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 monomers and decreased zinc content in eNOS derived from BAECs...
Increased monomers and decreased zinc content in eNOS derived from BAECs exposed to high glucose are associated with increased O2.– and ONOO–. (a) Increased eNOS monomers in cells exposed to high glucose. Confluent BAECs were exposed to control (5 mmol/l D-glucose) or high glucose (30 mmol/l) for 3 days. The eNOS protein was separated by low-temperature SDS-PAGE under reducing conditions, blotted, and detected with a monoclonal antibody. Blot shown is representative of results from eight independent experiments. (b) Increased eNOS monomer and correlated O2.– release are associated with decreased zinc content in eNOS from cells exposed to high glucose (n = 10, #P < 0.05). The zinc content of eNOS from cells incubated in elevated glucose was assayed as described in Methods and expressed as a percentage of that in control cells exposed to 5 mmol/l glucose. Of note, both L-NAME (1 mmol/l) and SOD-PEG (500 U/ml) prevented high glucose–induced dissociation of eNOS dimers (n = 8, *P < 0.01), zinc loss (n = 5, *P < 0.01), and O2.– release (n = 6, #P < 0.05 compared with cells exposed to high glucose alone). (c) eNOS-dependent tyrosine nitration of prostacyclin synthase (PGIS) in cells exposed to high glucose. PGIS was immunoprecipitated with a polyclonal antibody against PGIS (4 μg/ml). The nitrated PGIS was detected with a monoclonal antibody against 3-nitrotyrosine (3-NT) (1:1,000; Upstate Biotechnology Inc.). Of note, SOD-PEG (500 U/ml) or L-NAME (1 mmol/l), but not oxypurinol (10 μmol/l) or rotenone (10 μmol/l), prevented the nitration of PGIS. IP, immunoprecipitation; IB, immunoblot; PGIS, prostacyclin synthase. Blot shown is representative of three independent experiments.