title: Neuronal nitric oxide synthase self-inactivates by forming a ferrous-nitrosyl complex during aerobic catalysis.
abstract: Neuronal NO synthase (NOS) is a flavin-containing hemeprotein that generates NO from L-arginine, NADPH, and O2. NO has recently been proposed to autoinhibit NOS. We have investigated whether a NOS heme-NO complex forms during aerobic steady-state catalysis. Visible and resonance Raman spectra recorded during steady-state NO synthesis by NOS showed that the majority of enzyme (70-90%) was present as its ferrous-nitrosyl complex. Ferrous-nitrosyl NOS formed only in the coincident presence of NADPH, L-arginine, and O2. Its level remained constant during NO synthesis until the NADPH was exhausted, after which the complex decayed to regenerate ferric resting NOS. Stopped-flow measurements revealed that the buildup of the ferrous-NO complex was rapid (< 2 s) and caused a 10-fold decrease in the rate of NADPH consumption by NOS. Complex formation and decay could occur several times with no adverse affect on its subsequent formation or on NOS catalytic activity. Neither enzyme dilution nor NO scavengers (superoxide and oxyhemoglobin) diminished formation of ferrous-nitrosyl NOS or prevented the catalytic inhibition attributed to its formation. The ferrous-nitrosyl complex also formed in unfractionated cell cytosol containing neuronal NOS upon initiating NO synthesis. We conclude that a majority of neuronal NOS is converted quickly to a catalytically inactive ferrous-nitrosyl complex during NO synthesis independent of the external NO concentration. Thus, NO binding to the NOS heme may be a fundamental feature of catalysis and functions to down-regulate NO synthesis by neuronal NOS.
authors.affiliation: Department of Immunology, Cleveland Clinic Research Institute, Ohio 44195, USA.
authors.forename: H M
authors.initials: HM
authors.lastname: Abu-Soud
authors.forename: J
authors.initials: J
authors.lastname: Wang
authors.forename: D L
authors.initials: DL
authors.lastname: Rousseau
authors.forename: J M
authors.initials: JM
authors.lastname: Fukuto
authors.forename: L J
authors.initials: LJ
authors.lastname: Ignarro
authors.forename: D J
authors.initials: DJ
authors.lastname: Stuehr
mesh_terms: D000332:Aerobiosis; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002384:Catalysis; D002460:Cell Line; D002956:Citrulline / Q000378:metabolism; D003198:Computer Simulation; D005296:Ferrous Compounds; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008962:Models, Theoretical; D009249:NADP / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D010108:Oxyhemoglobins / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013481:Superoxides / Q000378:metabolism; D014162:Transfection
chemical_list: D005296:Ferrous Compounds; D010108:Oxyhemoglobins; D011994:Recombinant Proteins; D013481:Superoxides; D002956:Citrulline; D009569:Nitric Oxide; D009249:NADP; D001120:Arginine; D019001:Nitric Oxide Synthase