A tetrahydrobiopterin radical forms and then becomes reduced during Nomega-hydroxyarginine oxidation by nitric-oxide synthase.
Nitric-oxide synthases are flavoheme enzymes that catalyze two sequential monooxygenase reactions to generate nitric oxide (NO) from l-arginine. We investigated a possible redox role for the enzyme-bound cofactor 6R-tetrahydrobiopterin (H4B) in the second reaction of NO synthesis, which is conversion of N-hydroxy-l-arginine (NOHA) to NO plus citrulline. We used stopped-flow spectroscopy and rapid-freeze EPR spectroscopy to follow heme and biopterin transformations during single-turnover NOHA oxidation reactions catalyzed by the oxygenase domain of inducible nitric-oxide synthase (iNOSoxy). Significant biopterin radical (>0.5 per heme) formed during reactions catalyzed by iNOSoxy that contained either H4B or 5-methyl-H4B. Biopterin radical formation was kinetically linked to conversion of a heme-dioxy intermediate to a heme-NO product complex. The biopterin radical then decayed within a 200-300-ms time period just prior to dissociation of NO from a ferric heme-NO product complex. Measures of final biopterin redox status showed that biopterin radical decay occurred via an enzymatic one-electron reduction process that regenerated H4B (or 5MeH4B). These results provide evidence of a dual redox function for biopterin during the NOHA oxidation reaction. The data suggest that H4B first provides an electron to a heme-dioxy intermediate, and then the H4B radical receives an electron from a downstream reaction intermediate to regenerate H4B. The first one-electron transition enables formation of the heme-based oxidant that reacts with NOHA, while the second one-electron transition is linked to formation of a ferric heme-NO product complex that can release NO from the enzyme. These redox roles are novel and expand our understanding of biopterin function in biology.
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D001708:Biopterins / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism*; D005615:Freezing; D007700:Kinetics; D051379:Mice; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D012996:Solutions
D005609:Free Radicals; D012996:Solutions; D001708:Biopterins; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; C003402:sapropterin