title: 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.
abstract: 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 14NO 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 KD of the cytochrome c' in the 1 microM range, and the second was to provide a redox potential that enhances the stability of the ferrous heme.
authors.affiliation: Department of Chemistry, Center for Biochemistry and Biophysics, University at Albany, State University of New York, Albany, New York 12222, USA.
authors.forename: Byunghoon
authors.initials: B
authors.lastname: Lee
authors.forename: Oleg M
authors.initials: OM
authors.lastname: Usov
authors.forename: Vladimir M
authors.initials: VM
authors.lastname: Grigoryants
authors.forename: William K
authors.initials: WK
authors.lastname: Myers
authors.forename: James P
authors.initials: JP
authors.lastname: Shapleigh
authors.forename: Charles P
authors.initials: CP
authors.lastname: Scholes
mesh_terms: D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D045305:Cytochromes c' / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012242:Rhodobacter sphaeroides / Q000737:chemistry* / Q000235:genetics; D013053:Spectrophotometry; D055672:Static Electricity
chemical_list: D001426:Bacterial Proteins; D045305:Cytochromes c'; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D001120:Arginine
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