Involvement of the reductase domain of neuronal nitric oxide synthase in superoxide anion production.
Neuronal nitric oxide synthase (nNOS) is a modular enzyme which consists of a flavin-containing reductase domain and a heme-containing oxygenase domain, linked by a stretch of amino acids which contains a calmodulin (CaM) binding site. CaM binding to nNOS facilitates the transfer of NADPH-derived electrons from the reductase domain to the oxygenase domain, resulting in the conversion of L-arginine to L-citrulline with the concomitant formation of a guanylate cyclase activating factor, putatively nitric oxide. Numerous studies have established that peroxynitrite-derived nitrogen oxides are present following nNOS turnover. Since peroxynitrite is formed by the diffusion-limited reaction between the two radical species, nitric oxide and O2.-, we employed the adrenochrome assay to examine whether nNOS was capable of producing O2.- during catalytic turnover in the presence of L-arginine. To differentiate between the role played by the reductase domain and that of the oxygenase domain in O2.- production, we compared its production by nNOS against that of a nNOS mutant (CYS-331), which was unable to transfer NADPH-derived electrons efficiently to the heme iron under special conditions, and against that of a flavoprotein module construct of nNOS. We report that O2.- production by nNOS and the CYS-331 mutant is CaM-dependent and that O2.- production can be modulated by substrates and inhibitors of nNOS. O2.- was also produced by the reductase domain of nNOS; however, it did not display the same CaM dependency. We conclude that both the reductase and oxygenase domains of nNOS produce O2.-, but that the reductase domain is both necessary and sufficient for O2.- production.
Miller
R T
RT
Department of Biochemistry, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-7760, USA.
Martásek
P
P
Roman
L J
LJ
Nishimura
J S
JS
Masters
B S
BS
D000323:Adrenochrome / Q000096:biosynthesis; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004837:Epinephrine / Q000494:pharmacology; D007700:Kinetics; D016296:Mutagenesis; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
D002147:Calmodulin; D013481:Superoxides; D000323:Adrenochrome; D010088:Oxidoreductases; D019001:Nitric Oxide Synthase; D003545:Cysteine; D002118:Calcium; D004837:Epinephrine