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.     

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.     

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.     

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.     

Structural and functional characterization of the dimerization region of soluble guanylyl cyclase

Soluble guanylyl cyclase (sGC) is a ubiquitous enzyme that functions as a receptor for nitric oxide. Despite the obligate heterodimeric nature of sGC, the sequence segments mediating subunit association have remained elusive. Our initial screening for relevant interaction site(s) in the most common sGC isoenzyme, alpha(1) beta(1), identified two regions in each subunit, i.e. the regulatory domains and the central regions, contributing to heterodimer formation. To map the relevant segments in the beta(1) subunit precisely, we constructed multiple N- and C-terminal deletion variants and cotransfected them with full-length alpha(1) in COS cells. Immunoprecipitation revealed that a sequence segment spanning positions 204-408 mediates binding of beta(1) to alpha(1) The same region of beta(1)[204-408] was found to promote beta /beta(1) homodimerization. Fusion of [204 beta(1)-408] to enhanced green fluorescent protein conferred binding activity to the recipient protein. Coexpression of beta(1)[204-408] with alpha(1) or beta(1) targeted the sGC subunits for proteasomal degradation, suggesting that beta(1)[204-408] forms structurally deficient complexes with alpha(1) and beta(1). Analysis of deletion constructs lacking portions of the beta(1) dimerization region identified two distinct segments contributing to alpha(1) binding, i.e. an N-terminal site covering positions 204-244 and a C-terminal site at 379-408. Both sites are crucial for sGC function because deletion of either site rendered sGC dimerization-deficient and thus functionally inactive. We conclude that the dimerization region of beta(1) extends over 205 residues of its regulatory and central domains and that two discontinuous sites of 41 and 30 residues, respectively, facilitate binding of beta(1) to the alpha(1) subunit of sGC.     

Inhibition of soluble guanylate cyclase by ODQ

The heme in soluble guanylate cyclases (sGC) as isolated is ferrous, high-spin, and 5-coordinate. [1H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one] (ODQ) has been used extensively as a specific inhibitor for sGC and as a diagnostic tool for identifying a role for sGC in signal transduction events. Addition of ODQ to ferrous sGC leads to a Soret shift from 431 to 392 nm and a decrease in nitric oxide (NO)-stimulated sGC activity. This Soret shift is consistent with oxidation of the ferrous heme to ferric heme. The results reported here further define the molecular mechanism of inhibition of sGC by ODQ. Addition of ODQ to the isolated sGC heme domain [beta1(1-385)] gave the same spectral changes as when sGC was treated with ODQ. EPR and resonance Raman spectroscopy was used to show that the heme in ODQ-treated beta1(1-385) is indeed ferric. Inhibition of the NO-stimulated sGC activity by ODQ is due to oxidation of the sGC heme and not to perturbation of the catalytic site, since the ODQ-treated sGC has the same basal activity as untreated sGC (68 +/- 12 nmol min(-)(1) mg(-)(1)). In addition, ODQ-oxidized sGC can be re-reduced by dithionite, and this re-reduced sGC has identical NO-stimulated activity as the original ferrous sGC. Oxidation of the sGC heme by ODQ is fast with a second-order rate constant of 8.5 x 10(3) M(-)(1) s(-)(1). ODQ can also oxidize hemoglobin, indicating that the reaction is not specific for the heme in sGC versus that in other hemoproteins.     

Ligand selectivity of soluble guanylyl cyclase: effect of the hydrogen-bonding tyrosine in the distal heme pocket on binding of oxygen, nitric oxide, and carbon monoxide

Although soluble guanylyl cyclase (sGC) functions in an environment in which O(2), NO, and CO are potential ligands for its heme moiety, the enzyme displays a high affinity for only its physiological ligand, NO, but has a limited affinity for CO and no affinity for O(2). Recent studies of a truncated version of the sGC beta(1)-subunit containing the heme-binding domain (Boon, E. M., Huang, S H., and Marletta, M. A. (2005) Nat. Chem. Biol., 1, 53-59) showed that introduction of the hydrogen-bonding tyrosine into the distal heme pocket changes the ligand specificity of the heme moiety and results in an oxygen-binding sGC. The hypothesis that the absence of hydrogen-bonding residues in the distal heme pocket is sufficient to provide oxygen discrimination by sGC was put forward. We tested this hypothesis in a context of a complete sGC heterodimer containing both the intact alpha(1)- and beta(1)-subunits. We found that the I145Y substitution in the full-length beta-subunit of the sGC heterodimer did not produce an oxygen-binding enzyme. However, this substitution impeded the association of NO and destabilized the NO.heme complex. The tyrosine in the distal heme pocket also impeded both the binding and dissociation of the CO ligand. We propose that the mechanism of oxygen exclusion by sGC not only involves the lack of hydrogen bonding in the distal heme pocket, but also depends on structural elements from other domains of sGC.     

Aspartate 102 in the heme domain of soluble guanylyl cyclase has a key role in NO activation

Nitric oxide (NO) is involved in the physiology and pathophysiology of the cardiovascular and neuronal systems via activation of soluble guanylyl cyclase (sGC), a heme-containing heterodimer. Recent structural studies have allowed a better understanding of the residues that dictate the affinity and binding of NO to the heme and the resulting breakage of the bond between the heme iron and histidine 105 (H105) of the β subunit of sGC. Still, it is unknown how the breakage of the iron-His bond translates into NO-dependent increased catalysis. Structural studies on homologous H-NOX domains in various states pointed to a role for movement of the H105 containing αF helix. Our modeling of the heme-binding domain highlighted conserved residues in the vicinity of H105 that could potentially regulate the extent to which the αF helix shifts and/or propagate the activation signal once the covalent bond with H105 has been broken. These include a direct interaction of αF helix residue aspartate 102 (D102) with the backbone nitrogen of F120. Mutational analysis of this region points to an essential role of the interactions in the vicinity of H105 for heme stability and identifies D102 as having a key role in NO activation following breakage of the iron-His bond.     

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.     

Identification of residues crucially involved in soluble guanylate cyclase activation

The ubiquitous heterodimeric nitric oxide (NO) receptor soluble guanylate cyclase (sGC) plays a key role in various signal transduction pathways. Binding of NO takes place at the prosthetic heme moiety at the N-terminus of the beta(1)-subunit of sGC. The induced structural changes lead to an activation of the catalytic C-terminal domain of the enzyme and to an increased conversion of GTP into the second messenger cyclic GMP (cGMP). In the present work we selected and substituted different residues of the sGC heme-binding pocket based on a sGC homology model. The generated sGC variants were tested in a cGMP reporter cell for their effect on the enzyme activation by heme-dependent (NO, BAY 41-2272) stimulators and heme-independent (BAY 58-2667) activators. The use of these experimental tools allows the enzyme's heme content to be explored in a non-invasive manner. Asp(44), Asp(45) and Phe(74) of the beta(1)-subunit were identified as being crucially important for functional enzyme activation. beta(1)Asp(45) may serve as a switch between different conformational states of sGC and point to a possible mechanism of action of the heme dependent sGC stimulator BAY 41-2272. Furthermore, our data shows that the activation profile of beta(1)IIe(145) Tyr is unchanged compared to the native enzyme, suggesting that Tyr(145) does not confer the ability to distinguish between NO and O(2). In summary, the present work further elucidated intramolecular mechanisms underlying the NO- and BAY 41-2272-mediated sGC activation and raises questions regarding the postulated role of Tyr(145) for ligand discrimination.     

A novel insight into the heme and NO/CO binding mechanism of the alpha subunit of human soluble guanylate cyclase

Human soluble guanylate cyclase (sGC), a critical heme-containing enzyme in the NO-signaling pathway of eukaryotes, is an αβ heterodimeric hemoprotein. Upon the binding of NO to the heme, sGC catalyzes the conversion of GTP to cyclic GMP, playing a crucial role in many physiological processes. However, the specific contribution of the α and β subunits of sGC in the intact heme binding remained intangible. The recombinant human sGC α1 subunit has been expressed in Escherichia coli and characterized for the first time. The heme binding and related NO/CO binding properties of both the α1 subunit and the β1 subunit were investigated via heme reconstitution, UV–vis spectroscopy, EPR spectroscopy, stopped-flow kinetics, and homology modeling. These results indicated that the α1 subunit of human sGC, lacking the conserved axial ligand, is likely to interact with heme noncovalently. On the basis of the equilibrium and kinetics of CO binding to sGC, one possible CO binding model was proposed. CO binds to human sGCβ195 by simple one-step binding, whereas CO binds to human sGCα259, possibly from both axial positions through a more complex process. The kinetics of NO dissociation from human sGC indicated that the NO dissociation from sGC was complex, with at least two release phases, and human sGCα259 has a smaller k ₁ but a larger k ₂. Additionally, the role of the cavity of the α1 subunit of human sGC was explored, and the results indicate that the cavity likely accommodates heme. These results are beneficial for understanding the overall structure of the heme binding site of the human sGC and the NO/CO signaling mechanism.     

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.     

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.     

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.     

Efficient expression of human soluble guanylate cyclase in Escherichia coli and its signaling-related interaction with nitric oxide

Soluble guanylate cyclase (sGC), as a nitric oxide (NO) sensor, is a critical heme-containing enzyme in NO-signaling pathway of eukaryotes. Human sGC is a heterodimeric hemoprotein, composed of a α-subunit (690 AA) and a heme-binding β-subunit (619 AA). Upon NO binding, sGC catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP). cGMP is a second messenger and initiates the nitric oxide signaling, triggering vasodilatation, smooth muscle relaxation, platelet aggregation, and neuronal transmission etc. The breakthrough of the bottle neck problem for sGC-mediated NO singling was made in this study. The recombinant human sGC β1 subunit (HsGCβ619) and its truncated N-terminal fragments (HsGCβ195 and HsGCβ384) were efficiently expressed in Escherichia coli and purified successfully in quantities. The three proteins in different forms (ferric, ferrous, NO-bound, CO-bound) were characterized by UV–vis and EPR spectroscopy. The homology structure model of the human sGC heme domain was constructed, and the mechanism for NO binding to sGC was proposed. The EPR spectra showed a characteristic of five-coordinated heme-nitrosyl species with triplet hyperfine splitting of NO. The interaction between NO and sGC was investigated and the schematic mechanism was proposed. This study provides new insights into the structure and NO-binding of human sGC. Furthermore, the efficient expression system of E. coli will be beneficial to the further studies on structure and activation mechanism of human sGC.     

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.     

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 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.     

Structure-function study of the amino-terminal stretch of the catalase subunit molecule in oligomerization,heme binding,and activity expression

Analysis of the protein structure of bovine liver catalase suggested that the N-terminal region containing two alpha-helices may function as a linker binding to another subunit. The number of amino-acid residues in catalase from the n-alkane-assimilating yeast Candida tropicalis (CTC) is the lowest of any eukaryotic catalase molecule hitherto investigated, and only one helix, corresponding to the helix alpha2 in bovine liver catalase, is estimated to be present in the same region. In the present study, N-terminal-deleted mutants of CTC were characterized to evaluate the role of the alpha-helix structure in the N-terminal region. CTCDelta1-4 and CTCDelta1-24, whose N-terminal regions were shortened by four and 24 amino-acid residues, respectively, showed an 80% decrease in specific activity compared to wild-type CTC in spite of containing the same amount of heme as in the wild-type. Polyacrylamide gel electrophoresis under nondenaturing conditions revealed that the mutants contained large amounts of oligomeric forms with molecular masses less than 220 kDa (tetramer assembly). Although the smaller oligomers were found to be bound with heme, only the tetramer exhibited catalase activity in activity staining on nondenaturing gel. CTCDelta1-49, a mutant with deletion of the N-terminal 49 amino-acid residues which contain the conserved helix alpha2, showed no catalase activity and no heme binding. However, the CD spectrum profiles of CTCDelta1-49, CTCDelta1-4, and CTCDelta1-24 indicated that these mutant subunits could attain secondary conformations similar to that of wild-type CTC, regardless of their binding with heme. From these results, it was concluded that the N-terminal stretch of catalase is significant for complete assembly into active tetramer and that the conserved helix alpha2, although it has little effect on the formation of the subunit secondary structure, is indispensable not only in assembling tetramer but also in binding heme.     

Structural and functional insights into the heme-binding domain of the human soluble guanylate cyclase α2 subunit and heterodimeric α2β1

Soluble guanylate cyclase (sGC) mediates NO signaling for a wide range of physiological effects in the cardiovascular system and the central nervous system. The α1β1 isoform is ubiquitously distributed in cytosolic fractions of tissues, whereas α2β1 is mainly found in the brain. The major occurrence and the unique characteristic of human sGC α2β1 indicate a special role in the mediation of neuronal communication. We have efficiently purified and characterized the recombinant heme-binding domain of the human sGC α2 subunit (hsGC α2(H)) and heterodimeric α2β1 (hsGC β1(H)-α2(H)) by UV-vis spectroscopy, circular dichrosim spectroscopy, EPR spectroscopy, and homology modeling. The heme dissociation and related NO/CO binding/dissociation of both hsGC α2(H) and hsGC β1(H)-α2(H) were investigated. The two truncated proteins interact with heme noncovalently. The CO binding affinity of hsGC α2(H) is threefold greater than that of human sGC α1(H), whereas the dissociation constant k (1) for dissociation of NO from hsGC α2(H) is sevenfold larger than that for dissociation of NO from hsGC α1(H), although k (2) is almost identical. The results indicate that in comparison with the α1β1 isoform, the brain α2β1 isoform exhibits a distinctly different CO/NO affinity and binding rate in favor of NO signaling, and this is consistent with its physiological role in the activation and desensitization. Molecular modeling and sequence alignments are consistent with the hypothesis that His105 contributes to the different CO/NO binding properties of different isoforms. This valuable information is helpful to understand the molecular mechanism by which human sGC α2β1 mediates NO/CO signaling.