Objective—The aim of the present study is to determine whether hypochlorous acid (HOCl), the major oxidant of leukocyte-derived myeloperoxidase (MPO), oxidizes the zinc-thiolate center of endothelial nitric oxide synthase (eNOS) and uncouples the enzyme.

Methods and Results—Exposure of purified recombinant eNOS to HOCl (≥ 100 μmol/L) released zinc and disrupted the enzyme-active eNOS dimers. In parallel with increased detections of both O 2·− and ONOO−, clinically relevant concentrations of HOCl disrupted eNOS dimers in cultured human umbilical vein endothelial cells (HUVEC) at concentration 10-to 100-fold lower than those required for recombinant eNOS. In HUVEC, HOCl increased the translocation of both p67 phox and p47 phox of NAD (P) H oxidase and the phosphorylation of atypical protein kinase C-ζ. Further, genetic or pharmacological inhibition of either NAD (P) H oxidase–derived O 2·− or PKC-ζ or NOS abolished the effects of HOCl on eNOS dimers. Consistently, HOCl increased both O 2·− and ONOO− and eNOS dimer oxidation in isolated mouse aortas from C57BL/6 but less in those of gp91 phox knock-out mice. Finally, in human carotid atherosclerotic arteries, eNOS predominantly existed as monomers in parallel with increased staining of both MPO and 3-nitrotyrosine.

Conclusions—We conclude that HOCl uncouples eNOS by ONOO− generated from PKC-ζ–dependent NAD (P) H oxidase.