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, O-2, 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 O-2 response of NOS-1, me measured rates of NO synthesis and NADPH oxidation versus O-2 concentration in the presence and absence of L-arginine, In the absence of L-arginine, NOS-1 catalyzed simple O-2 reduction, and its heme iron displayed a typical affinity for O-2 (estimated K-m O-2 less than or equal to 40 mu M, saturation at similar to 100 mu M). In the presence of L-arginine, the rates of NO synthesis and NADPH oxidation were proportional to the O-2 concentration over a much broader range (estimated KmO2 similar to 400 mu M, saturation at similar to 800 mu M), indicating that ferrous-NO complex formation altered the O-2 response of NOS-1, Stopped-flow experiments revealed that the rate of ferrous-NO complex formation was relatively independent of the O-2 concentration between 100 and 700 mu M, while the rate of complex breakdown was directly proportional to O-2 concentration. We conclude that the O-2 sensitivity of the ferrous-NO complex governs the O-2 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 O-2 concentration throughout the physiologic range.