Spectroscopic characterization of the soluble guanylate cyclase-like heme domains from Vibrio cholerae and Thermoanaerobacter tengcongensis

Soluble guanylate cyclase (sGC) is a nitric oxide- (NO-) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. Characterization of two of these proteins is reported here. The first is a 181 amino acid protein cloned from Vibrio cholerae (VCA0720) that is encoded in a histidine kinase-containing operon. The ferrous unligated form of VCA0720 is 5-coordinate, high-spin. The CO complex is low-spin, 6-coordinate, and the NO complex is high-spin and 5-coordinate. These ligand-binding properties are very similar to those of sGC. The second protein is the N-terminal 188 amino acids of Tar4 (TtTar4H), a predicted methyl-accepting chemotaxis protein (MCP) from the strict anaerobe Thermoanaerobacter tengcongensis. TtTar4H forms a low-spin, 6-coordinate ferrous-oxy complex, the first of this sGC-related family that binds O2. TtTar4H has ligand-binding properties similar to those of the heme-containing O2 sensors such as AxPDEA1. sGC does not bind O2 despite having a porphyrin with a histidyl ligand like the globins. The results reported here, with sequence-related proteins from prokaryotes but in the same family as the sGC heme domain, show that these proteins have evolved to discriminate between ligands such as NO and O2; hence, we term this family H-NOX domains (heme-nitric oxide/oxygen).     

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.     

CO as a vibrational probe of heme protein active sites

Carbon monoxide is a useful vibrational probe of heme binding sites in proteins, because FeCO backbonding is modulated by polar interactions with protein residues, and by variations in the donor strength of the trans ligand. This modulation is sensitively monitored by the CO and FeC stretching frequencies, which are readily detectable in infrared and resonance Raman spectra. The two frequencies are anticorrelated, and the nuFeC/nuCO position along the correlation line reflects the type and strength of distal polar interactions. Changes in the trans ligand donor strength shift the correlation to higher or lower positions. Illustrative applications of the nuFeC/nuCO diagram are reviewed for proteins bearing histidine and thiolate axial ligands. Steric crowding has not been found to affect the nuFeC/nuCO correlations significantly, except in the special case of cytochrome oxidase, where the heme-bound CO may interact with the nearby CuB center.     

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

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

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.     

Nitric oxide binding to prokaryotic homologs of the soluble guanylate cyclase beta1 H-NOX domain

The heme cofactor in soluble guanylate cyclase (sGC) is a selective receptor for NO, an important signaling molecule in eukaryotes. The sGC heme domain has been localized to the N-terminal 194 amino acids of the beta1 subunit of sGC and is a member of a family of conserved hemoproteins, called the H-NOX family (Heme-Nitric Oxide and/or OXygen-binding domain). Three new members of this family have now been cloned and characterized, two proteins from Legionella pneumophila (L1 H-NOX and L2 H-NOX) and one from Nostoc punctiforme (Np H-NOX). Like sGC, L1 H-NOX forms a 5-coordinate Fe(II)-NO complex. However, both L2 H-NOX and Np H-NOX form temperature-dependent mixtures of 5- and 6-coordinate Fe(II)-NO complexes; at low temperature, they are primarily 6-coordinate, and at high temperature, the equilibrium is shifted toward a 5-coordinate geometry. This equilibrium is fully reversible with temperature in the absence of free NO. This process is analyzed in terms of a thermally labile proximal Fe(II)-His bond and suggests that in both the 5- and 6-coordinate Fe(II)-NO complexes of L2 H-NOX and Np H-NOX, NO is bound in the distal heme pocket of the H-NOX fold. NO dissociation kinetics for L1 H-NOX and L2 H-NOX have been determined and support a model in which NO dissociates from the distal side of the heme in both 5- and 6-coordinate complexes.     

Mechanism of the CO-sensing heme protein CooA: new insights from the truncated heme domain and UVRR spectroscopy

The bacterial CO-sensing heme protein CooA activates expression of genes whose products perform CO-metabolism by binding its target DNA in response to CO binding. The required conformational change has been proposed to result from CO-induced displacement of the heme and of the adjacent C-helix, which connects the sensory and DNA-binding domains. Support for this proposal comes from UV Resonance Raman (UVRR) spectroscopy, which reveals a more hydrophobic environment for the C-helix residue Trp110 when CO binds. In addition, we find a tyrosine UVRR response, which is attributable to weakening of a Tyr55-Glu83 H-bond that anchors the proximal side of the heme. Both Trp and Tyr responses are augmented in the heme domain when the DNA-binding domain has been removed, apparently reflecting loss of the inter-domain restraint. This augmentation is abolished by a Glu83Gln substitution, which weakens the anchoring H-bond. The CO recombination rate following photolysis of the CO adduct is similar for truncated and full-length protein, though truncation does increase the rate of CO association in the absence of photolysis; together these data indicate that truncation causes a faster dissociation of the endogenous Pro2 ligand. These findings are discussed in the light of structural evidence that the N-terminal tail, once released from the heme, selects the proper orientation of the DNA-binding domain, via docking interactions.     

Ligand specificity of H-NOX domains: from sGC to bacterial NO sensors

Soluble guanylate cyclase (sGC) is a nitric oxide (NO) sensing hemoprotein that has been found in eukaryotes from Drosophila to humans. Prokaryotic proteins with significant homology to the heme domain of sGC have recently been identified through genomic analysis. This family of heme proteins has been named the H-NOX domain, for Heme-Nitric oxide/OXygen binding domain. The key observation from initial studies in this family is that some members, those proteins from most eukaryotes and facultative aerobic prokaryotes, bind NO in a five-coordinate heme complex, but do not bind oxygen (O(2)), the same ligand binding characteristics as sGC. H-NOX family members from obligate aerobic prokaryotes bind O(2) and NO in six-coordinate complexes, similar to the globins and other O(2)-sensing heme proteins. The molecular factors that contribute to these differences in ligand specificity, within a family of sequence related proteins, are the subject of this review.     

Shewanella oneidensis MR-1 H-NOX regulation of a histidine kinase by nitric oxide

Nitric oxide (NO) signaling in animals controls processes such as smooth muscle relaxation and neurotransmission by activation of soluble guanylate cyclase (sGC). Prokaryotic homologues of the sGC heme domain, called H-NOX domains, have been identified and are generally found in a predicted operon in conjunction with a histidine kinase. Here, we show that an H-NOX protein (SO2144) from Shewanella oneidensis directly interacts with the sensor histidine kinase (SO2145), binds NO in a 5-coordinate complex similar to mammalian sGC, and in that form inhibits the activity of a histidine kinase (SO2145). We also describe the first account of NO formation by S. oneidensis under anaerobic growth conditions derived from nitrate and nitrite. These observations suggest that the S. oneidensis H-NOX and histidine kinase pair function as part of a novel two-component signaling pathway that is responsive to NO formation from higher nitrogen oxides used as electron acceptors when oxygen is low and thereby functioning as an environmental sensor.     

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.     

High pressure fourier transform infrared (FT-IR) spectroscopic studies on inducible nitric oxide (NO) synthase active site: a comparison to cytochrome p450CAM.

High pressure Fourier transform infrared (FT-IR) spectroscopy is performed for the first time to analyse the active site of inducible nitric oxide synthase (iNOSox) using the carbon monoxide (CO) heme iron ligand stretch mode (nuCO) as spectroscopic probe. A membrane-driven sapphire anvil high-pressure cell is used. Three major conformational substates exist in substrate-free iNOSox which are characterized by nuCO at approximately 1936, 1945 and 1952 cm(-1). High pressure favors the 1936 cm(-1) substate with a volume difference to the 1945 substate of approximately -21 cm3/mol. The pressure induced cytochrome P420 formation with a reaction volume of approximately -80 cm3/mol is observed. Arginine binding produces a very low nuCO at approximately 1905 cm(-1) caused by the H-bond from the substrate to CO. nuCO for the substates in the substrate-free and arginine-bound proteins shift linearly with pressure which is qualitatively similar to the observation on cytochrome P450cam. The slightly smaller positive slope of the shift in substrate-free iNOSox compared to substrate-free P450cam is interpreted as a slightly lesser compressible heme pocket. In contrast, the significant slower negative slope for arginine-bound iNOSox compared to camphor-bound P450cam results from the different kind of interactions to the CO ligand (electrostatic interaction in P450cam, H-bond in iNOSox).     

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.