MUTATION OF GLU-361 IN HUMAN ENDOTHELIAL NITRIC-OXIDE SYNTHASE SELECTIVELY ABOLISHES L-ARGININE BINDING WITHOUT PERTURBING THE BEHAVIOR OF HEME AND OTHER REDOX CENTERS

Nitric oxide (NO) and L citrulline are formed from the oxidation of L- arginine by three different isoforms of NO synthase (NOS). Defining am ino acid residues responsible for L arginine binding and oxidation is a primary step toward a detailed understanding of the NOS reaction mec hanisms and designing strategies for the selective inhibition of the i ndividual isoform. We have altered Glu-361 in human endothelial NOS to Gin or Leu by site-directed mutagenesis and found that these mutation s resulted in a complete loss of L citrulline formation without disrup tion of the cytochrome c reductase and NADPH oxidase activities. Optic al and EPR spectroscopic studies demonstrated that the Glu-361 mutants had similar spectra either in resting state or reduced GO-complex as the wild type. The heme ligand, imidazole, could induce a low spin sta te in both wild-type and Glu-361 mutants. However, unlike the wild-typ e enzyme, the low spin imidazole complex of Glu-361 mutants was not re versed to a high spin state by addition of either L-arginine, acetylgu anidine, or a-aminothiazole. Direct L-arginine binding could not be de tected in the mutants either. These results strongly indicate that Glu -361 in human endothelial NOS is specifically involved in the interact ion with L arginine. Mutation of this residue abolished the L-arginine binding without disruption of other functional characteristics.