Substrate and substrate analog binding to endothelial nitric oxide synthase: Electron paramagnetic resonance as an isoform-specific probe of the binding mode of substrate analogs

John C. Salerno, Pavel Martásek, Robert F. Williams, Bettie Sue S. Masters

Research output: Contribution to journalArticle

15 Scopus citations

Abstract

The binding of arginine analogs to endothelial nitric oxide synthase (eNOS, NOSIII) perturbs the environment of the high-spin ferriheme in a highly ligand-specific manner. Using electron paramagnetic resonance as a probe of heme ligation geometry, four categories of high-spin complex could be distinguished. These are analogous to the four classes of high-spin complexes, stabilized individually by the binding of L-arginine, N-hydroxy- L-arginine (NHA), N-methyl-L-arginine (NMA), and N-nitro-L-arginine (NNA), which we have previously reported for the other two isoforms. Each of these species is five-coordinate and retains the axial thiolate ligand but each differs in its ligation geometry. N-Methyl-L-arginine is a relatively poor inhibitor of eNOS, and the NMA complex of eNOS differs from the N-methyl-L- arginine complexes of inducible nitric oxide synthase (iNOS, NOSII) and neuronal nitric oxide synthase (nNOS, NOSI) in that it is of lower rhombicity. We previously showed that inducible nitric oxide synthase, which binds NNA less tightly than eNOS and nNOS, could not form the lower rhombicity NNA complex characteristic of nNOS. Endothelial nitric oxide synthase readily forms such lower rhombicity complexes, which correlates with the tight binding of NNA to this isoform. Arginine and tetrahydrobiopterin promote loss of the flavin free radical EPR signal, while arginine analog inhibitors stabilize the radical; this suggests that the residual flavin radicals can serve as a source of reducing equivalents for slow turnover in the absence of endogenous reductant.

Original languageEnglish (US)
Pages (from-to)11821-11827
Number of pages7
JournalBiochemistry
Volume36
Issue number39
DOIs
StatePublished - Sep 30 1997

ASJC Scopus subject areas

  • Biochemistry

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