Incorporation of Tyrosine and Glutamine Residues into the Soluble Guanylate Cyclase Heme Distal Pocket Alters NO and O2 Binding

Nitric oxide (NO) is the physiologically relevant activator of the mammalian hemoprotein soluble guanylate cyclase (sGC). The heme cofactor of α1β1 sGC has a high affinity for NO but has never been observed to form a complex with oxygen. Introduction of a key tyrosine residue in the sGC heme binding domain β1(1–385) is sufficient to produce an oxygen-binding protein, but this mutation in the full-length enzyme did not alter oxygen affinity. To evaluate ligand binding specificity in full-length sGC we mutated several conserved distal heme pocket residues (β1 Val-5, Phe-74, Ile-145, and Ile-149) to introduce a hydrogen bond donor in proximity to the heme ligand. We found that the NO coordination state, NO dissociation, and enzyme activation were significantly affected by the presence of a tyrosine in the distal heme pocket; however, the stability of the reduced porphyrin and the proteins affinity for oxygen were unaltered. Recently, an atypical sGC from Drosophila, Gyc-88E, was shown to form a stable complex with oxygen. Sequence analysis of this protein identified two residues in the predicted heme pocket (tyrosine and glutamine) that may function to stabilize oxygen binding in the atypical cyclase. The introduction of these residues into the rat β1 distal heme pocket (Ile-145 → Tyr and Ile-149 → Gln) resulted in an sGC construct that oxidized via an intermediate with an absorbance maximum at 417 nm. This absorbance maximum is consistent with globin Fe^II-O₂ complexes and is likely the first observation of a Fe^II-O₂ complex in the full-length α1β1 protein. Additionally, these data suggest that atypical sGCs stabilize O₂ binding by a hydrogen bonding network involving tyrosine and glutamine.     

A molecular basis for NO selectivity in soluble guanylate cyclase

Soluble guanylate cyclases (sGCs) function as heme sensors that selectively bind nitric oxide (NO), triggering reactions essential to animal physiology. Recent discoveries place sGCs in the H-NOX family (heme nitric oxide/oxygen-binding domain), which includes bacterial proteins from aerobic and anaerobic organisms. Some H-NOX proteins tightly bind oxygen (O2), whereas others show no measurable affinity for O2, providing the basis for selective NO signaling in aerobic cells. Using a series of wild-type and mutant H-NOXs, we established a molecular basis for ligand discrimination. A distal pocket tyrosine is requisite for O2 binding in the H-NOX family. These data suggest that sGC uses a kinetic selection against O2; we propose that the O2 dissociation rate in the absence of this tyrosine is fast and that a stable O2 complex does not form.     

Ligand discrimination in soluble guanylate cyclase and the H-NOX family of heme sensor proteins

Soluble guanylate cyclases (s GC s) are eukaryotic heme sensor proteins that selectively bind NO in the presence of a large excess of the similar diatomic gas, O(2); this discrimination is essential for NO signaling. Recent discoveries place sGC in the H-NOX (heme nitric oxide and/or oxygen binding domain) family that includes bacterial proteins. The defining characteristic of this family is that some H-NOX proteins tightly bind O(2) whereas others, such as sGC, show no measurable affinity for O(2). A molecular basis for this ligand selectivity has now been established. A distal pocket tyrosine is requisite for O(2) binding and is used to kinetically distinguish between NO and O(2). In the absence of this tyrosine, the O(2) dissociation rate is so fast that the O(2) complex is never formed, whereas the rate of NO dissociation remains essentially unchanged, thus providing discrimination.     

Instability in a coiled‐coil signaling helix is conserved for signal transduction in soluble guanylyl cyclase

How nitric oxide (NO) activates its primary receptor, α1/β1 soluble guanylyl cyclase (sGC or GC‐1), remains unknown. Likewise, how stimulatory compounds enhance sGC activity is poorly understood, hampering development of new treatments for cardiovascular disease. NO binding to ferrous heme near the N‐terminus in sGC activates cyclase activity near the C‐terminus, yielding cGMP production and physiological response. CO binding can also stimulate sGC, but only weakly in the absence of stimulatory small‐molecule compounds, which together lead to full activation. How ligand binding enhances catalysis, however, has yet to be discovered. Here, using a truncated version of sGC from Manduca sexta, we demonstrate that the central coiled‐coil domain, the most highly conserved region of the ~150,000 Da protein, not only provides stability to the heterodimer but is also conformationally active in signal transduction. Sequence conservation in the coiled coil includes the expected heptad‐repeating pattern for coiled‐coil motifs, but also invariant positions that disfavor coiled‐coil stability. Full‐length coiled coil dampens CO affinity for heme, while shortening of the coiled coil leads to enhanced CO binding. Introducing double mutation αE447L/βE377L, predicted to replace two destabilizing glutamates with leucines, lowers CO binding affinity while increasing overall protein stability. Likewise, introduction of a disulfide bond into the coiled coil results in reduced CO affinity. Taken together, we demonstrate that the heme domain is greatly influenced by coiled‐coil conformation, suggesting communication between heme and catalytic domains is through the coiled coil. Highly conserved structural imperfections in the coiled coil provide needed flexibility for signal transduction.     

Crystal structures of ferrous and CO-, CN(-)-, and NO-bound forms of rat heme oxygenase-1 (HO-1) in complex with heme: structural implications for discrimination between CO and O2 in HO-1

Heme oxygenase (HO) catalyzes heme degradation by utilizing O(2) and reducing equivalents to produce biliverdin IX alpha, iron, and CO. To avoid product inhibition, the heme[bond]HO complex (heme[bond]HO) is structured to markedly increase its affinity for O(2) while suppressing its affinity for CO. We determined the crystal structures of rat ferrous heme[bond]HO and heme[bond]HO bound to CO, CN(-), and NO at 2.3, 1.8, 2.0, and 1.7 A resolution, respectively. The heme pocket of ferrous heme-HO has the same conformation as that of the previously determined ferric form, but no ligand is visible on the distal side of the ferrous heme. Fe[bond]CO and Fe[bond]CN(-) are tilted, whereas the Fe[bond]NO is bent. The structure of heme[bond]HO bound to NO is identical to that bound to N(3)(-), which is also bent as in the case of O(2). Notably, in the CO- and CN(-)-bound forms, the heme and its ligands shift toward the alpha-meso carbon, and the distal F-helix shifts in the opposite direction. These shifts allow CO or CN(-) to bind in a tilted fashion without a collision between the distal ligand and Gly139 O and cause disruption of one salt bridge between the heme and basic residue. The structural identity of the ferrous and ferric states of heme[bond]HO indicates that these shifts are not produced on reduction of heme iron. Neither such conformational changes nor a heme shift occurs on NO or N(3)(-) binding. Heme[bond]HO therefore recognizes CO and O(2) by their binding geometries. The marked reduction in the ratio of affinities of CO to O(2) for heme[bond]HO achieved by an increase in O(2) affinity [Migita, C. T., Matera, K. M., Ikeda-Saito, M., Olson, J. S., Fujii, H., Yoshimura, T., Zhou, H., and Yoshida, T. (1998) J. Biol. Chem. 273, 945-949] is explained by hydrogen bonding and polar interactions that are favorable for O(2) binding, as well as by characteristic structural changes in the CO-bound form.     

Identification of residues crucially involved in the binding of the heme moiety of soluble guanylate cyclase

Soluble guanylate cyclase (sGC), a heterodimeric hemeprotein, is the only receptor for the biological messenger nitric oxide (NO) identified to date and is intimately involved in various signal transduction pathways. By using the recently discovered NO- and heme-independent sGC activator BAY 58-2667 and a novel cGMP reporter cell, we could distinguish between heme-containing and heme-free sGC in an intact cellular system. Using these novel tools, we identified the invariant amino acids tyrosine 135 and arginine 139 of the beta(1)-subunit as crucially important for both the binding of the heme moiety and the activation of sGC by BAY 58-2667. The heme is displaced by BAY 58-2667 due to a competition between the carboxylic groups of this compound and the heme propionic acids for the identified residues tyrosine 135 and arginine 139. This displacement results in the release of the axial heme ligand histidine 105 and to the observed activation of sGC. Based on these findings we postulate a signal transmission triad composed of histidine 105, tyrosine 135, and arginine 139 responsible for the enzyme activation by this compound and probably also for transducing changes in heme status and porphyrin geometry upon NO binding into alterations of sGC catalytic activity.     

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.     

Unprecedented proximal binding of nitric oxide to heme: implications for guanylate cyclase

Microbial cytochromes c' contain a 5-coordinate His-ligated heme that forms stable adducts with nitric oxide (NO) and carbon monoxide (CO), but not with dioxygen. We report the 1.95 and 1.35 A resolution crystal structures of the CO- and NO-bound forms of the reduced protein from Alcaligenes xylosoxidans. NO disrupts the His-Fe bond and binds in a novel mode to the proximal face of the heme, giving a 5-coordinate species. In contrast, CO binds 6-coordinate on the distal side. A second CO molecule, not bound to the heme, is located in the proximal pocket. Since the unusual spectroscopic properties of cytochromes c' are shared by soluble guanylate cyclase (sGC), our findings have potential implications for the activation of sGC induced by the binding of NO or CO to the heme domain.     

Theoretical investigation on the diatomic ligand migration process and ligand binding properties in non‐O2‐binding H‐NOX domain

The Nostoc sp (Ns) H‐NOX (heme‐nitric oxide or OXygen‐binding) domain shares 35% sequence identity with soluble guanylate cyclase (sGC) and exhibits similar ligand binding property with the sGC. Previously, our molecular dynamic (MD) simulation work identified that there exists a Y‐shaped tunnel system hosted in the Ns H‐NOX interior, which servers for ligand migration. The tunnels were then confirmed by Winter et al. [PNAS 2011;108(43):E 881–889] recently using x‐ray crystallography with xenon pressured conditions. In this work, to further investigate how the protein matrix of Ns H‐NOX modulates the ligand migration process and how the distal residue composition affects the ligand binding prosperities, the free energy profiles for nitric oxide (NO), carbon monooxide (CO), and O₂ migration are explored using the steered MDs simulation and the ligand binding energies are calculated using QM/MM schemes. The potential of mean force profiles suggest that the longer branch of the tunnel would be the most favorable route for NO migration and a second NO trapping site other than the distal heme pocket along this route in the Ns H‐NOX was identified. On the contrary, CO and O₂ would prefer to diffuse via the shorter branch of the tunnel. The QM/MM (quantum mechanics/molecular mechanics) calculations suggest that the hydrophobic distal pocket of Ns H‐NOX would provide an approximately vacuum environment and the ligand discrimination would be determined by the intrinsic binding properties of the diatomic gas ligand to the heme group. Proteins 2013; 81:1363–1376. © 2013 Wiley Periodicals, Inc.     

Preparation of heme-free soluble guanylate cyclase

Soluble guanylate cyclase (sGC), a heterodimer consisting of alpha- and beta-subunit, is the key enzyme of the NO/cGMP signaling pathway. The heme moiety ligated to the beta-subunit via His(105) is crucial for the activation of the enzyme by NO. In addition to this NO binding capability, the heme status of the enzyme influences the activity of non-NO sGC activators and sGC inhibitors. Different sGC activity profiles were observed in the presence, absence, or the oxidized form of heme. Modulating the heme status is therefore crucial for the investigation of the mechanism of sGC activation. Here, we present a simple and reliable procedure for the removal of the heme moiety of sGC that is capable of eliminating any traces of unbound heme and detergent from the sample mixture in one single step. Samples containing 15 microg sGC and the non-ionic detergent Tween 20 (2%) were incubated at 37 degrees C for 10 min and loaded onto centrifugal ion exchange columns. After centrifugation, heme was bound entirely to the ion exchanger and could not be eluted, even after incubation with 1M NaCl. Tween 20 was found completely within the flowthrough. Heme-free sGC was eluted from the ion exchanger after application of 300 mM NaCl. The absence of the heme moiety was confirmed by UV/Vis spectra and determination of the enzymatic activity. In summary, the described procedure is suitable for the preparation of very small amounts of highly purified heme-free sGC for the investigation of the mechanism of action of different types of sGC activators.     

A comparative resonance Raman analysis of heme-binding PAS domains: heme iron coordination structures of the BjFixL, AxPDEA1, EcDos, and MtDos proteins

The heme-PAS is a specialized domain with which a broad class of signal-transducing heme proteins detect physiological heme ligands. Such domains exhibit a wide range of ligand binding parameters, yet they are all expected to feature an alpha-beta heme binding fold and a predominantly hydrophobic heme distal pocket without a distal histidine. We have compared, for the first time, the resonance Raman spectra of several heme-PASs: the heme-binding domains of Bradyrhizobium japonicum FixL, Escherichia coli Dos, Acetobacter xylinum PDEA1, and Methanobacterium thermoautotrophicum Dos. In all cases, the nu(Fe)-(CO) and nu(C-O) values of the carbonmonoxy forms were consistent with coordination of the heme iron to histidine on the proximal side and binding of the CO without electrostatic interaction with the heme distal pocket. EcDos was unusual in having predominantly hexacoordinate heme iron in the deoxy and met forms. Despite an evident lack of CO interaction with the EcDos heme pocket, relatively low Fe-O(2) (562 cm(-1)) and N-O (1576 cm(-1)) stretching frequencies indicated that strong polar interactions with that heme distal pocket are possible for highly bent ligands such as O(2) or NO. None of the newly studied NO adducts exhibited evidence of the Fe-His rupture and pentacoordination previously noted for Sinorhizobium meliloti FixL. A low Fe-His stretching frequency, formerly interpreted as a strained Fe-His bond, and the slow association of O(2) with S. meliloti FixL failed to correlate with the newly studied proteins having low association rate or low equilibrium association constants for binding of O(2). We conclude that although heme-PASs share some features, they represent distinct signal transduction mechanisms.     

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.     

Structural bases for heme binding and diatomic ligand recognition in truncated hemoglobins

Truncated hemoglobins (trHbs) are low-molecular-weight oxygen-binding heme-proteins distributed in eubacteria, cyanobacteria, unicellular eukaryotes, and in higher plants, constituting a distinct group within the hemoglobin (Hb) superfamily. TrHbs display amino acid sequences 20-40 residues shorter than classical (non)vertebrate Hbs and myoglobins, to which they are scarcely related by sequence similarity. The trHb tertiary structure is based on a 2-on-2 alpha-helical sandwich, which represents a striking editing of the highly conserved 3-on-3 alpha-helical globin fold, achieved through deletion/truncation of alpha-helices and specific residue substitutions. Despite their 'minimal' polypeptide chain span, trHbs display an inner tunnel/cavity system held to support ligand diffusion to/from the heme distal pocket, accumulation of heme ligands within the protein matrix, and/or multiligand reactions. Moreover, trHbs bind and effectively stabilize the heme and recognize diatomic ligands (i.e., O2, CO, NO, and cyanide), albeit with varying thermodynamic and kinetic parameters. Here, structural bases for heme binding and diatomic ligand recognition by trHbs are reviewed.     

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.     

Rearrangement of the distal pocket accompanying E7 His----Gln substitution in elephant carbonmonoxy- and oxymyoglobin: 1H NMR identification of a new aromatic residue in the heme pocket

Two-dimensional 1H NMR methods have been used to assign side-chain resonances for the residues in the distal heme pocket of elephant carbonmonoxymyoglobin (MbCO) and oxymyoglobin (MbO2). It is shown that, while the other residues in the heme pocket are minimally perturbed, the Phe CD4 residue in elephant MbCO and MbO2 resonates considerably upfield compared to the corresponding residue in sperm whale MbCO. The new NOE connectivities to Val E11 and heme-induced ring current calculations indicate that Phe CD4 has been inserted into the distal heme pocket by reorienting the aromatic side chain and moving the CD corner closer to the heme. The C zeta H proton of the Phe CD4 was found to move toward the iron of the heme by approximately 4 A relative to the position of sperm whale MbCO, requiring minimally a 3-A movement of the CD helical backbone. The significantly altered distal conformation in elephant myoglobin, rather than the single distal E7 substitution, forms a plausible basis for its altered functional properties of lower autoxidation rate, higher redox potential, and increased affinity for CO ligand. These results demonstrate that one-to-one interpretation of amino acid residue substitution (E7 His----Gln) is oversimplified and that conformational changes of substituted proteins which are not readily predicted have to be considered for interpretation of their functional properties.     

Mechanism of binding of NO to soluble guanylyl cyclase: implication for the second NO binding to the heme proximal site

Soluble guanylyl cyclase (sGC), the key enzyme for the formation of second messenger cyclic GMP, is an authentic sensor for nitric oxide (NO). Binding of NO to sGC leads to strong activation of the enzyme activity. Multiple molecules and steps of binding of NO to sGC have been implicated, but the target of the second NO and the detailed binding mechanism remain controversial. In this study, we used (15)NO and (14)NO and anaerobic sequential mixing-freeze-quench electron paramagnetic resonance to unambiguously confirm that the heme Fe is the target of the second NO. The linear dependence on NO concentration up to 600 s(-1) for the observed rate of the second step of NO binding not only indicates that the binding site of the second NO is different from that in the first step, i.e., the proximal site of the heme, but also supports a concerted mechanism in which the dissociation of the His105 proximal ligand occurs simultaneously with the binding of the second NO molecule. Computer modeling successfully predicts the kinetics of formation of a set of five-coordinate NO complexes with the ligand on either the distal or proximal site and supports the selective release of NO from the distal side of the transient bis-NO-sGC complex. Thus, as has been demonstrated with cytochrome c', a five-coordinate NO-sGC complex containing a proximal NO is formed after the binding of the second NO.     

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.     

NO and CO differentially activate soluble guanylyl cyclase via a heme pivot-bend mechanism

Diatomic ligand discrimination by soluble guanylyl cyclase (sGC) is paramount to cardiovascular homeostasis and neuronal signaling. Nitric oxide (NO) stimulates sGC activity 200-fold compared with only four-fold by carbon monoxide (CO). The molecular details of ligand discrimination and differential response to NO and CO are not well understood. These ligands are sensed by the heme domain of sGC, which belongs to the heme nitric oxide oxygen (H-NOX) domain family, also evolutionarily conserved in prokaryotes. Here we report crystal structures of the free, NO-bound, and CO-bound H-NOX domains of a cyanobacterial homolog. These structures and complementary mutational analysis in sGC reveal a molecular ruler mechanism that allows sGC to favor NO over CO while excluding oxygen, concomitant to signaling that exploits differential heme pivoting and heme bending. The heme thereby serves as a flexing wedge, allowing the N-terminal subdomain of H-NOX to shift concurrent with the transition of the six- to five-coordinated NO-bound state upon sGC activation. This transition can be modulated by mutations at sGC residues 74 and 145 and corresponding residues in the cyanobacterial H-NOX homolog.     

Resonance Raman studies of cytochrome c' support the binding of NO and CO to opposite sides of the heme: implications for ligand discrimination in heme-based sensors

Resonance Raman (RR) studies have been conducted on Alcaligenes xylosoxidans cytochrome c', a mono-His ligated hemoprotein which reversibly binds NO and CO but not O(2). Recent crystallographic characterization of this protein has revealed the first example of a hemoprotein which can utilize both sides of its heme (distal and proximal) for binding exogenous ligands to its Fe center. The present RR investigation of the Fe coordination and heme pocket environments of ferrous, carbonyl, and nitrosyl forms of cytochrome c' in solution fully supports the structures determined by X-ray crystallography and offers insights into mechanisms of ligand discrimination in heme-based sensors. Ferrous cytochrome c' reacts with CO to form a six-coordinate heme-CO complex, whereas reaction with NO results in cleavage of the proximal linkage to give a five-coordinate heme-NO adduct, despite the relatively high stretching frequency (231 cm(-1)) of the ferrous Fe-N(His) bond. RR spectra of the six-coordinate CO adduct indicate that CO binds to the Fe in a nonpolar environment in line with its location in the hydrophobic distal heme pocket. On the other hand, RR data for the five-coordinate NO adduct suggest a positively polarized environment for the NO ligand, consistent with its binding close to Arg 124 on the opposite (proximal) side of the heme. Parallels between certain physicochemical properties of cytochrome c' and those of heme-based sensor proteins raise the possibility that the latter may also utilize both sides of their hemes to discriminate between NO and CO binding.