Control of Nitric Oxide Dynamics by Guanylate Cyclase in Its Activated State

Abstract

are assumed to induce conformational changes, which are the origin of the catalytic activation. At the molecular level, the activation and deactivation mechanisms are unknown, as is the dynamics of NO once in the heme pocket. Using ultrafast time-resolved absorption spectroscopy, we measured the kinetics of NO rebinding to sGC after photodissociation. The main spectral transient in the Soret band does not match the equilibrium difference spectrum of NO-liganded minus unliganded sGC, and the geminate rebinding was found to be monoexponential and ultrafast ( ‫؍‬ 7.5 ps), with a relative amplitude close to unity (0.97). These characteristics, so far not observed in other hemoproteins, indicate that NO encounters a high energy barrier for escaping from the heme pocket once the His-Fe 2؉ bond has been cleaved; this bond does not reform before NO recombination. The deactivation of isolated sGC cannot occur by only simple diffusion of NO from the heme; therefore, several allosteric states may be inferred, including a desensitized one, to induce NO release. Thus, besides the structural change leading to activation, a consequence of the decoupling of the proximal histidine may also be to induce a change of the heme pocket distal geometry, which raises the energy barrier for NO escape, optimizing the efficiency of NO trapping. The nonsingle exponential character of the NO picosecond rebinding coexists only with the presence of the protein structure surrounding the heme, and the single exponential rate observed in sGC is very likely to be due to a closed conformation of the heme pocket. Our results emphasize the physiological importance of NO geminate recombination in hemoproteins like nitric-oxide synthase and sGC and show that the protein structure controls NO dynamics in a manner adapted to their function. This control of ligand dynamics provides a regulation at molecular level in the function of these enzymes.Guanylate cyclase is involved in signal transduction pathways in which cGMP 1 acts as a second messenger in several types of cells (for reviews, see Refs. 1-4). In endothelial cells, sGC is activated by NO produced by the constitutive enzyme nitric-oxide synthase. The binding of NO induces a several hundred-fold increase of the sGC catalytic activity, leading to cGMP production from GTP. This heterodimeric enzyme possesses a heme in the ␤-subunit where NO binding takes place, while the ␣-subunit harbors the catalytic site, which binds the precursor substrate GTP. The second messenger cGMP then triggers the downstream events leading to smooth muscle relaxation.Regarding diatomic ligand binding, sGC differs from other hemoproteins like myoglobin or hemoglobin; while sGC displays a very high affinity for NO, its affinity for O 2 is very low, but the structural features giving rise to this property are unknown. As in Hb and Mb, the heme iron of sGC is bound to a His side chain (5, 6) but much more weakly. Upon binding of NO to the heme, the His-Fe 2ϩ bond is cleaved, as reflected by the change in Soret absorption posi...

Effects of Distal Pocket Mutations on the Geminate Recombination of NO with Leghemoglobin on the Picosecond Time Scale

“…It is remarkable that for H61G, H61V, and H61L the rebinding is more than 80% complete within 80 ps after NO photolysis. Such efficient geminate recombination is comparable to that of protoheme-NO in ethylene glycol water, 8 myoglobin at pH 4, 25 and soluble guanylate cyclase, 21 which afford the most rapid and complete rebinding of which we are aware. Tables 1-3 summarize these geminate and bimolecular rebinding parameters.…”

“…Such an analysis has been applied on several occasions to geminate rebinding of NO. 17,21,30 This method obtains the rate distributions from the kinetic curves by using the maximum entropy method (MEM). In this approach, an entropy function is maximized and a distribution of probabilities of the underlying rate components is obtained.…”

“…Négrerie et al 21 have studied NO geminate recombination in Mb in which the proximal histidine is mutated to glycine, thus furnishing a protein without covalent attachment to the heme iron. At sufficiently high imidazole concentration, the H93G mutant coordinates an imidazole at the proximal position of the heme, which is denoted as H93G(Im).…”

“…Négrerie et al 21 have recently studied soluble guanylate cyclase (sGC), in which the binding of NO to the heme iron induces a cleavage of the bond between the proximal histidine and the iron. sGC-NO is an example of a heme protein where there is no covalent attachment between the Fe and the proximal side of the protein, and its kinetics may thus be compared against other systems in evaluating distal and proximal effects on geminate rebinding.…”

Ligand Dynamics in an Electron Transfer Protein

“…For dissociated NO, which has a very high intrinsic affinity to heme, there is no effective competing pathway for rebinding to the heme. Indeed the 7-ps time constant of the unique phase of NO rebinding in the M80A mutant corresponds to the fastest phase of NO rebinding observed in many other heme proteins, including Mb (22,45,46), the NO sensor guanylate cyclase (47), and the sensor protein EcDos (41) (where external ligands replace an internal methionine heme ligand), and presumably corresponds to the intrinsic activationless rate of NO binding (48).…”