Nitric oxide binding to the heme of neuronal nitric-oxide synthase links its activity to changes in oxygen tension.
Neuronal nitric-oxide synthase (NOS-1) is a hemeprotein that generates NO and citrulline from L-arginine, O2, and NADPH. During catalysis, a majority of NOS-1 binds self-generated NO and converts to a ferrous-NO complex, which causes it to operate at a fraction of its maximum possible activity during the steady state (Abu-Soud, H. M., Wang, J., Rousseau, D. L., Fukuto, J., Ignarro, L. J., and Stuehr, D. J. (1995) J. Biol. Chem. 270, 22997-23006). To examine how NO complex formation affects the O2 response of NOS-1, we measured rates of NO synthesis and NADPH oxidation versus O2 concentration in the presence and absence of L-arginine. In the absence of L-arginine, NOS-1 catalyzed simple O2 reduction, and its heme iron displayed a typical affinity for O2 (estimated KmO2 </= 40 microM, saturation at approximately 100 microM). In the presence of L-arginine, the rates of NO synthesis and NADPH oxidation were proportional to the O2 concentration over a much broader range (estimated KmO2 approximately 400 microM, saturation at approximately 800 microM), indicating that ferrous-NO complex formation altered the O2 response of NOS-1. Stopped-flow experiments revealed that the rate of ferrous-NO complex formation was relatively independent of the O2 concentration between 100 and 700 microM, while the rate of complex breakdown was directly proportional to O2 concentration. We conclude that the O2 sensitivity of the ferrous-NO complex governs the O2 response of NOS-1 and thus its activity during the steady state. This enables NOS-1 to couple its rate of NO synthesis to the O2 concentration throughout the physiologic range.
Abu-Soud
H M
HM
Department of Immunology, The Cleveland Clinic Research Institute, Cleveland, Ohio 44195, USA.
Rousseau
D L
DL
Stuehr
D J
DJ
D002147:Calmodulin / Q000378:metabolism; D003198:Computer Simulation; D005290:Ferric Compounds / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009474:Neurons / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism
D002147:Calmodulin; D005290:Ferric Compounds; D013481:Superoxides; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D019001:Nitric Oxide Synthase; D010100:Oxygen