| Abstract: | It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols.Nitric oxide (NO)2 is the most studied of the endogenously generated nitrogen oxides and is well known to mediate many aspects of cardiovascular function including the regulation of vascular tone and platelet aggregation (for example, see Ref. 1). These responses are in large part due to the interaction of NO with its most established endogenous receptor, soluble guanylate cyclase (sGC) (2). This 150-kDa heterodimeric heme protein catalyzes the production of the second messenger molecule cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP) (3). The basal activity of sGC is enhanced several hundred fold upon binding of NO to the single regulatory heme site. This stimulation of activity is a result of a conformational change induced by cleavage of the proximal histidine heme ligand upon formation of the ferrous nitrosyl complex, which is preferentially pentacoordinate (4). In addition to heme site regulation of sGC, there are numerous reports indicating that oxidation of cysteine thiol residues on this protein can also alter/regulate both the basal activity and the degree of NO-mediated activation (5–10).Recently, the one-electron reduced and protonated congener of NO, nitroxyl (HNO) has received significant interest as a cardiovascular agent whose actions are independent of NO formation (11). For example, a study by Ellis and co-workers (12) suggests that HNO is a vital component of endothelium-derived relaxing factor along with NO in rat aorta. HNO is also able to mediate murine aorta vasorelaxation even in the presence of NO scavengers (13). Furthermore, the vasodilation produced by HNO was inhibited by the sGC heme site inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one implicating sGC activation in this HNO-mediated effect. In addition to its effects on large conduit vessels like the aorta, HNO also dilates rat small mesenteric resistance-like arteries through sGC-dependent and voltage-dependent K+ channel-dependent mechanisms (14). Nitroxyl (derived from the HNO-donor Angeli''s salt) is also a potent dilator of feline pulmonary vasculature equal to that of the NO donors SPER/NO, DETA/NO, and SULFI/NO (15). Most recently, HNO was found to be a potent dilator of rat coronary arteries through an sGC-mediated mechanism (16). The evidence presented in these studies suggests that HNO is able to modulate cGMP levels through an interaction with sGC, an idea in conflict with a previous report showing that NO is the only nitrogen oxide capable of directly activating sGC (17).HNO forms a stable adduct with the ferrous heme of deoxymyoglobin (18, 19) providing precedence for a possible interaction between HNO and sGC that is akin to the interaction of NO with ferrous sGC. In light of all the reports indicating possible HNO-mediated activation of sGC, an examination of the direct interaction of HNO with purified sGC was carried out to evaluate the possibility that HNO may be capable of directly interacting with sGC to elicit activation. Moreover, due to the previously reported thiol redox regulation of sGC (see above) and the known thiophilicity of HNO (20), we also examined the effects of HNO-mediated thiol modification on enzyme activity. |
