CA2+ CALMODULIN-DEPENDENT NO SYNTHASE TYPE-I - A BIOPTEROFLAVOPROTEINWITH 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 cha racterized to date, the soluble and Ca2+/calmodulin-regulated type I f rom rat cerebellum (homodimer of 160-kDa subunits) is dependent on NAD PH for catalytic activity. The enzyme also possesses NADPH diaphorase activity in the presence of the electron acceptor nitroblue tetrazoliu m (NBT). We investigated the requirements of NOS and its content of th e proposed additional cofactors tetrahydrobiopterin (H4biopterin) and flavins, further characterized the NADPH diaphorase activity, and quan tified the NADPH binding site(s). Purified NOS type I Ca2+/calmodulin- independently bound the [P-32]2',3'-dialdehyde analogue of NADPH (dNAD PH), which, at near K(m) concentrations during 3-min incubations was u tilized as a substrate and at higher concentrations or after prolonged incubations and cross-linking inhibited NOS activity. The NADPH diaph orase activity was Ca2+/calmodulin-independent, required higher NADPH concentrations than NOS activity, and was affected by dNADPH to a less er 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 riboflav in was detected. These findings suggest that NOS may share cofactors b etween two identical subunits via high-affinity binding sites. They al so explain why different preparations of NOS have different requiremen ts for exogenous flavins and H-4biopterin for maximal catalytic activi ty. Furthermore, H-4biopterin alone reduced NBT, and purified dihydrop teridine 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 nonco mpetitive inhibition of NOS activity by NBT suggest also that NOS type I may have a novel methotrexate-insensitive quinoid-H-2biopterin redu ctase activity.