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 K(m) O₂ ≤ 40 μM, saturation at ~100 μM). 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 K(m) O₂ ~400 μM, saturation at ~800 μM), 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 μM, 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.