title: Characterization of C415 mutants of neuronal nitric oxide synthase.
abstract: Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. C415H and C415A mutants of the neuronal isoform of NOS (nNOS) were expressed in a baculovirus system and purified to homogeneity for spectral analysis and activity measurements. UV-visible spectra of each mutant lacked an observable Soret peak, suggesting that neither mutant contained heme. When reduced in the presence of CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axial ligand to the heme. In addition to a deficiency in bound heme, neither mutant had any detectable bound tetrahydrobiopterin, as compared to wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine:1 mol of nNOS 160 kDa subunit. The C415H mutant contained bound FAD and FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit, respectively. UV-visible spectra of both nNOS mutants retained the distinctive absorbance due to tightly associated oxidized flavin prosthetic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone. Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 reducing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially identical to the spectrum of wild-type nNOS. Neither mutant could synthesize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrome c reduction by each mutant was found to be slightly less, but very similar to the rate (approximately 20 mumol mg-1 min-1) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction increased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme ligand and that a point mutation at C415 prevents binding of heme and tetrahydrobiopterin without interfering with the global folding or the reductase function of nNOS.
authors.affiliation: Department of Biological Chemistry, School of Medicine, University of Michigan, Ann Arbor 48109-1065, USA.
authors.forename: M K
authors.initials: MK
authors.lastname: Richards
authors.forename: M J
authors.initials: MJ
authors.lastname: Clague
authors.forename: M A
authors.initials: MA
authors.lastname: Marletta
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D017124:Conserved Sequence; D003574:Cytochrome c Group / Q000378:metabolism; D005292:Ferricyanides; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D018411:Spodoptera; D014162:Transfection
chemical_list: D003574:Cytochrome c Group; D005292:Ferricyanides; D007527:Isoenzymes; D011994:Recombinant Proteins; C007931:hexacyanoferrate III; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; D019001:Nitric Oxide Synthase