title: Neutralizing a surface charge on the FMN subdomain increases the activity of neuronal nitric-oxide synthase by enhancing the oxygen reactivity of the enzyme heme-nitric oxide complex.
abstract: Nitric-oxide synthases (NOSs) are calmodulin-dependent flavoheme enzymes that oxidize l-Arg to nitric oxide (NO) and l-citrulline. Their catalytic behaviors are complex and are determined by their rates of heme reduction (k(r)), ferric heme-NO dissociation (k(d)), and ferrous heme-NO oxidation (k(ox)). We found that point mutation (E762N) of a conserved residue on the enzyme's FMN subdomain caused the NO synthesis activity to double compared with wild type nNOS. However, in the absence of l-Arg, NADPH oxidation rates suggested that electron flux through the heme was slower in E762N nNOS, and this correlated with the mutant having a 60% slower k(r). During NO synthesis, little heme-NO complex accumulated in the mutant, compared with approximately 50-70% of the wild-type nNOS accumulating as this complex. This suggested that the E762N nNOS is hyperactive because it minimizes buildup of an inactive ferrous heme-NO complex during NO synthesis. Indeed, we found that k(ox) was 2 times faster in the E762N mutant than in wild-type nNOS. The mutational effect on k(ox) was independent of calmodulin. Computer simulation and experimental measures both indicated that the slower k(r) and faster k(ox) of E762N nNOS combine to lower its apparent K(m,O(2)) for NO synthesis by at least 5-fold, which in turn increases its V/K(m) value and enables it to be hyperactive in steady-state NO synthesis. Our work underscores how sensitive nNOS activity is to changes in the k(ox) and reveals a novel means for the FMN module or protein-protein interactions to alter nNOS activity.
authors.affiliation: Department of Pathobiology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio 44195, USA.
authors.forename: Mohammad Mahfuzul
authors.initials: MM
authors.lastname: Haque
authors.forename: Mohammed
authors.initials: M
authors.lastname: Fadlalla
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Wang
authors.forename: Sougata Sinha
authors.initials: SS
authors.lastname: Ray
authors.forename: Koustubh
authors.initials: K
authors.lastname: Panda
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D005296:Ferrous Compounds / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D017354:Point Mutation; D013696:Temperature; D013997:Time Factors
chemical_list: D002147:Calmodulin; D005296:Ferrous Compounds; D050505:Mutant Proteins; D005182:Flavin-Adenine Dinucleotide; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D005486:Flavin Mononucleotide; D052248:Nitric Oxide Synthase Type I; D010100:Oxygen