Nitric oxide (NO) is an important physiological mediator of vasodilation, platelet aggregation, and neurotransmission. An NO signal regulates these processes by signal transduction through the enzyme soluble guanylate cyclase (sGC), and dysfunction in this pathway manifests in human disease. NO activates sGC several hundred fold to produce the second messenger cGMP, and the mechanism of activation was previously thought to result exclusively from NO binding to the sGC heme. However, recent studies have shown that heme-bound NO only partially activates sGC and additional NO binding to a nonheme site is required for maximal NO activation. Therefore, understanding the nature the nonheme-NO coordination is crucial to understanding the mechanism of enzyme activation.
Thiol oxidation of sGC cysteines results in the loss of enzyme activity, therefore, the role of cysteines in NO-stimulated sGC activity was investigated. We found that the thiol modifying reagent methyl methanethiosulfonate specifically inhibits NO activation of sGC by blocking the non-heme site, which defines a role for sGC cysteine(s) in mediating NO binding. The nature of the NO/cysteine interaction was probed by examining the effects of redox active reagents on NO-stimulated activity. These results show that NO binding to, and dissociation from, the critical cysteine(s) does not involve a change in the thiol redox state. Evidence is provided for non-heme NO in the physiological activation of sGC in context of a primary cell culture of human umbilical vein endothelial cells. These findings have relevance to diseases involving the NO/cGMP signaling pathway.
Heme-oxidation also results in the loss enzyme activity and may play a role in the NO-desensitization of sGC in cardiovascular disease. In vitro, the sGC heme is typically found in the ferrous [Fe(II)] state, which is activated several hundred fold by NO. Comparatively, the ferric [Fe(III)] form of sGC is only weakly activated by NO, but the mechanism of this blunted response is not well understood. Specifically, sGC undergoes a reaction with NO termed reductive nitrosylation, where the sGC heme is reduced to the ferrous [Fe(II)] state; but despite the recovery of the initial heme state, NO-stimulated activity does not recover. We found that the reduction of the sGC heme occurs via a coupled oxidation of an sGC cysteine to a nitrosothiol, and that the S-nitrosation of this key cysteine inhibits the enzymatic response to NO. The data from chapter 2 suggests the mechanism of inhibition may be the oxidation of a cysteine that constitutes the nonheme NO binding site. Thus, thiol-oxidation is at the root of the NO-desensitization of heme-oxidized sGC and the implications to human health and disease are discussed.
