Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite
Ming-Hui Zou, … , Chaomei Shi, Richard A. Cohen
Ming-Hui Zou, … , Chaomei Shi, Richard A. Cohen
Published March 15, 2002
Citation Information: J Clin Invest. 2002;109(6):817-826. https://doi.org/10.1172/JCI14442.
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Categories: Article Vascular biology

Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite

Abstract

Nitric oxide (NO) is produced by NO synthase (NOS) in many cells and plays important roles in the neuronal, muscular, cardiovascular, and immune systems. In various disease conditions, all three types of NOS (neuronal, inducible, and endothelial) are reported to generate oxidants through unknown mechanisms. We present here the first evidence that peroxynitrite (ONOO–) releases zinc from the zinc-thiolate cluster of endothelial NOS (eNOS) and presumably forms disulfide bonds between the monomers. As a result, disruption of the otherwise SDS-resistant eNOS dimers occurs under reducing conditions. eNOS catalytic activity is exquisitely sensitive to ONOO–, which decreases NO synthesis and increases superoxide anion (O2.–) production by the enzyme. The reducing cofactor tetrahydrobiopterin is not oxidized, nor does it prevent oxidation of eNOS by the same low concentrations of OONO–. Furthermore, eNOS derived from endothelial cells exposed to elevated glucose produces more O2.–, and, like eNOS purified from diabetic LDL receptor–deficient mice, contains less zinc and fewer SDS-resistant dimers. Hence, eNOS exposure to oxidants including ONOO– causes increased enzymatic uncoupling and generation of O2.– in diabetes, contributing further to endothelial cell oxidant stress. Regulation of the zinc-thiolate center of NOS by ONOO– provides a novel mechanism for modulation of the enzyme function in disease.

Authors

Ming-Hui Zou, Chaomei Shi, Richard A. Cohen

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Figure 1

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ONOO– dissociates SDS-resistant dimers and alters recombinant eNOS activ...
ONOO– dissociates SDS-resistant dimers and alters recombinant eNOS activity. Purified eNOS was treated with ONOO– (0–100 μmol/l), decomposed ONOO–, or NaOH vehicle (100 mmol/l) as described in Methods. Five minutes after treatment, eNOS dimers and monomers were separated by low-temperature SDS-PAGE (6%) under reducing or nonreducing conditions. The proteins were visualized by Coomassie staining. (a) Representative blots of eNOS dimers and monomers in reducing (left) or nonreducing gels (right). (b) ONOO– dissociated eNOS dimers into monomers under reducing conditions. The intensity (area times density) of dimers and monomers was determined by densitometry as described in Methods. The results were expressed as percent change compared with untreated enzyme (n = 10, *P < 0.05). (c) Inhibition by ONOO– (0.1–50 μmol/l) of the rate of eNOS-dependent L-citrulline formation (n = 12) was associated with an increase in NADPH oxidation (n = 14). L-citrulline formation and NADPH oxidation by purified recombinant eNOS were each assayed as described in Methods. *P < 0.01. Di-eNOS, eNOS dimer; eNOS, eNOS monomer.