The ferrous-dioxy complex of neuronal nitric oxide synthase - Divergent effects of L-arginine and tetrahydrobiopterin on its stability

Nitric oxide synthases (NOS) are hemeproteins that catalyze oxidation of L-arginine to nitric oxide (NO) and citrulline. The NOS heme iron is expected to participate in oxygen activation during catalysis, but its interac tions with O-2 are not characterized. We utilized the heme-containing oxygenase domain of neuronal NOS (nNOSoxy) and stopped-flow methods to study forma tion and autooxidative decomposition of the nNOSoxy oxygenated complex at 10 degrees C. Mixing ferrous nNOSoxy with air-saturated buffer generated a transient species with absorption maxima at 427 and similar to 560 nm. This species decayed within 1 s to form ferric nNOSoxy. Its formation was first order with respect to O-2, monophasic, and gave rate constants for k(on) = 9 x 10(5) M-1 s(-1) and k(off) = 108 s(-1) for an L-arginine- and tetrahydrobiopterin (H4B)-saturated nNOSoxy. Omission of L-arginine and/or H4B did not greatly effect O-2 binding and dissociation rates. Decomposition of the oxygenated intermediate was independent of O-2 concentration and was either biphasic or monophasic depending on sample conditions, L-Arginine stabilized the oxygenated intermediate (decay rate = 0.14 s(-1)), while H4B accelerated its decay by a factor of 70 irrespective of L-arginine. The spectral and kinetic properties of the intermediate identify it as the (FeO2)-O-II complex of nNOSoxy. Destabilization of a metallo-oxy species by H4B is unprecedented and may be important regarding the role of this cofactor in NO synthesis.