CA2+/CALMODULIN-DEPENDENT NO SYNTHASE TYPE-I - A BIOPTEROFLAVOPROTEIN WITH CA2+/CALMODULIN-INDEPENDENT DIAPHORASE AND REDUCTASE ACTIVITIES

NO synthase (NOS; EC 1. 14.23) catalyzes the conversion Of L-arginine into L-citrulline and a guanylyl cyclase-activating factor (GAF) that is chemically identical with nitric oxide or a nitric oxide-releasing compound (NO). Similar to the other isozymes of NOS that have been characterized to date, the soluble and Ca2+/calmodulin-regulated type I from rat cerebellum (homodimer of 160-kDa subunits) is dependent on NADPH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazolium (NBT). We investigated the requirements of NOS and its content of the proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quantified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin-independently bound the [P-32]2',3'-dialdehyde analogue of NADPH (dNADPH), which, at near K(m) concentrations during 3-min incubations was utilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaphorase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a lesser degree. Divalent cations interfered with the diaphorase assay. Per dimer, native NOS contained about 1 mol each of H-4biopterin, FAD, and FMN, classifying it as a biopteroflavoprotein, and incorporated 1 mol of dNADPH. No dihydrobiopterin (H-2biopterin), biopterin, or riboflavin was detected. These findings suggest that NOS may share cofactors between two identical subunits via high-affinity binding sites. They also explain why different preparations of NOS have different requirements for exogenous flavins and H-4biopterin for maximal catalytic activity. Furthermore, H-4biopterin alone reduced NBT, and purified dihydropteridine reductase was found to have diaphorase activity. The absence of enzyme-bound biopterins other than H-4biopterin, the reported lack of effect of methotrexate on enzyme activity, and the potent and noncompetitive inhibition of NOS activity by NBT suggest also that NOS type I may have a novel methotrexate-insensitive quinoid-H-2biopterin reductase activity.