Electron transfer in the nitric-oxide synthases. Characterization of L-arginine analogs that block heme iron...

Heme iron reduction in the nitric-oxide synthases (NOSs) requires calmodulin binding and is associated with increased NO synthesis and NADPH oxidation (Abu-Soud, H. M., and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci., U. S. A. 90, 10769-10772). Here, we examined how L-arginine and the analogs N omega-methyl-L-arginine (NMA), N omega-nitro-L-arginine methyl ester (NAME), and d-(thioureido)-L-norvaline (thiocitrulline) affect electron flux through neuronal and macrophage NOS. L-Arginine and NMA increased or decreased NOS NADPH consumption depending on the isoform, while thiocitrulline and NAME decreased NADPH oxidation in both NOS by 73-86% relative to their ligand-free rates. Kinetic studies showed that thiocitrulline and NAME inhibited NOS NADPH consumption through binding within the substrate binding site. Thiocitrulline and NAME did not affect the NADPH-dependent reduction of NOS flavins nor NOS cytochrome c reduction, indicating that they blocked electron flux at a point beyond the flavins in the electron transfer sequence. Thiocitrulline and NAME inhibited both NADPH-dependent and dithionite-mediated heme iron reduction in the NOS isoforms relative to the substrate-free NOS, whereas L-arginine and NMA did not. Thus, L-arginine and NMA increase or decrease electron flux through the NOS by coupling NADPH oxidation to NO synthesis (L-arginine), or by occupying the substrate binding site with minimal catalytic coupling (NMA). In contrast, thiocitrulline and NAME decrease electron flux through both NOS isoforms by decreasing the reduction potential of the heme iron. Inhibition of heme iron reduction by substrate analogs is unusual and represents a new means to modulate electron flow through the NOS.