Flavin-dependent superoxide production by nitric oxide synthase

S. Kurz, R. Venema, H. Sayegh, J. D. Kent, D. G. Harrison

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Abstract

The neuronal NO synthase transfers electrons from NADPH to flavins and then in the presence of calcium and calmodulin (Cam) to heme and subsequently to either L-arginine (Larg) {to produce nitric oxide) or to oxygen to produce Superoxide (62") and H%O2. In vivo studies have suggested that the endothelial isoform (eNOS) produces H-4Oz via a similar mechanism, but direct evidence for Oa" production by the endothelial enzyme is lacking. eNOS was expressed using a baculovirus/Sf-9 insect cell system and lucigenin chemiluminesence was employed to quantify 02" production. In the presence of co-factors {NADPH, flavins and Cam) and absence of L-arg, wild-type eNOS generated 105 ±3 nmol of 02" per min-mg-1 protein. Surprisingly, 02" production persisted in the absence of Cam (27± 0.4 nmolmin"1mg"1 protein). We performed additional studies to examine this Camindependent source of 02" production by eNOS. To assure that this was not due to residual insect Cam binding to the enzyme, we studied a mutant eNOS lacking the Cam binding domain (eNOSiCAM)- This enzyme produced quantities of 02" similar those produced by the wildtype enzyme in the absence of Cam. These findings raised the possibility that residual electron transfer from the flavins to heme occurred in the absence of Cam, and that subsequent electron transfer to oxygen from heme could account for 02" production. To examine this possibility we studied an enzyme mutated to lack the heme binding cysteine (eNOSci84)- eNOScis4 produced O2" in a fashion identical to the wild-type enzyme in the absence of Cam and similar to eNOS-4cAM- These findings suggested that Cam-independent 02" production can occur in eNOS, likely via transfer of electrons from the flavins to oxygen. To confirm this, we demonstrated that the flavin-inhibitor diphenylene iodonium completely abolished 02' production by eNOScis4- Studies of Sf-9 expressed neuronal NOS also revealed Cam independent 62" production. These studies demonstrate a new source of 02" from both brain and endothelial cell NOS which is flavin dependent and not regulated by Cam or L-arginine.

Original languageEnglish (US)
Pages (from-to)314a
JournalJournal of Investigative Medicine
Volume44
Issue number3
StatePublished - Jan 1 1996

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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    Kurz, S., Venema, R., Sayegh, H., Kent, J. D., & Harrison, D. G. (1996). Flavin-dependent superoxide production by nitric oxide synthase. Journal of Investigative Medicine, 44(3), 314a.