S-glutathionylation reshapes our understanding of endothelial nitric oxide synthase uncoupling and nitric oxide/reactive oxygen species-mediated signaling

Abstract

Oxidative stress has been shown to convert endothelial nitric oxide synthase (eNOS) from an NO-producing enzyme to an enzyme that generates superoxide, a process termed NOS uncoupling. This uncoupling of eNOS converts it to function as an NADPH oxidase with superoxide and hydrogen peroxide generation. eNOS uncoupling has been associated with many pathophysiologic conditions, such as heart failure, ischemia/reperfusion injury, hypertension, atherosclerosis, and diabetes. The mechanisms implicated in the uncoupling of eNOS include oxidation of the critical NOS cofactor tetrahydrobiopterin, depletion of L-arginine, and accumulation of methylarginines. All of these prior mechanisms of eNOS-derived reactive oxygen species formation occur primarily at the heme of the oxygenase domain and are blocked by heme blockers or the NOS inhibitor N-nitro-L-arginine methylester. Recently, we have identified another unique mechanism of redox regulation of eNOS through S-glutathionylation that was shown to be important in cell signaling and vascular disease. Herein, we briefly review the mechanisms of eNOS uncoupling as well as their interrelationships and the evidence for their importance in disease.

FIG. 1.

Summary of physiological action of eNOS S-glutathionylation. eNOS present in endothelial cells, indicated by the green coloration, generates NO in the vasculature, which is known to induce vasodilation. When eNOS is S-gutathionylated, indicated by the yellow-colored portion of the endothelium (provided from immunohistology), the enzyme switches to produce superoxide (depicted as white bilobed spheres) instead of NO (blue/white bilobed spheres), which leads to vasoconstriction. eNOS, endothelial nitric oxide synthase. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).

FIG. 2.

Molecular model of the human NOS reductase domain showing sites of S-glutathionylation. The three-dimensional structure of human NOS reductase was obtained by use of the Swiss-Model First Approach Mode. PyMOL was used to view and construct the three-dimensional structure. Cys689 and Cys908 are both located on the surface of reductase domain and surrounded by several positively charged residues, including Lys, Arg, and His. Cys689 is surrounded by Arg898, Lys904, and Arg1174. Cys908 is located in a pocket that contains His972, Arg1174, and the adenine ring of FAD. Because of this positively charged environment, both Cys689 and 908 are thought to be in deprotonated states with lower pK_a and susceptible to oxidation by oxidized glutathione. Green and red regions represent residues at the interface of FMN and FAD domains, respectively. Cys689 is located at the FMN domain interface and Cys908 is near the FAD domain interface. (To see this illustration in color the reader is referred to the web version of this article at www.liebertonline.com/ars).