BAY 41-2272 activates two isoforms of nitric oxide-sensitive guanylyl cyclase

Soluble guanylyl cyclase is an important target for endogenous nitric oxide and the guanylyl cyclase modulator, YC-1. Recently BAY 41-2272 was identified as a similar but more potent and more specific substance. While YC-1 also acts as non-specific phosphodiesterase inhibitor, BAY 41-2272 is devoid of an effect on phosphodiesterases. BAY 41-2272 has so far only been tested on the alpha(1)/beta(1) heterodimeric isoform of soluble guanylyl cyclase and its binding site has been mapped to a region in the alpha(1) subunit amino-terminal sequence. Although this region is poorly conserved in the alpha(2) subunit, we show in the current study that the alpha(2)/beta(1) heterodimeric enzyme isoform is activated by BAY 41-2272. Deletion analysis of the alpha(2) subunit and co-expression with the beta(1) subunit in the baculovirus/Sf9 system is consistent with the amino-terminal amino acids 104 to 401 of the alpha(2) subunit as binding site for BAY 41-2272.     

Nitric oxide activates the beta 2 subunit of soluble guanylyl cyclase in the absence of a second subunit.

Previously characterized mammalian soluble guanylyl cyclases form alpha/beta heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named alpha(1), beta(1), alpha(2), and beta(2). The alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzyme isoforms have been rigorously characterized. The role of the beta(2) subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the beta(2) subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the beta(3) subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mm free Mn(2+). The EC(50) values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the alpha(1)/beta(1) heterodimeric form. In the presence of the detergent Tween, NO sensitivity of beta(2) was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the beta(2) subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.     

Inhibition of deactivation of NO-sensitive guanylyl cyclase accounts for the sensitizing effect of YC-1

Many of the physiological effects of the signaling molecule nitric oxide are mediated by the stimulation of the NO-sensitive guanylyl cyclase. Activation of the enzyme is achieved by binding of NO to the prosthetic heme group of the enzyme and the initiation of conformational changes. So far, the rate of NO dissociation of the purified enzyme has only been determined spectrophotometrically, whereas the respective deactivation, i.e. the decline in enzymatic activity, has only been determined in cytosolic fractions and intact cells. Here, we report on the deactivation of purified NO-sensitive guanylyl cyclase determined after addition of the NO scavenger oxyhemoglobin or dilution. The deactivation rate corresponded to a half-life of the NO/guanylyl cyclase complex of approximately 4 s, which is in good agreement with the spectrophotometrically measured NO dissociation rate of the enzyme. The deactivation rate of the enzyme determined in platelets yielded a much shorter half-life indicating either partial damage of the enzyme during the purification procedure or the existence of endogenous deactivation accelerating factors. YC-1, a component causing sensitization of guanylyl cyclase toward NO, inhibited deactivation of guanylyl cyclase, resulting in an extremely prolonged half-life of the NO/guanylyl cyclase complex of more than 10 min. The deactivation of an ATP-utilizing guanylyl cyclase mutant was almost unaffected by YC-1, indicating the existence of a special structure within the catalytic domain required for YC-1 binding or for the transduction of the YC-1 effect. In contrast to the wild type enzyme, YC-1 did not increase NO sensitivity of this mutant, clearly establishing inhibition of deactivation as the underlying mechanism of the NO sensitizer YC-1.     

Studying the structure and regulation of soluble guanylyl cyclase

Soluble guanylyl cyclase acts as the receptor for the signaling molecule nitric oxide. The enzyme consists of two different subunits. Each subunit shows the cyclase catalytic domain, which is also conserved in the membrane-bound guanylyl cyclases and the adenylyl cyclases. The N-terminal regions of the subunits are responsible for binding of the prosthetic heme group of the enzyme, which is required for the stimulatory effect of nitric oxide (NO). The five-coordinated ferrous heme displays a histidine as the axial ligand; activation of soluble guanylyl cyclase by NO is initiated by binding of NO to the heme iron and proceeds via breaking of the histidine-to-iron bond. Recently, a novel pharmacological and possibly physiological principle of guanylyl cyclase sensitization was demonstrated. The substance YC-1 has been shown to activate the enzyme independent of NO, to potentiate the effect of submaximally effective NO concentrations, and to turn carbon monoxide into an effective activator of soluble guanylyl cyclase.     

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.     

Guanylyl cyclase: NO hits its target

The NO receptor, NO-sensitive guanylyl cyclase, plays a key role in the NO/cGMP signal-transduction cascade. Two isoforms of the enzyme are currently known, the widely distributed vascular alpha1beta1 isoform and the neuronal alpha2beta1 isoform predominantly expressed in brain. Interaction with the PSD-95 (postsynaptic density protein-95) family of scaffolding proteins targets the neuronal alpha2beta1 isoform to synaptic membranes. The NO sensor of the guanylyl cyclase is formed by the prosthetic haem group, where NO binding takes place and induces the up to 200-fold activation of the enzyme. The haem group allows tight regulation of enzymic activity by NO and represents the most striking feature of the enzyme, as it differs in many aspects from the well-characterized haem groups of other haemoproteins. The new NO sensitizers such as YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole] affect activation by NO and CO by mechanisms that are currently subject to intense research.     

Nitric oxide-sensitive guanylyl cyclase: structure and regulation

By the formation of the second messenger cGMP, NO-sensitive guanylyl cyclase (GC) plays a key role within the NO/cGMP signaling cascade which participates in vascular regulation and neurotransmission. The enzyme contains a prosthetic heme group that acts as the acceptor site for NO. High affinity binding of NO to the heme moiety leads to an up to 200-fold activation of the enzyme. Unexpectedly, NO dissociates with a half-life of a few seconds which appears fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogs act as NO sensitizers and led to the discovery of a novel pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme. The two isoforms of the heterodimeric enzyme (alpha1beta1, alpha2beta1) are known that are functionally indistinguishable. The alpha2beta1-isoform mainly occurs in brain whereas the alpha1beta1-enzyme shows a broader distribution and represents the predominantly expressed form of NO-sensitive GC. Until recently, the enzyme has been thought to occur in the cytosol. However, latest evidence suggests that the alpha2-subunit mediates the membrane association of the alpha2beta1-isoform via interaction with a PDZ domain of the post-synaptic scaffold protein PSD-95. Binding to PSD-95 locates this isoform in close proximity to the NO-generating synthases thereby enabling the NO sensor to respond to locally elevated NO concentrations. In sum, the two known isoforms may stand for the neuronal and vascular form of NO-sensitive GC reflecting a possible association to the neuronal and endothelial NO-synthase, respectively.     

Dimerization of nitric oxide-sensitive guanylyl cyclase requires the alpha 1 N terminus

The enzyme nitric oxide-sensitive guanylyl cyclase is an obligate heterodimer consisting of an alpha and a beta subunit. Whereas the C-terminal parts of the subunits have been shown to be sufficient for catalysis, regulation was assigned to the N termini. The central domains have been postulated to be responsible for the formation of alphabeta heterodimers. Here, we have analyzed dimerization by precipitation of various N- and C-terminally truncated alpha(1) mutants with beta(1) wild type or deletion mutants thereof after coexpression in the baculovirus/Sf9 system. In contrast to the current hypothesis, our analysis revealed that an N-terminal region of the alpha(1) subunit (amino acids 61-128) is mandatory for quantitative dimerization. The central domain (amino acids 367-462) contributes but is not sufficient to mediate robust alphabeta interaction. Wild type-like binding of the identified minimum dimerization region of alpha(1) (amino acids 61-462) requires the N-terminal and central region of beta(1) (amino acids 1-385). Furthermore, we observed an unequal stability of the alpha(1) and beta(1) subunit. Whereas beta(1) forms heme containing homodimers and is stable, alpha(1) appears to be prone to misfolding and degradation when heterodimerization is impaired by deletion of important sequences.     

Post-transcriptional regulation of soluble guanylyl cyclase expression in rat aorta

We investigated the molecular mechanism of cyclic GMP-induced down-regulation of soluble guanylyl cyclase expression in rat aorta. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1), an allosteric activator of this enzyme, decreased the expression of soluble guanylyl cyclase alpha(1) subunit mRNA and protein. This effect was blocked by the enzyme inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b-1,4)oxazin-1-one (NS2028) and by actinomycin D. Guanylyl cyclase alpha(1) mRNA-degrading activity was increased in protein extracts from YC-1-exposed aorta and was attenuated by pretreatment with actinomycin D and NS2028. Gelshift and supershift analyses using an adenylate-uridylate-rich ribonucleotide from the 3'-untranslated region of the alpha(1) mRNA and a monoclonal antibody directed against the mRNA-stabilizing protein HuR revealed HuR mRNA binding activity in aortic extracts, which was absent in extracts from YC-1-stimulated aortas. YC-1 decreased the expression of HuR, and this decrease was prevented by NS2028. Similarly, down-regulation of HuR by RNA interference in cultured rat aortic smooth muscle cells decreased alpha(1) mRNA and protein expression. We conclude that HuR protects the guanylyl cyclase alpha(1) mRNA by binding to the 3'-untranslated region. Activation of guanylyl cyclase decreases HuR expression, inducing a rapid degradation of guanylyl cyclase alpha(1) mRNA and lowering alpha(1) subunit expression as a negative feedback response.     

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.     

A-350619: a novel activator of soluble guanylyl cyclase

Nitric oxide (NO) is a key mediator in many physiological processes and one of the major receptors through which NO exerts its effects is soluble guanylyl cyclase. Guanylyl cyclase converts GTP to cyclic GMP as part of the cascade that results in physiological processes such as smooth muscle relaxation, neurotransmission, inhibition of platelet aggregation and immune response. The properties of A-350619, a novel soluble guanylyl cyclase activator, were examined to determine the modulatory effect on the catalytic properties of soluble guanylyl cyclase. A-350619 increased V(max) from 0.1 to 14.5 micromol/min/mg (145 fold increase), and lowered K(m) from 300 to 50 microM (6 fold decrease). When YC-1 (another sGC activator) and A-350619 were combined, a 156 fold increase in V(max) and a 5 fold decrease in Km were observed, indicating that the modulation of the enzyme brought about by YC-1 and A-350619 are not additive, suggesting a common binding site. Activation of soluble guanylyl cyclase by A-350619 was partially inhibited by ODQ, a specific inhibitor of soluble guanylyl cyclase by oxidation of the enzyme heme. YC-1 and A-350619 after pre-treatment with N-omega-nitro-L-arginine, an NO-synthase inhibitor, relaxed cavernosum tissue strips in a dose-dependent manner with EC(50) of 50 microM and 80 microM, respectively. Addition of SNP potentiated the relaxation effect of YC-1 and A-350619, shifting the dose-response curve to the left to 3 microM and 10 microM, respectively. Consistent with its biochemical activity, A-350619 (1 micromol/kg) alone induced penile erection in a conscious rat model. Activation of soluble guanylyl cyclase in cavernosum tissue as an alternate method of enhancing the effect of NO may provide a novel treatment of sexual dysfunction.     

Characterization of functional heme domains from soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, alpha1 and beta1. NO binds to the heme cofactor in the beta1 subunit, forming a five-coordinate NO complex that activates the enzyme several hundred-fold. In this paper, the heme domain has been localized to the N-terminal 194 residues of the beta1 subunit. This fragment represents the smallest construct of the beta1 subunit that retains the ligand-binding characteristics of the native enzyme, namely, tight affinity for NO and no observable binding of O(2). A functional heme domain from the rat beta2 subunit has been localized to the first 217 amino acids beta2(1-217). These proteins are approximately 40% identical to the rat beta1 heme domain and form five-coordinate, low-spin NO complexes and six-coordinate, low-spin CO complexes. Similar to sGC, these constructs have a weak Fe-His stretch [208 and 207 cm(-)(1) for beta1(1-194) and beta2(1-217), respectively]. beta2(1-217) forms a CO complex that is very similar to sGC and has a high nu(CO) stretching frequency at 1994 cm(-)(1). The autoxidation rate of beta1(1-194) was 0.073/min, while the beta2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003/min at 37 degrees C. This paper has identified and characterized the minimum functional ligand-binding heme domain derived from sGC, providing key details toward a comprehensive characterization.     

The alpha2beta1 isoform of guanylyl cyclase mediates plasma membrane localized nitric oxide signalling

Nitric oxide (NO) is a mediator of copious biological processes, in many cases through the production of cGMP from the enzyme nitric oxide-sensitive guanylyl cyclase. Natriuretic peptides also elevate cGMP, often with distinct biological effects, raising the issue of how specificity is achieved. Here we show that a recently described alpha(2)beta(1) isoform of guanylyl cyclase is expressed in a number of epithelia, where it is localized to the apical plasma membrane. We measured the functional properties of the alpha(2)beta(1) isoform by utilizing the NO-dependent activation of the ion channel cystic fibrosis transmembrane conductance regulator (CFTR), which occurs by phosphorylation via the membrane-bound type II isoform of cGMP-dependent protein kinase. We found that cGMP generated by NO activation of the alpha(2)beta(1) isoform of guanylyl cyclase is an exceptionally efficient mediator of nitric oxide action on membrane targets, activating CFTR far more effectively than the cytoplasmically located alpha(1)beta(1) guanylyl cyclase isoform. Targeting the alpha(1)beta(1) isoform of guanylyl cyclase to the membrane also dramatically enhanced the effects of nitric oxide on CFTR within the membrane. This was not due to increased enzymatic activity of guanylyl cyclase in a membrane location, but to production of a localised membrane pool of cGMP by membrane-localized NO-dependent guanylyl cyclase that was resistant to degradation by phosphodiesterases. Selective effects of cGMP produced from this enzyme in response to NO are directed at membrane targets and suggest that drugs selectively activating or inhibiting this alpha(2)beta(1) isoform of guanylyl cyclase may have unique pharmacological properties.     

Mapping of heme-binding domains in soluble guanylyl cyclase beta1 subunit.

Soluble guanylyl cyclase (sGC) is activated upon the interaction of NO with heme in the sGC beta1 subunit. To identify the domains contributing to heme-binding, we constructed a series of deletion mutants of the beta1 subunit, and evaluated their heme-binding capability. Deletion mutants consisting of residues 1-120 [beta1(1-120)] and 80-385 [beta1(80-385)] were the shortest mutants exhibiting heme binding among the C-terminal and N-terminal-truncated mutants, respectively. The region common to both beta1(1-120) and beta1(80-385), i.e., residues 80-120, is therefore essential for heme binding, although the residues 341-385 play an auxiliary role in heme binding. Two deletion mutants, beta1(80-195) and beta1(60-195), which include only the essential region, exhibited strong heme binding and spectral properties similar to those of the nitrosyl complex of native sGC. Thus, these heme-binding core proteins may serve as model proteins for future studies on the tertiary structure of the nitrosyl complex of sGC.     

A point-mutated guanylyl cyclase with features of the YC-1-stimulated enzyme: implications for the YC-1 binding site?

Guanylyl cyclases (GCs) and adenylyl cyclases (ACs) play key roles in various signaling cascades and are structurally closely related. The crystal structure of a soluble AC revealed one binding site each for the substrate ATP and the activator forskolin. Recently, YC-1, a novel activator of the heterodimeric soluble GC (sGC), has been identified which acts like forskolin on AC. Here, we investigated the respective substrate and potential activator domains of sGC using point-mutated subunits. Whereas substitution of the conserved Cys-541 of the beta(1) subunit with serine led to an almost complete loss of activity, mutation of the respective homologue (Cys-596) in the alpha(1) subunit yielded an enzyme with an increased catalytic rate and higher sensitivity toward NO. This phenotype exhibits characteristics similar to those of the YC-1-treated wild-type enzyme. Conceivably, this domain which corresponds to the forskolin site of the ACs may comprise the binding site for YC-1.     

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.     

Major occurrence of the new alpha2beta1 isoform of NO-sensitive guanylyl cyclase in brain

NO-sensitive guanylyl cyclase (GC) acts as the effector molecule for NO and therefore plays a key role in the NO/cGMP signalling cascade. Besides the long known GC isoform (alpha(1)beta(1)), another heterodimer (alpha(2)beta(1)) has recently been identified to be associated with PSD-95 in brain.Here, we report on the tissue distribution of all known guanylyl cyclase subunits to elucidate the isoform content in different tissues of the mouse. The guanylyl cyclase subunit levels were assessed with quantitative real-time PCR, and the most important results were verified in Western blots. We demonstrate the major occurrence of the alpha(2)beta(1) heterodimer in brain, find a significant amount in lung and lower amounts in all other tissues tested. In brain, the levels of the alpha(2)beta(1) and alpha(1)beta(1) isoforms were comparable; in all other tissues, the alpha(1)beta(1) heterodimer was the predominating isoform. The highest guanylyl cyclase content was found in lung; here the GC amounted to approximately twice as much as in brain.In sum, the major occurrence of the alpha(2)beta(1) heterodimer suggests a special role in synaptic transmission; whether this isoform outside the brain also occurs in neuronal networks has to be addressed in future studies.     

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.     

Elongation factor 2 as a target for selective inhibition of protein synthesis in vitro by the novel aromatic biasamidine

By the formation of cGMP the NO-sensitive guanylyl cyclase plays a key role within the NO/cGMP signaling cascade involved in vascular regulation and neurotransmission. The prosthetic heme group of the enzyme acts as the NO sensor, and binding of NO induces conformational changes leading to an up to 200-fold activation of the enzyme. The unexpected fast dissociation half-life of NO of a few seconds is fast enough to account for the deactivation of the enzyme in biological systems. YC-1 and its analogues acting as NO sensitizers uncovered a new pharmacologically and conceivably physiologically relevant regulatory principle of the enzyme.Two existing isoforms of the heterodimeric guanylyl cyclase (₁₁, ₂₁) are known that are functionally indistinguishable. Up to now, the NO-sensitive guanylyl cyclase has been considered as a soluble enzyme. However, recent evidence about the ₂₁ isoform interacting with a PDZ domain of the postsynaptic scaffold protein PSD-95 suggests that the ₂ subunit directs a membrane association of this isoform. The interaction with PSD-95 locates the ₂₁ isoform in close proximity to the NO-generating NO synthase thereby enabling the NO sensor to respond to locally raised NO concentrations.     

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.