclear double resonance), and increase the maximal electronic g-value. The larger isotropic NO hyperfine and the smaller maximal g-value of the R127A Mutant together predicted that the Fe-N-O bond angle in the mutant is larger than that of the Arg-127-containing wild-type protein. Deuterium ENDOR provided evidence for exchangeable H/D consistent with hydrogen bonding of Arg-127, but not Ala-127, to the O of the NO. Proton ENDOR features previously assigned to Phe-14 on the distal side of the heme were unperturbed by the proximal side R127A mutation, implying the localized nature of that mutational perturbation at the proximal, NO-binding side of the heme. From this work two functions of positively charged Arg-127 emerged: the first was to maintain the K-D of the cytochrome c' in the 1 mu M range, and the second was to provide a redox potential that enhances the stability of the ferrous heme.
