CHARACTERIZATION OF C415 MUTANTS OF NEURONAL NITRIC-OXIDE SYNTHASE

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to c itrulline and nitric oxide. C415H and C415A mutants of the neuronal is oform of NOS (nNOS) were expressed in a baculovirus system and purifie d to homogeneity for spectral analysis and activity measurements, UV-v isible spectra of each mutant lacked an observable Soret peak, suggest ing that neither mutant contained heme. When reduced in the presence o f CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axi al ligand to the heme. In addition to a deficiency in bound heme, neit her mutant had any detectable bound tetrahydrobiopterin, as compared t o wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine: 1 mol of nNOS 160 kDa subunit, The C415H mutant contained bound FAD an d FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit , respectively. UV-visible spectra of both nNOS mutants retained the d istinctive absorbance due to tightly associated oxidized flavin prosth etic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone, Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 redu cing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially ide ntical to the spectrum of wild-type nNOS. Neither mutant could synthes ize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrom e c reduction by each mutant was found to be slightly less, but very s imilar to the rate (similar to 20 mu mol mg(-1) min(-1)) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction incr eased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme l igand and that a point mutation at C415 prevents binding of heme and t etrahydrobiopterin without interfering with the global folding or the reductase function of nNOS.