Nitric oxide activation of soluble guanylyl cyclase reveals high and low affinity sites that mediate allosteric inhibition by calcium

Cyclic GMP (cGMP) and Ca(2+) regulate opposing mechanisms in (patho)physiological processes reflected in the reciprocal regulation of their intracellular concentrations. Although mechanisms by which cGMP regulates [Ca(2+)](i) have been described, those by which Ca(2+) regulates [cGMP](i) are less well understood. In the present study, Ca(2+) inhibited purified sGC activated by sodium nitroprusside (SNP), a precursor of nitric oxide (NO), employing Mg-GTP as substrate in a concentration-dependent fashion, but was without effect on basal enzyme activity. Ca(2+) inhibited sGC stimulated by protoporphyrin IX or YC-1 suggesting that inhibition was not NO-dependent. In contrast, Ca(2+) was without effect on sGC activated by SNP employing Mn-GTP as substrate, demonstrating that inhibition did not reflect displacement of heme from sGC. Ligand activation of sGC unmasked negative allosteric sites of high (K(i) similar 10(-7) M) and low (K(i) approximately 10(-5) M) affinity for Ca(2+) that mediated noncompetitive and uncompetitive inhibition, respectively. Free Mg(2+) in excess of substrate did not alter the concentration-response relationship of Ca(2+) inhibition at high affinity sites, but produced a rightward shift in that relationship at low affinity sites. Similarly, Ca(2+) inhibition at high affinity sites was noncompetitive, whereas inhibition at low affinity sites was competitive, with respect to free Mg(2+). Purified sGC specifically bound (45)Ca(2+) in the presence of a 1000-fold excess of Mg(2+) and in the absence of activating ligands. These data suggest that sGC is a constitutive Ca(2+) binding protein whose allosteric function is conditionally dependent upon ligand activation.     

Functional characterization of nitric oxide and YC-1 activation of soluble guanylyl cyclase: structural implication for the YC-1 binding site?

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme formed by an alpha subunit and a beta subunit, the latter containing the heme where nitric oxide (NO) binds. When NO binds, the basal activity of sGC is increased several hundred fold. sGC activity is also increased by YC-1, a benzylindazole allosteric activator. In the presence of NO, YC-1 synergistically increases the catalytic activity of sGC by enhancing the affinity of NO for the heme. The site of interaction of YC-1 with sGC is unknown. We conducted a mutational analysis to identify the binding site and to determine what residues were involved in the propagation of NO and/or YC-1 activation. Because guanylyl cyclases (GCs) and adenylyl cyclases (ACs) are homologous, we used the three-dimensional structure of AC to guide the mutagenesis. Biochemical analysis of purified mutants revealed that YC-1 increases the catalytic activity not only by increasing the NO affinity but also by increasing the efficacy of NO. Effects of YC-1 on NO affinity and efficacy were dissociated by single-point mutations implying that YC-1 has, at least, two types of interaction with sGC. A structural model predicts that YC-1 may adopt two configurations in one site that is pseudosymmetric with the GTP binding site and equivalent to the forskolin site in AC.     

Allostery in recombinant soluble guanylyl cyclase from Manduca sexta

Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric full-length and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO off-rates for the approximately 100-kDa N-terminal heterodimeric fragment and increases the CO affinity by approximately 50-fold to 1.7 microm. Binding of NO leads to a transient six-coordinate intermediate, followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex (k(6-5) = 12.8 s(-1)). The conversion rate is insensitive to nucleotides, YC-1, and changes in NO concentration up to approximately 30 microm. NO release is biphasic in the absence of YC-1 (k(off1) = 0.10 s(-1) and k(off2) = 0.0015 s(-1)); binding of YC-1 eliminates the fast phase but has little effect on the slower phase. Our data are consistent with a model for allosteric activation in which sGC undergoes a simple switch between two conformations, with an open or a closed heme pocket, integrating the influence of numerous effectors to give the final catalytic rate. Importantly, YC-1 binding occurs in the N-terminal two-thirds of the protein. Homology modeling and mutagenesis experiments suggest the presence of an H-NOX domain in the alpha subunit with importance for heme binding.     

Potentiation of NO-dependent activation of soluble guanylate cyclase by 5-nitroisatin and the antiviral agent arbidol

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. The effect of isatin derivatives, 5-nitroisatin and arbidol (an antiviral agent) on spermine NONO-induced activation of human platelet soluble guanylate cyclase has been investigated. 5-Nitroisatin and arbidol had no effect on basal activity, but synergistically increased in a concentration-dependent manner the spermine NONO-induced activation of this enzyme. 5-Nitroisatin and arbidol, like YC-1, sensitized guanylate cyclase towards nitric oxide (NO) and produced a leftward shift of the spermine NONO concentration response curve. However, both compounds did not influence the activation of guanylate cyclase by YC-1 and did not change the synergistic increase of spermine NONO-induced activation of soluble guanylate cyclase in the presence of YC-1. This suggests that 5-nitroisanin and arbidol did not compete with YC-1. Addition of isatin did not change the synergistic increase in the spermine NONO-induced guanylate cyclase activation by 5-nitroisatin and arbidol and did not influence a leftward shift of the spermine NONO concentration response curve produced by these compounds. These data suggest lack of competitive interaction between isatin and both its derivatives used.     

Butyl isocyanide as a probe of the activation mechanism of soluble guanylate cyclase. Investigating the role of non-heme nitric oxide

Nitric oxide (NO) is a physiologically relevant activator of the hemoprotein soluble guanylate cyclase (sGC). In the presence of NO, sGC is activated several hundredfold above the basal level by a mechanism that remains to be elucidated. The heme ligand n-butyl isocyanide (BIC) was used to probe the mechanism of NO activation of sGC. Electronic absorption spectroscopy was used to show that BIC binds to the sGC heme, forming a 6-coordinate complex with an absorbance maximum at 429 nm. BIC activates sGC 2-5-fold, and synergizes with the allosteric activator YC-1, to activate the enzyme 15-25-fold. YC-1 activates the sGC-BIC complex, and leads to an increase in both the V(max) and K(m). BIC was also used to probe the mechanism of NO activation. The activity of the sGC-BIC complex increases 15-fold in the presence of NO, without displacing BIC at the heme, which is consistent with previous reports that proposed the involvement of a non-heme NO binding site in the activation process.     

Binding of YC-1/BAY 41-2272 to soluble guanylate cyclase: A new perspective to the mechanism of activation

Soluble guanylate cyclase (sGC), a heterodimeric heme protein, catalyses the conversion of GTP in to cyclic GMP, which acts as a second messenger in cellular signaling. Nitric oxide activates this enzyme several hundred folds over its basal level. Carbon monoxide, along with some activator molecules like YC-1 and BAY, also synergistically activate sGC. Mechanism of this synergistic activation is a matter of debate. Here we review the existing literature to identify the possible binding site for YC-1 and BAY on bovine lung sGC and its mechanism of activation. These two exogenous compounds bind sGC on α subunit inside a pocket and thus exert allosteric effect via subunit interface, which is relayed to the catalytic site. We used docking studies to further validate this hypothesis. We propose that the binding of YC-1/BAY inside the sensory domain of the α subunit modulates the interactions on the subunit interface resulting in rearrangements in the catalytic site into active conformation and this partly induces the cleavage of Fe-His bond.     

Interaction of soluble guanylate cyclase with YC-1: kinetic and resonance Raman studies

The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 microM YC-1, the V(max)/K(m GTP) increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm(-1). Similarly, YC-1 has no effect on the RR spectrum of ferrous beta1(1-385), the isolated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and beta1(1-385) in the presence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of beta1(1-385). The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket.     

Role of conformational changes in the heme-dependent regulation of human soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is a receptor for endogenous and exogenous nitric oxide (NO) and is activated many fold upon its binding, making it a core enzyme in the nitric oxide signal transduction pathway. Much effort has been made to understand the link between binding of NO at the sGC heme and activation of the cyclase activity. We report here the first direct evidence for the role of conformational changes in transmitting the signal between the heme and cyclase domains. Using both circular dichroism (CD) and fluorescence spectroscopies, we have probed the effect that the sGC activators NO and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole (YC-1) and the inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) have on the structure of the protein. Surprisingly, binding of either ODQ or YC-1 to NO-bound sGC cause virtually identical changes in the far-UV CD spectra of sGC, reflecting a perturbation in the secondary structure of the enzyme. This change is absent upon binding of NO, YC-1 or ODQ alone. Using this and previous data, we propose a working model for the mechanism of activation of sGC by NO and YC-1 and inhibition by ODQ.     

Characterization of two different five-coordinate soluble guanylate cyclase ferrous-nitrosyl complexes

Soluble guanylate cyclase (sGC), a hemoprotein, is the primary nitric oxide (NO) receptor in higher eukaryotes. The binding of NO to sGC leads to the formation of a five-coordinate ferrous-nitrosyl complex and a several hundred-fold increase in cGMP synthesis. NO activation of sGC is influenced by GTP and the allosteric activators YC-1 and BAY 41-2272. Electron paramagnetic resonance (EPR) spectroscopy shows that the spectrum of the sGC ferrous-nitrosyl complex shifts in the presence of YC-1, BAY 41-2272, or GTP in the presence of excess NO relative to the heme. These molecules shift the EPR signal from one characterized by g 1 = 2.083, g 2 = 2.036, and g 3 = 2.012 to a signal characterized by g 1 = 2.106, g 2 = 2.029, and g 3 = 2.010. The truncated heme domain constructs beta1(1-194) and beta2(1-217) were compared to the full-length enzyme. The EPR spectrum of the beta2(1-217)-NO complex is characterized by g 1 = 2.106, g 2 = 2.025, and g 3 = 2.010, indicating the protein is a good model for the sGC-NO complex in the presence of the activators, while the spectrum of the beta1(1-194)-NO complex resembles the EPR spectrum of sGC in the absence of the activators. Low-temperature resonance Raman spectra of the beta1(1-194)-NO and beta2(1-217)-NO complexes show that the Fe-NO stretching vibration of the beta2(1-217)-NO complex (535 cm (-1)) is significantly different from that of the beta1(1-194)-NO complex (527 cm (-1)). This shows that sGC can adopt different five-coordinate ferrous nitrosyl conformations and suggests that the Fe-NO conformation characterized by this unique EPR signal and Fe-NO stretching vibration represents a highly active sGC state.     

Nitric oxide. Potentiation of NO-dependent activation of soluble guanylate cyclase: (Patho)physiological and pharmacotherapeutic importance

The review highlights the molecular mechanism underlying the physiological effects of nitric oxide (NO), the role of signaling system: NO-soluble guanylate cyclase-cyclic 3′,5′-guanosine monophosphate (cGMP) in the realization of NO action. This review considers data on basic chemical characteristics of guanylate cyclase, such as the subunits structure, isoforms, modern concepts of the catalytic and regulatory centers of this enzyme. Realization of physiological effects of NO by guanylate cyclase depends on its heme prostetic group. NO-dependent activation of guanylate cyclase may be synergistically increased by a new NO-independent, allosteric activator of soluble guanylate cyclase-YC-1-(benzyl indasol derivative). Special attention is paid to the data on guanylate cyclase sites responcible for binding of the enzyme with YC-1 and the possible molecular mechanism underlying the synergistic increase of NO-dependent activation of soluble guanylate cyclase by YC-1. New compounds of endogenous nature capable to potentiate and synergistically increase the activation of guanylate cyclase by NO-donors have been found and investigated. The important physiological, pharmacotherapeutical and pathophysiological significance of this new fact is discussed.     

Identification of residues in the heme domain of soluble guanylyl cyclase that are important for basal and stimulated catalytic activity

Nitric oxide signals through activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. NO binds to the heme domain located in the N-terminal part of the β subunit of sGC resulting in increased production of cGMP in the catalytic domain located at the C-terminal part of sGC. Little is known about the mechanism by which the NO signaling is propagated from the receptor domain (heme domain) to the effector domain (catalytic domain), in particular events subsequent to the breakage of the bond between the heme iron and Histidine 105 (H105) of the β subunit. Our modeling of the heme-binding domain as well as previous homologous heme domain structures in different states point to two regions that could be critical for propagation of the NO activation signal. Structure-based mutational analysis of these regions revealed that residues T110 and R116 in the αF helix-β1 strand, and residues I41 and R40 in the αB-αC loop mediate propagation of activation between the heme domain and the catalytic domain. Biochemical analysis of these heme mutants allows refinement of the map of the residues that are critical for heme stability and propagation of the NO/YC-1 activation signal in sGC.     

Probing domain interactions in soluble guanylate cyclase

Eukaryotic nitric oxide (NO) signaling involves modulation of cyclic GMP (cGMP) levels through activation of the soluble isoform of guanylate cyclase (sGC). sGC is a heterodimeric hemoprotein that contains a Heme-Nitric oxide and OXygen binding (H-NOX) domain, a Per/ARNT/Sim (PAS) domain, a coiled-coil (CC) domain, and a catalytic domain. To evaluate the role of these domains in regulating the ligand binding properties of the heme cofactor of NO-sensitive sGC, we constructed chimeras by swapping the rat β1 H-NOX domain with the homologous region of H-NOX domain-containing proteins from Thermoanaerobacter tengcongensis, Vibrio cholerae, and Caenorhabditis elegans (TtTar4H, VCA0720, and Gcy-33, respectively). Characterization of ligand binding by electronic absorption and resonance Raman spectroscopy indicates that the other rat sGC domains influence the bacterial and worm H-NOX domains. Analysis of cGMP production in these proteins reveals that the chimeras containing bacterial H-NOX domains exhibit guanylate cyclase activity, but this activity is not influenced by gaseous ligand binding to the heme cofactor. The rat-worm chimera containing the atypical sGC Gcy-33 H-NOX domain was weakly activated by NO, CO, and O(2), suggesting that atypical guanylate cyclases and NO-sensitive guanylate cyclases have a common molecular mechanism for enzyme activation. To probe the influence of the other sGC domains on the mammalian sGC heme environment, we generated heme pocket mutants (Pro118Ala and Ile145Tyr) in the β1 H-NOX construct (residues 1-194), the β1 H-NOX-PAS-CC construct (residues 1-385), and the full-length α1β1 sGC heterodimer (β1 residues 1-619). Spectroscopic characterization of these proteins shows that interdomain communication modulates the coordination state of the heme-NO complex and the heme oxidation rate. Taken together, these findings have important implications for the allosteric mechanism of regulation within H-NOX domain-containing proteins.     

In vitro activation of soluble guanylyl cyclase and nitric oxide release: a comparison of NO donors and NO mimetics

Nitric oxide (NO) performs a central role in biological systems, binding to the heme site of soluble guanylyl cyclase (sGC), leading to enzyme activation and elevation of intracellular levels of cGMP. Organic nitrates, in particular, nitroglycerin (GTN), are clinically important nitrovasodilators that function as NO-mimetics in biological systems. Comparison of sGC activation data with electrochemically measured rates of NO release for genuine NO donors, NONOates and nitrosothiols, yields an excellent correlation between the EC(50) for sGC activation and the rate constant for NO release, k(NO). However, activation of sGC by GTN and the nitrates has very different characteristics, including the requirement for specific added thiols, for example, cysteine. The reaction of GTN with cysteine in anaerobic solution yields NO slowly, and NO release, measured by chemiluminescence detection, is quenched by added metal ion chelator. The generation of NO under aerobic conditions is 100-fold slower than the anaerobic reaction. Furthermore, NO release from the reaction of GTN with cysteine in phosphate buffer is too slow to account for sGC activation by GTN/cysteine. The slow rate of the chemical reaction to release NO suggests that nitrates can activate sGC by an NO-independent mechanism. In contrast to the genuine NO donors, GTN behaves as a partial agonist with respect to sGC activation, but in the presence of the allosteric sGC activator, YC-1, GTN exhibits full agonist activity.     

Interactions of soluble guanylate cyclase with diatomics as probed by resonance Raman spectroscopy

Soluble guanylate cyclase (sGC, EC 4.6.1.2) acts as a sensor for nitric oxide (NO), but is also activated by carbon monoxide in the presence of an allosteric modulator. Resonance Raman studies on the structure-function relations of sGC are reviewed with a focus on the CO-adduct in the presence and absence of allosteric modulator, YC-1, and substrate analogues. It is demonstrated that the sGC isolated from bovine lung contains one species with a five-coordinate (5c) ferrous high-spin heme with the Fe-His stretching mode at 204 cm(-1), but its CO adduct yields two species with different conformations about the heme pocket with the Fe-CO stretching (nuFe-CO) mode at 473 and 489 cm(-1), both of which are His- and CO-coordinated 6c ferrous adducts. Addition of YC-1 to it changes their population and further addition of GTP yields one kind of 6c (nuFe-CO=489 cm(-1)) in addition to 5c CO-adduct (nuFe-CO=521 cm(-1)). Under this condition the enzymatic activity becomes nearly the same level as that of NO adduct. Addition of gamma-S-GTP yields the same effect as GTP does but cGMP and GDP gives much less effects. Unexpectedly, ATP cancels the effects of GTP. The structural meaning of these spectroscopic observations is discussed in detail.     

CCTeta, a novel soluble guanylyl cyclase-interacting protein

Nitric oxide (NO) transduces most of its biological effects through activation of the heterodimeric enzyme, soluble guanylyl cyclase (sGC). Activation of sGC results in the production of cGMP from GTP. In this paper, we demonstrate a novel protein interaction between CCT (chaperonin containing t-complex polypeptide) subunit eta and the alpha1beta1 isoform of sGC. CCTeta was found to interact with the beta1 subunit of sGC via a yeast-two-hybrid screen. This interaction was then confirmed in vitro with a co-immunoprecipitation from mouse brain. The interaction between these two proteins was further supported by a co-localization of the proteins within rat brain. Using the yeast two-hybrid system, CCTeta was found to bind to the N-terminal portion of sGC. In vitro assays with purified CCTeta and Sf9 lysate expressing sGC resulted in a 30-50% inhibition of diethylamine diazeniumdiolate-NO-stimulated sGC activity. The same assays were then performed using BAY41-2272, an NO-independent allosteric sGC activator, and CCTeta had no effect on this activity. Furthermore, CCTeta had no effect on basal or sodium nitroprusside-stimulated alphabeta(Cys-105) sGC, a constitutively active mutant that only lacks the heme group. The N-terminal 94 amino acids of CCTeta seem to be critical for the mediation of this inhibition. Lastly, a 45% inhibition of sGC activity by CCTeta was seen in vivo in BE2 cells stably transfected with CCTeta and treated with sodium nitroprusside. These data suggest that CCTeta binds to sGC and, in cooperation with some other factor, inhibits its activity by modifying the binding of NO to the heme group or the subsequent conformational changes.     

Resonance Raman study on synergistic activation of soluble guanylate cyclase by imidazole, YC-1 and GTP

Soluble guanylate cyclase (sGC), a physiological nitric oxide (NO) receptor, is a heme-containing protein and catalyzes the conversion of GTP to cyclic GMP. We found that 200 mM imidazole moderately activated sGC in the coexistence with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), although imidazole or YC-1 alone had little effect for activation. GTP facilitated this process. Resonance Raman spectra of imidazole complex of native sGC and CO-bound sGC (CO-sGC) have demonstrated that a simple heme adduct with imidazole at the sixth coordination position is not present for both sGC and CO-sGC below 200 mM of the imidazole concentration and that the Fe-CO stretching band (nuFe-CO)) appears at 492 cm(-1) in the presence of imidazole compared with 473 cm(-1) in its absence. Both frequencies fall on the line of His-coordinated heme proteins in the nuFe-CO vs nuC-O plot. However, it is stressed that the CO-heme of sGC becomes apparently photo-inert in a spinning cell in the presence of imidazole, suggesting the formation of five-coordinate CO-heme or of six-coordinate heme with a very weak trans ligand. These observations suggest that imidazole alters not only the polarity of heme pocket but also the coordination structure at the fifth coordination side presumably by perturbing the heme-protein interactions at propionic side chains. Despite the fact that the isolated sGC stays in the reduced state and is not oxidized by O(2), sGC under the high concentration of imidazole (1.2 M) yielded nu4 at 1373 cm(-1) even after its removal by gel-filtration, but addition of dithionite gave the strong nu4 band at 1360 cm(-1). This indicated that imidazole caused autoxidation of sGC.     

Thrombospondin-1 and angiotensin II inhibit soluble guanylyl cyclase through an increase in intracellular calcium concentration

Nitric oxide (NO) regulates cardiovascular hemostasis by binding to soluble guanylyl cyclase (sGC), leading to cGMP production, reduced cytosolic calcium concentration ([Ca(2+)](i)), and vasorelaxation. Thrombospondin-1 (TSP-1), a secreted matricellular protein, was recently discovered to inhibit NO signaling and sGC activity. Inhibition of sGC requires binding to cell-surface receptor CD47. Here, we show that a TSP-1 C-terminal fragment (E3CaG1) readily inhibits sGC in Jurkat T cells and that inhibition requires an increase in [Ca(2+)](i). Using flow cytometry, we show that E3CaG1 binds directly to CD47 on the surface of Jurkat T cells. Using digital imaging microscopy on live cells, we further show that E3CaG1 binding results in a substantial increase in [Ca(2+)](i), up to 300 nM. Addition of angiotensin II, a potent vasoconstrictor known to increase [Ca(2+)](i), also strongly inhibits sGC activity. sGC isolated from calcium-treated cells or from cell-free lysates supplemented with Ca(2+) remains inhibited, while addition of kinase inhibitor staurosporine prevents inhibition, indicating inhibition is likely due to phosphorylation. Inhibition is through an increase in K(m) for GTP, which rises to 834 μM for the NO-stimulated protein, a 13-fold increase over the uninhibited protein. Compounds YC-1 and BAY 41-2272, allosteric stimulators of sGC that are of interest for treating hypertension, overcome E3CaG1-mediated inhibition of NO-ligated sGC. Taken together, these data suggest that sGC not only lowers [Ca(2+)](i) in response to NO, inducing vasodilation, but also is inhibited by high [Ca(2+)](i), providing a fine balance between signals for vasodilation and vasoconstriction.     

Control of nitric oxide dynamics by guanylate cyclase in its activated state

Soluble guanylate cyclase (sGC) is the target of nitric oxide (NO) released by nitric-oxide synthase in endothelial cells, inducing an increase of cGMP synthesis in response. This heterodimeric protein possesses a regulatory subunit carrying a heme where NO binding occurs, while the second subunit harbors the catalytic site. The binding of NO and the subsequent breaking of the bond between the proximal histidine and the heme-Fe(2+) 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 (tau = 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 non-single 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.     

A functional domain of the alpha1 subunit of soluble guanylyl cyclase is necessary for activation of the enzyme by nitric oxide and YC-1 but is not involved in heme binding

Soluble guanylyl cyclase is a heterodimeric enzyme consisting of an alpha(1) and a beta(1) subunit and is an important target for endogenous nitric oxide and the guanylyl cyclase modulator YC-1. The activation of the enzyme by both substances is dependent on the presence of a prosthetic heme group. It has been unclear whether this prosthetic heme group is sandwiched between the alpha(1) and beta(1) subunits or whether it exclusively binds to the beta(1) subunit. Here we analyze progressive amino-terminal deletion mutants of the human alpha(1) subunit after co-expression with the human beta(1) subunit in the baculovirus/Sf9 system. Spectral, biochemical, and pharmacological analysis shows that the first 259 amino acids of the alpha(1) subunit can be deleted without loss of sensitivity to nitric oxide (NO) or YC-1 or loss of heme binding of the respective enzyme complex with the beta(1) subunit. This is in contrast to previous data indicating that NO sensitivity and a functional heme binding site requires full-length amino termini of bovine alpha(1) and beta(1) subunits. Further deletion of the first 364 amino acids of the alpha(1) subunit leads to an enzyme complex with preserved heme binding but loss of sensitivity to NO or YC-1 despite induction of the typical spectral shift by NO binding to the prosthetic heme group. We conclude that 1) the amino-terminal part of the alpha(1) subunit is not involved in heme binding and 2) amino acids 259-364 of the alpha(1) subunit represent an important functional domain for the transduction of the NO activation signal and likely represent the target for NO-sensitizing substances like YC-1.