Role of reductase domain cluster 1 acidic residues in neuronal nitric-oxide synthase. Characterization of the FMN-FREE enzyme.
The nNOS reductase domain is homologous to cytochrome P450 reductase, which contains two conserved clusters of acidic residues in its FMN module that play varied roles in its electron transfer reactions. To study the role of nNOS reductase domain cluster 1 acidic residues, we mutated two conserved acidic (Asp(918) and Glu(919)) and one conserved aromatic residue (Phe(892)), and investigated the effect of each mutation on flavin binding, conformational change, electron transfer reactions, calmodulin regulation, and catalytic activities. Each mutation destabilized FMN binding without significantly affecting other aspects including substrate, cofactor or calmodulin binding, or catalytic activities upon FMN reconstitution, indicating the mutational effect was restricted to the FMN module. Characterization of the FMN-depleted mutants showed that bound FMN was essential for reduction of the nNOS heme or cytochrome c, but not for ferricyanide or dichlorophenolindolphenol, and established that the electron transfer path in nNOS is NADPH to FAD to FMN to heme. Steady-state and stopped-flow kinetic analysis revealed a novel role for bound FMN in suppressing FAD reduction by NADPH. The suppression could be relieved either by FMN removal or calmodulin binding. Calmodulin binding induced a conformational change that was restricted to the FMN module. This increased the rate of FMN reduction and triggered electron transfer to the heme. We propose that the FMN module of nNOS is the key positive or negative regulator of electron transfer at all points in nNOS. This distinguishes nNOS from other related flavoproteins, and helps explain the mechanism of calmodulin regulation.
Adak
S
S
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Ghosh
S
S
Abu-Soud
H M
HM
Stuehr
D J
DJ
D015086:2,6-Dichloroindophenol / Q000378:metabolism; D000595:Amino Acid Sequence; D000600:Amino Acids, Dicarboxylic / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D017124:Conserved Sequence; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport; D004789:Enzyme Activation; D005292:Ferricyanides / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism*; D005420:Flavoproteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D017386:Sequence Homology, Amino Acid
D000600:Amino Acids, Dicarboxylic; D002147:Calmodulin; D003574:Cytochrome c Group; D005292:Ferricyanides; D005420:Flavoproteins; D009419:Nerve Tissue Proteins; C007931:hexacyanoferrate III; D001224:Aspartic Acid; D018698:Glutamic Acid; D005486:Flavin Mononucleotide; D015086:2,6-Dichloroindophenol; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I