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Tetrahydrobiopterin-dependent inhibition of superoxide generation from neuronal nitric oxide synthase. J Biol Chem 1999 Sep 17;274(38):26736-42



Pubmed ID




Scopus ID

2-s2.0-0040886300   159 Citations


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.

Author List

Vásquez-Vivar J, Hogg N, Martásek P, Karoui H, Pritchard KA Jr, Kalyanaraman B


Neil Hogg PhD Associate Dean, Professor in the Biophysics department at Medical College of Wisconsin
Balaraman Kalyanaraman PhD Chair, Professor in the Biophysics department at Medical College of Wisconsin
Kirkwood A. Pritchard PhD Professor in the Surgery department at Medical College of Wisconsin
Jeannette M. Vasquez-Vivar PhD Professor in the Biophysics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Electron Spin Resonance Spectroscopy
Models, Chemical
Nerve Tissue Proteins
Nitric Oxide Synthase
Nitric Oxide Synthase Type I
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a