title: Unusual oxidative chemistry of N(omega)-hydroxyarginine and N-hydroxyguanidine catalyzed at an engineered cavity in a heme peroxidase.
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.
authors.affiliation: Department of Molecular Biology, Scripps Research Institute, La Jolla, California 92037, USA.
authors.forename: J
authors.initials: J
authors.lastname: Hirst
authors.forename: D B
authors.initials: DB
authors.lastname: Goodin
mesh_terms: D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D003578:Cytochrome-c Peroxidase / Q000235:genetics / Q000378:metabolism*; D019791:Guanidine / Q000378:metabolism; D006146:Guanidines / Q000737:chemistry / Q000378:metabolism*; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006898:Hydroxylamines; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism; D013055:Spectrophotometry, Infrared; D013379:Substrate Specificity
chemical_list: D006146:Guanidines; D006420:Hemeproteins; D006898:Hydroxylamines; D011994:Recombinant Proteins; C068309:N(omega)-hydroxyarginine; C029604:hydroxyguanidine; D001120:Arginine; D006861:Hydrogen Peroxide; D003578:Cytochrome-c Peroxidase; D019791:Guanidine