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 (O 2 .-) 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 O 2 .-, 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 O 2 .-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.

Oxidant stress from nitric oxide synthase–3 uncoupling stimulates cardiac pathologic remodeling from chronic pressure load

“…To test this possibility, NOS3 was immunoprecipitated and separated using nondenaturing gels to assess dimer (coupled) versus monomer (uncoupled) forms (20). Both monomer and dimer bands were present in WT sham controls, whereas the monomer primarily existed in TAC hearts ( Figure 6A).…”

“…ROS can be generated by mitochondrial electron transport leakage (10,15), NADPH oxidases (3,16,17), xanthine oxidase (18), and nitric oxide synthase (NOS) (19,20). Among these, NOS is intriguing because NO and its downstream target, protein kinase G, are generally considered to blunt hypertrophy (21)(22)(23)(24).…”

“…Among these, NOS is intriguing because NO and its downstream target, protein kinase G, are generally considered to blunt hypertrophy (21)(22)(23)(24). However, NOS can be converted to a ROS generator, as demonstrated in vascular endothelium exposed to increased oxidant or hemodynamic stress (19,20,25). NOS3 is the dominant isoform in the endothelium, as well as in cardiac myocytes, where it is an important regulator of adrenergic, muscarinic, and rate-mediated reserve (26,27).…”

“…When exposed to oxidant stress, including peroxynitrite (ONOO -), or deprived of its reducing cofactor tetrahydrobiopterin (BH4) or substrate L-arginine, NOS3 uncouples to the monomeric form that generates O 2 -rather than NO (25,28,29). Uncoupled NOS3 is thought to be a prominent source of endothelial ROS in hypertension (19), neurohormonal stimulation, and hyperglycemia (8), and upon exposure to ONOO - (20).…”

Adiponectin deficiency increases leukocyte-endothelium interactions via upregulation of endothelial cell adhesion molecules in vivo

“…Relevant to this view, cytokines such as TNF-α are closely linked to the endothelial production of ROS (superoxide and H 2 O 2 ), a central component of the inflammatory milieu that contributes to atherogenesis in some pathologic conditions (52)(53)(54). ROS can quickly quench NO, a process that results in the formation of peroxynitrite, which has also been postulated to alter the catalytic activity of eNOS, diverting its synthesis from NO toward increased superoxide production (55). In addition, TNF-α directly downregulates basal and insulin-stimulated eNOs expression (56).…”