SUPEROXIDE GENERATION BY ENDOTHELIAL NITRIC-OXIDE SYNTHASE - THE INFLUENCE OF COFACTORS

The mechanism of superoxide generation by endothelial nitric oxide syn thase (eNOS) was investigated by the electron spin resonance spin-trap ping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide . In the absence of calcium/calmodulin, eNOS produces low amounts of s uperoxide. Upon activating eNOS electron transfer reactions by calcium /calmodulin binding, superoxide formation is increased, Heme-iron liga nds, cyanide, imidazole, and the phenyl(diazene)-derived radical inhib it superoxide generation, No inhibition is observed after addition of L-arginine, N-G-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomet hyl arginine to activated eNOS, These results demonstrate that superox ide is generated from the oxygenase domain by dissociation of the ferr ous-dioxygen complex and that occupation of the L-arginine binding sit e does Plot inhibit this process, However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generat ion by eNOS, Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidat ion to NADPH consumption and prevents dissociation of the ferrous-diox ygen complex, Under these conditions, eNOS does not generate superoxid e. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase doma in. Our data indicate that modulation of BH4 concentration may regulat e the ratio of superoxide to nitric oxide generated by eNOS.