Characterization of the mechanism of cytochrome P450 reductase-cytochrome P450-mediated nitric oxide and nitrosothiol generation from organic nitrates

Haitao Li, Xiaoping Liu, Hongmei Cui, Yeong Renn Chen, Arturo J. Cardounel, Jay L. Zweier

Research output: Contribution to journalArticlepeer-review

Abstract

Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to NO2-. The CPR flavin site inhibitor diphenyleneiodonium inhibited this NO2- generation, whereas the CP inhibitor clotrimazole did not. However, clotrimazole greatly inhibited NO2--dependent NO generation. Therefore, CPR catalyzes organic nitrate reduction, producing nitrite, whereas CP can mediate further nitrite reduction to NO. Nitrite-dependent NO generation contributed 2- is not the main precursor of NO. CPR-CP-mediated NO generation was largely thiol-dependent. Studies suggested that organic nitrite (R-O-NO) was produced from organic nitrate reduction by CPR. Further reaction of organic nitrite with free or microsome-associated thiols led to NO or nitrosothiol generation and thus stimulated the activation of sGC. Thus, organic nitrite is the initial product in the process of CRP-CP-mediated organic nitrate activation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.

Original languageEnglish (US)
Pages (from-to)12546-12554
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number18
DOIs
StatePublished - May 5 2006
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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