Tonic and acute nitric oxide signaling through soluble guanylate cyclase is mediated by nonheme nitric oxide, ATP, and GTP

Abstract

Nitric oxide (NO) affects many physiological systems by activating cGMP signaling cascades through soluble guanylate cyclase (sGC). In the accepted model, NO binds to the sGC heme, activating the enzyme. Here, we report that in the presence of physiological concentrations of ATP and GTP, NO dissociation from the sGC heme is Ϸ160 times slower than the rate of enzyme deactivation in vitro. Deactivated sGC still has NO bound to the heme, and full activation requires additional NO. We propose an activation model where, in the presence of both ATP and GTP, tonic NO forms a stable heme complex with low sGC activity; acute production of NO transiently and fully activates this NO-bound sGC.heme ͉ nucleotide regulation N itric oxide (NO) mediates blood vessel relaxation, complex aspects of myocardial function, perfusion and function of all major organs, synaptic plasticity in the brain, platelet aggregation, skin function, and numerous other physiological processes, by targeting and activating soluble guanylate cyclase (sGC) (reviewed in refs. 1-5). Dysregulation of NO signaling, then, contributes to many types of disease state, from erectile dysfunction and heart disease, to neurodegeneration, stroke, hypertension, and gastrointestinal disease, to name a few (reviewed in refs. 6-8).In vivo and ex vivo tissue studies of NO have revealed two fundamentally distinct and paradoxical signaling modes: tonic and acute. Tonic NO describes the continual low-level production of NO that elicits a long-lasting low-level cGMP signal (9). Under resting conditions such as normotension, inhibition of nitric oxide synthase (NOS) or sGC results in vasoconstriction; therefore, tonic NO-elicited cGMP production maintains homeostatic vascular tone. Relaxation of smooth muscle requires an acute burst of NO synthesis, typically triggered by acetylcholine, and cGMP levels rise rapidly (10). These data, which describe two separate effects of NO, cannot be explained by the established binary model for NO regulation of sGC: that NO activates sGC solely by binding to its heme cofactor.The N-terminal Ϸ180 aa of the ␤1 subunit of sGC form an evolutionarily conserved protoporphyrin-IX heme domain with spectral properties similar to the holoenzyme (11-13). NO binding to the sGC heme is diffusion-limited; however, oxygen does not bind to sGC, and carbon monoxide (CO) binding is at least 10 6 -fold weaker than NO. Thus, the sGC heme environment is a specific NO sensor. The C-terminal domains of each subunit are homologous to the catalytic domains of adenylate cyclase and fold together to form the active site of the enzyme (14). In the existing activation model (Fig. 1, black scheme), NO binds to the heme of sGC (15), forming a six-coordinate intermediate (16)(17)(18). The rate of conversion of this intermediate to the final five-coordinate ferrous-nitrosyl species depends on the concentration of NO; thus, nonheme NO accelerates rupture of the proximal histidine-iron bond. Breaking of this bond is thought to result in a conformational change in the cataly...

Dynamic Ligand Exchange in Soluble Guanylyl Cyclase (sGC)

“…When the activity was probed ϳ4 min after exposure to NO, the sGC-NO complex was only a few -fold more active than the resting sGC (Fig. 4), consistent with previous findings that stoichiometric NO does not lead to full activation of sGC (21,22,26). This low activity was not due to sGC protein aggregation during preincubation with NO.…”

“…S1). Previous reports demonstrated that sGC-NO complexes formed over several minutes with excess NO donor (21,26) or with stoichiometric NO donor in the presence of GTP (21,22) have the high cGMPforming activity expected from fully activated sGC. Our measurements corroborated these observations and demonstrated that high cGMP-forming activity was retained for up to 260 s after the sGC-NO complex was formed with excess NO gas or with stoichiometric NO in the presence of GTP (Fig.…”

“…Effect of the GTP Substrate on the Dynamics of sGC/NO Interaction-A number of reports have indicated that the GTP substrate affects the stability (21,25) of the 5C sGC-NO complex. Thus, we monitored the effect of GTP on the kinetics of the 5C sGC-NO complex generated with stoichiometric NO.…”

“…The reaction rates determined in these studies are shown in blue, the number in red is the theoretical estimation of the minimal reaction rate, and the numbers in black are from the literature: *, values from Refs. 21 bacterium Rhodospirillum rubrum is coordinated by Cys-75 in the ferric form, but, upon reduction to the ferrous form, His-77 becomes the ligand (38,39). Similarly, ligand switching was described for the bacterial phosphodiesterase EcDOS (40) and the transcriptional regulator RcoM-2 from the bacterium Burkholderia xenovorans (41).…”

“…Accumulating experimental evidence suggests that a number of environmental factors affect the dynamics of formation of the sGC-NO complex and its activity. Several studies reported that the sGC-NO complex formed with a stoichiometric amount of NO has a much lower cGMP-forming activity in comparison with sGC-NO formed with excess NO (21,22). It was also reported that if sGC reacts with stoichiometric NO in the presence of GTP, the high cGMP-forming activity is restored (22).…”