Unusual oxidative chemistry of N(omega)-hydroxyarginine and N-hydroxyguanidine catalyzed at an engineered cavity in a heme peroxidase.

TitleUnusual oxidative chemistry of N(omega)-hydroxyarginine and N-hydroxyguanidine catalyzed at an engineered cavity in a heme peroxidase.
Publication TypeJournal Article
Year of Publication2000
AuthorsHirst, J, Goodin, DB
JournalJ Biol Chem
Volume275
Issue12
Pagination8582-91
Date Published2000 Mar 24
ISSN0021-9258
KeywordsArginine, Catalytic Domain, Crystallography, X-Ray, Cytochrome-c Peroxidase, Guanidine, Guanidines, Hemeproteins, Hydrogen Peroxide, Mass Spectrometry, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Oxidation-Reduction, Protein Engineering, Recombinant Proteins, Spectrophotometry, Infrared, Substrate Specificity
Abstract

Heme enzymes are capable of catalyzing a range of oxidative chemistry with high specificity, depending on the surrounding protein environment. We describe here a reaction catalyzed by a mutant of cytochrome c peroxidase, which is similar but distinct from those catalyzed by nitric-oxide synthase. In the R48A mutant, an expanded water-filled cavity was created above the distal heme face. N-hydroxyguanidine (NHG) but not guanidine was shown to bind in the cavity with K(d) = 8.5 mM, and coordinate to the heme to give a low spin state. Reaction of R48A with peroxide produced a Fe(IV)=O/Trp(.+) center capable of oxidizing either NHG or N(omega)-hydroxyarginine (NHA), but not arginine or guanidine, by a multi-turnover catalytic process. Oxidation of either NHG or NHA by R48A did not result in the accumulation of NO, NO(2)(-), NO(3)(-), urea, or citrulline, but instead afforded a yellow product with absorption maxima of 257 and 400 nm. Mass spectrometry of the derivatized NHA products identified the yellow species as N-nitrosoarginine. We suggest that a nitrosylating agent, possibly derived from HNO, is produced by the oxidation of one molecule of substrate. This then reacts with a second substrate molecule to form the observed N-nitroso products. This complex chemistry illustrates how the active sites of enzymes such as nitric-oxide synthase may serve to prevent alternative reactions from occurring, in addition to enabling those desired.

DOI10.1074/jbc.275.12.8582
Alternate JournalJ. Biol. Chem.
Citation Key10.1074/jbc.275.12.8582
PubMed ID10722697
Grant ListR01 GM041049 / GM / NIGMS NIH HHS / United States
GM41049 / GM / NIGMS NIH HHS / United States