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

• DOI: 10.1074/jbc.272.10.6114
• Publication Date: 2002-07-26T14:57:46Z
• Authors: Pei-Feng Chen; Ah-Lim Tsai; Vladimir Berka; Kenneth K. Wu
• Keywords: Hemeproteins; 0301 basic medicine; Binding Sites; Molecular Sequence Data; Electron Spin Resonance Spectroscopy; Heme; Arginine; Molecular Weight; Structure-Activity Relationship; 03 medical and health sciences; Glutamates; Humans; Amino Acid Sequence; Endothelium, Vascular; Nitric Oxide Synthase; Oxidation-Reduction; NADP

Nitric oxide (NO) and L-citrulline are formed from the oxidation of L-arginine by three different isoforms of NO synthase (NOS). Defining amino acid residues responsible for L-arginine binding and oxidation is a primary step toward a detailed understanding of the NOS reaction mechanisms and designing strategies for the selective inhibition of the individual isoform. We have altered Glu-361 in human endothelial NOS to Gln or Leu by site-directed mutagenesis and found that these mutations resulted in a complete loss of L-citrulline formation without disruption of the cytochrome c reductase and NADPH oxidase activities. Optical and EPR spectroscopic studies demonstrated that the Glu-361 mutants had similar spectra either in resting state or reduced CO-complex as the wild type. The heme ligand, imidazole, could induce a low spin state in both wild-type and Glu-361 mutants. However, unlike the wild-type enzyme, the low spin imidazole complex of Glu-361 mutants was not reversed to a high spin state by addition of either L-arginine, acetylguanidine, or 2-aminothiazole. Direct L-arginine binding could not be detected in the mutants either. These results strongly indicate that Glu-361 in human endothelial NOS is specifically involved in the interaction with L-arginine. Mutation of this residue abolished the L-arginine binding without disruption of other functional characteristics.