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
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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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Increased eNOS monomer and decreased eNOS dimer and zinc content are ass...
Increased eNOS monomer and decreased eNOS dimer and zinc content are associated with decreased eNOS activity in tissues of diabetic LDLR-KO mice. (a) Representative Western blot of eNOS protein in tissues from control and diabetic LDLR-KO mice. Tissue homogenate proteins were separated by normal-temperature SDS-PAGE under reducing conditions, blotted, and stained with a polyclonal antibody against eNOS. Blot shown is representative of three independent experiments. (b) Increased eNOS monomers are accompanied by a decrease in eNOS dimer in the tissues obtained from diabetic LDLR-KO mice. The eNOS protein in control or diabetic tissue homogenates was purified, separated by low-temperature SDS-PAGE under reducing conditions (without boiling), and detected by Coomassie staining. Results shown represent four independent experiments. (c) Decreased zinc content is associated with inhibition of NO synthesis and decreased eNOS dimer in diabetic LDLR-KO mice. The zinc content of eNOS from diabetic LDLR-KO mouse tissues was expressed as a percentage of that in normoglycemic control LDLR-KO animals. L-citrulline formation was determined in homogenates from control and diabetic animals and expressed as a percentage of that in tissues from control animals (n = 7, *P < 0.05). The assay was performed as described in Methods, and the rates of L-citrulline formation were determined to be 47 ± 11, 56 ± 19, and 43 ± 16 pmol/mg/min in liver, heart, and kidneys, respectively, of normoglycemic control LDLR-KO animals.