EPR SPECTROSCOPIC CHARACTERIZATION OF NEURONAL NO SYNTHASE

Neuronal NO synthase (nNOS) consists of a reductase domain that binds FAD, FMN, NADPH, and calmodulin, and an oxygenase domain that binds he me, tetrahydrobiopterin, and the substrate L-arginine. One flavin in r esting nNOS exists as an air-stable semiquinone radical. During NO syn thesis, electron transfer occurs between the flavins and heme iron. We have characterized the nNOS heme iron and flavin semiquinone radical by electron paramagnetic resonance (EPR) spectroscopy. Under anaerobic conditions, the flavin radical spin relaxation was very slow (8 Hz at 22 K) and was enhanced 13-fold by dissolved dioxygen via spin-spin co upling. The flavin radical, probably the semiquinone FMNH(.), was show n by progressive microwave power saturation and EPR saturation recover y under anaerobic conditions to be spin-spin coupled with the heme iro n located in the nNOS oxygenase domain. Analysis of an nNOS preparatio n that was devoid of heme but contained the flavin radical revealed th at spin-spin coupling increased the rate of flavin radical relaxation by a factor of 15. The presence of bound substrate (L-arginine) or the substrate analogue N-omega-nitro-L-arginine methyl ester (NAME) had n o effect on the flavin spin relaxation kinetics. The observed g values of the nNOS heme were 7.68, 4.15, and 1.81 and were unchanged by occu pation of the substrate binding site by L-arginine or NAME. The substr ate also had no effect on the heme zero-field splitting parameter, D = 5.2 cm(-1). Together, the data indic;lte that the flavin and heme red ox centers are positioned near each other in nNOS, consistent with the ir participating in an interdomain electron transfer. The flavin radic al is affected by dissolved oxygen, suggesting that its binding site w ithin the reductase domain is partially exposed to solvent, but is una ffected when substrate binds to the oxygenase domain. Substrate bindin g also appears to take place outside the first coordination shell of t he nNOS heme iron.