Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase.
The binding of calcium/calmodulin stimulates electron transfer between the reductase and oxygenase domains of neuronal nitric oxide synthase (nNOS). Here, we demonstrate using electron spin resonance spin-trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide that pterin-free nNOS generates superoxide from the reductase and the oxygenase domain by a calcium/calmodulin-dependent mechanism. Tetrahydrobiopterin (BH(4)) diminishes the formation of superoxide by a mechanism that does not cause inhibition of NADPH consumption. In contrast, BH(4) analogs 7,8-dihydrobiopterin and sepiapterin do not affect superoxide yields. L-Arginine alone inhibits the generation of superoxide by nNOS but not by C331A-nNOS mutant that has a low affinity for L-arginine. A greater decrease in superoxide yields is observed when nNOS is preincubated with L-arginine. This effect is in accordance with the slow binding rates of L-arginine to NOS in the absence of BH(4). L-Arginine alone or in combination with BH(4) decreases the rates of NADPH consumption. The effect of L-arginine on superoxide yields, however, was less dramatic than that caused by BH(4) as much higher concentrations of L-arginine are necessary to attain the same inhibition. In combination, L-arginine and BH(4) inhibit the formation of superoxide generation and stimulate the formation of L-citrulline. We conclude that, in contrast to L-arginine, BH(4) does not inhibit the generation of superoxide by controlling electron transfer through the enzyme but by stimulating the formation of the heme-peroxo species.
Vásquez-Vivar
J
J
Department of Pathology, Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Hogg
N
N
Martásek
P
P
Karoui
H
H
Pritchard
K A
KA
Kalyanaraman
B
B
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002417:Cattle; D002956:Citrulline / Q000096:biosynthesis; D004578:Electron Spin Resonance Spectroscopy; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052248:Nitric Oxide Synthase Type I; D010100:Oxygen / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D013481:Superoxides; D001708:Biopterins; D002956:Citrulline; D009249:NADP; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C003402:sapropterin; D010100:Oxygen