Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A 1998 Aug 04;95(16):9220-5



Pubmed ID


Pubmed Central ID




Scopus ID

2-s2.0-0032482975   1156 Citations


The mechanism of superoxide generation by endothelial nitric oxide synthase (eNOS) was investigated by the electron spin resonance spin-trapping technique using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide. In the absence of calcium/calmodulin, eNOS produces low amounts of superoxide. Upon activating eNOS electron transfer reactions by calcium/calmodulin binding, superoxide formation is increased. Heme-iron ligands, cyanide, imidazole, and the phenyl(diazene)-derived radical inhibit superoxide generation. No inhibition is observed after addition of L-arginine, NG-hydroxy-L-arginine, L-thiocitrulline, and L-NG-monomethyl arginine to activated eNOS. These results demonstrate that superoxide is generated from the oxygenase domain by dissociation of the ferrous-dioxygen complex and that occupation of the L-arginine binding site does not inhibit this process. However, the concomitant addition of L-arginine and tetrahydrobiopterin (BH4) abolishes superoxide generation by eNOS. Under these conditions, L-citrulline production is close to maximal. Our data indicate that BH4 fully couples L-arginine oxidation to NADPH consumption and prevents dissociation of the ferrous-dioxygen complex. Under these conditions, eNOS does not generate superoxide. The presence of flavins, at concentrations commonly employed in NOS assay systems, enhances superoxide generation from the reductase domain. Our data indicate that modulation of BH4 concentration may regulate the ratio of superoxide to nitric oxide generated by eNOS.

Author List

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


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
NADPH Oxidases
Nitric Oxide Synthase
Nitric Oxide Synthase Type III
Spin Labels
jenkins-FCD Prod-482 91ad8a360b6da540234915ea01ff80e38bfdb40a