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

• Published in: Biochemistry (Easton) Vol. 48; no. 38; pp. 8985 - 8993
• Main Authors: Lee, Byunghoon, Usov, Oleg M, Grigoryants, Vladimir M, Myers, William K, Shapleigh, James P, Scholes, Charles P
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 29.09.2009
• Subjects: Amino Acid Substitution; Arginine - chemistry; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Binding Sites - genetics; Cytochromes c' - chemistry; Cytochromes c' - genetics; Cytochromes c' - metabolism; Electron Spin Resonance Spectroscopy; Heme - chemistry; Kinetics; Models, Molecular; Mutagenesis, Site-Directed; Nitric Oxide - chemistry; Nitric Oxide - metabolism; Oxidation-Reduction; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rhodobacter sphaeroides; Rhodobacter sphaeroides - chemistry; Rhodobacter sphaeroides - genetics; Spectrophotometry; Static Electricity; USA, Washington
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi900833f

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 K D of the cytochrome c′ in the 1 μM range, and the second was to provide a redox potential that enhances the stability of the ferrous heme.