Gaseous ligand selectivity of the H-NOX sensor protein from Shewanella oneidensis and comparison to those of other bacterial H-NOXs and soluble guanylyl cyclase.
ABSTRACT: To delineate the commonalities and differences in gaseous ligand discrimination among the heme-based sensors with Heme Nitric oxide/OXygen binding protein (H-NOX) scaffold, the binding kinetic parameters for gaseous ligands NO, CO, and O2, including KD, kon, and koff, of Shewanella oneidensis H-NOX (So H-NOX) were characterized in detail in this study and compared to those of previously characterized H-NOXs from Clostridium botulinum (Cb H-NOX), Nostoc sp. (Ns H-NOX), Thermoanaerobacter tengcongensis (Tt H-NOX), Vibrio cholera (Vc H-NOX), and human soluble guanylyl cyclase (sGC), an H-NOX analogue. The KD(NO) and KD(CO) of each bacterial H-NOX or sGC follow the "sliding scale rule"; the affinities of the bacterial H-NOXs for NO and CO vary in a small range but stronger than those of sGC by at least two orders of magnitude. On the other hand, each bacterial H-NOX exhibits different characters in the stability of its 6c NO complex, reactivity with secondary NO, stability of oxyferrous heme and autoxidation to ferric heme. A facile access channel for gaseous ligands is also identified, implying that ligand access has only minimal effect on gaseous ligand selectivity of H-NOXs or sGC. This comparative study of the binding parameters of the bacterial H-NOXs and sGC provides a basis to guide future new structural and functional studies of each specific heme sensor with the H-NOX protein fold.
Project description: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 ?1 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 ?1 H-NOX construct (residues 1-194), the ?1 H-NOX-PAS-CC construct (residues 1-385), and the full-length ?1?1 sGC heterodimer (?1 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.
Project description:Vc H-NOX (or VCA0720) is an H-NOX (heme-nitric oxide and oxygen binding) protein from facultative aerobic bacterium Vibrio cholerae. It shares significant sequence homology with soluble guanylyl cyclase (sGC), a NO sensor protein commonly found in animals. Similar to sGC, Vc H-NOX binds strongly to NO and CO with affinities of 0.27 nM and 0.77 ?M, respectively, but weakly to O2. When positioned on a "sliding scale" plot [Tsai, A.-l., et al. (2012) Biochemistry 51, 172-186], the line connecting log K(D)(NO) and log K(D)(CO) of Vc H-NOX can almost be superimposed with that of Ns H-NOX. Therefore, the measured affinities and kinetic parameters of gaseous ligands to Vc H-NOX provide more evidence to validate the "sliding scale rule" hypothesis. Like sGC, Vc H-NOX binds NO in multiple steps, forming first a six-coordinate heme-NO complex at a rate of 1.1 × 10(9) M(-1) s(-1), and then converts to a five-coordinate heme-NO complex at a rate that is also dependent on NO concentration. Although the formation of oxyferrous Vc H-NOX cannot be detected at a normal atmospheric oxygen level, ferrous Vc H-NOX is oxidized to the ferric form at a rate of 0.06 s(-1) when mixed with O2. Ferric Vc H-NOX exists as a mixture of high- and low-spin states and is influenced by binding to different ligands. Characterization of both ferric and ferrous Vc H-NOX and their complexes with various ligands lays the foundation for understanding the possible dual roles in gas and redox sensing of Vc H-NOX.
Project description: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.
Project description:Soluble guanylate cyclase (sGC) is a heterodimeric heme protein and the primary nitric oxide receptor. NO binding stimulates cyclase activity, leading to regulation of cardiovascular physiology and making sGC an attractive target for drug discovery. YC-1 and related compounds stimulate sGC both independently and synergistically with NO and CO binding; however, where the compounds bind and how they work remain unknown. Using linked equilibrium binding measurements, surface plasmon resonance, and domain truncations in Manduca sexta and bovine sGC, we demonstrate that YC-1 binds near or directly to the heme-containing domain of the ? subunit. In the absence of CO, YC-1 binds with a Kd of 9-21 ?M, depending on the construct. In the presence of CO, these values decrease to 0.6-1.1 ?M. Pfizer compound 25 bound ?10-fold weaker than YC-1 in the absence of CO, whereas compound BAY 41-2272 bound particularly tightly in the presence of CO (Kd = 30-90 nM). Additionally, we found that CO binds much more weakly to heterodimeric sGC proteins (Kd = 50-100 ?M) than to the isolated heme domain (Kd = 0.2 ?M for Manduca ? H-NOX/PAS). YC-1 greatly enhanced binding of CO to heterodimeric sGC, as expected (Kd ? 1 ?M). These data indicate the ? subunit induces a heme pocket conformation with a lower affinity for CO and NO. YC-1 family compounds bind near the heme domain, overcoming the ? subunit effect and inducing a heme pocket conformation with high affinity. We propose this high-affinity conformation is required for the full-length protein to achieve high catalytic activity.
Project description:The H-NOX (Heme-Nitric oxide/OXygen binding) family of diatomic gas sensing hemoproteins has attracted great interest. Soluble guanylate cyclase (sGC), the well-characterized eukaryotic nitric oxide (NO) sensor is an H-NOX family member. When NO binds sGC at the ferrous histidine-ligated protoporphyrin-IX, the proximal histidine ligand dissociates, resulting in a 5-coordinate (5c) complex; formation of this 5c complex is viewed as necessary for activation of sGC. Characterization of other H-NOX family members has revealed that while most also bind NO in a 5c complex, some bind NO in a 6-coordinate (6c) complex or as a 5c/6c mixture. To gain insight into the heme pocket structural differences between 5c and 6c Fe(ii)-NO H-NOX complexes, we investigated the extended X-ray absorption fine structure (EXAFS) of the Fe(II)-unligated and Fe(II)-NO complexes of H-NOX domains from three species, Thermoanaerobacter tengcongensis, Shewanella woodyi, and Pseudoalteromonas atlantica. Although the Fe(II)-NO complex of TtH-NOX is formally 6c, we found the Fe-N(His) bond is substantially lengthened. Furthermore, although NO binds to SwH-NOX and PaH-NOX as a 5c complex, consistent with histidine dissociation, the EXAFS data do not exclude a very weakly associated histidine. Regardless of coordination number, upon NO-binding, the Fe-N(porphyrin) bond lengths in all three H-NOXs contract by ~0.07 Å. This study reveals that the overall heme structure of 5c and 6c Fe(II)-NO H-NOX complexes are substantially similar, suggesting that formal histidine dissociation may not be required to trigger NO/H-NOX signal transduction. The study has refined our understanding of the molecular mechanisms underlying NO/H-NOX signaling.
Project description:Ligand selectivity for dioxygen (O(2)), carbon monoxide (CO), and nitric oxide (NO) is critical for signal transduction and is tailored specifically for each heme-protein sensor. Key NO sensors, such as soluble guanylyl cyclase (sGC), specifically recognized low levels of NO and achieve a total O(2) exclusion. Several mechanisms have been proposed to explain the O(2) insensitivity, including lack of a hydrogen bond donor and negative electrostatic fields to selectively destabilize bound O(2), distal steric hindrance of all bound ligands to the heme iron, and restriction of in-plane movements of the iron atom.Crystallographic structures of the gas sensors, Thermoanaerobacter tengcongensis heme-nitric oxide/oxygen-binding domain (Tt H-NOX(1)) or Nostoc puntiforme (Ns) H-NOX, and measurements of O(2) binding to site-specific mutants of Tt H-NOX and the truncated ? subunit of sGC suggest the need for a H-bonding donor to facilitate O(2) binding.However, the O(2) insensitivity of full length sGC with a site-specific replacement of isoleucine by a tyrosine on residue 145 and the very slow autooxidation of Ns H-NOX and cytochrome c' suggest that more complex mechanisms have evolved to exclude O(2) but retain high affinity NO binding. A combined graphical analysis of ligand binding data for libraries of heme sensors, globins, and model heme shows that the NO sensors dramatically inhibit the formation of six-coordinated NO, CO, and O(2) complexes by direct distal steric hindrance (cyt c'), proximal constraints of in-plane iron movement (sGC), or combinations of both following a sliding scale rule. High affinity NO binding in H-NOX proteins is achieved by multiple NO binding steps that produce a high affinity five-coordinate NO complex, a mechanism that also prevents NO dioxygenation.Knowledge advanced by further extensive test of this "sliding scale rule" hypothesis should be valuable in guiding novel designs for heme based sensors.
Project description:Nostoc sp. (Ns) H-NOX is a heme protein found in symbiotic cyanobacteria, which has approximately 35% sequence identity and high structural homology to the beta subunit of soluble guanylyl cyclase (sGC), suggesting a NO sensing function. However, UV-vis, EPR, NIR MCD, and ligand binding experiments with ferrous and ferric Ns H-NOX indicate significant functional differences between Ns H-NOX and sGC. (1) After NO binding to sGC, the proximal histidine dissociates from the heme iron, causing a conformational change that triggers activation of sGC. In contrast, formation of pentacoordinate (5c) NO heme occurs to only a limited extent in Ns H-NOX, even at >1 mM NO. (2) Unlike sGC, two different hexacoordinate (6c) NO complexes are formed in Ns H-NOX with initial and final absorbance peaks at 418 and 414 nm, and the conversion rate is linearly dependent on [NO], indicating that a second NO binds transiently to catalyze formation of the 414 nm species. (3) sGC is insensitive to oxygen, and ferric sGC prepared by ferricyanide oxidation has a 5c high-spin heme complex. In contrast, Ns H-NOX autoxidizes in 24 h if exposed to air and forms a 6c ferric heme complex, indicating a major conformational change after oxidation and coordination by a second histidine side chain. Such a large conformational transition suggests that Ns H-NOX could function as either a redox or a NO sensor in the cyanobacterium.
Project description:Soluble guanylyl/guanylate cyclase (sGC) converts GTP to cGMP after binding nitric oxide, leading to smooth muscle relaxation and vasodilation. Impaired sGC activity is common in cardiovascular disease, and sGC stimulatory compounds are vigorously sought. sGC is a 150 kDa heterodimeric protein with two H-NOX domains (one with heme, one without), two PAS domains, a coiled-coil domain, and two cyclase domains. Binding of NO to the sGC heme leads to proximal histidine release and stimulation of catalytic activity. To begin to understand how binding leads to activation, we examined truncated sGC proteins from Manduca sexta (tobacco hornworm) that bind NO, CO, and stimulatory compound YC-1 but lack the cyclase domains. We determined the overall shape of truncated M. sexta sGC using analytical ultracentrifugation and small-angle X-ray scattering (SAXS), revealing an elongated molecule with dimensions of 115 Å × 90 Å × 75 Å. Binding of NO, CO, or YC-1 had little effect on shape. Using chemical cross-linking and tandem mass spectrometry, we identified 20 intermolecular contacts, allowing us to fit homology models of the individual domains into the SAXS-derived molecular envelope. The resulting model displays a central parallel coiled-coil platform upon which the H-NOX and PAS domains are assembled. The ?1 H-NOX and ?1 PAS domains are in contact and form the core signaling complex, while the ?1 H-NOX domain can be removed without a significant effect on ligand binding or overall shape. Removal of 21 residues from the C-terminus yields a protein with dramatically increased proximal histidine release rates upon NO binding.
Project description:The Heme Nitric oxide/OXygen binding (H-NOX) family of proteins have important functions in gaseous ligand signaling in organisms from bacteria to humans, including nitric oxide (NO) sensing in mammals, and provide a model system for probing ligand selectivity in hemoproteins. A unique vibrational feature that is ubiquitous throughout the H-NOX family is the presence of a high C-O stretching frequency. To investigate the cause of this spectroscopic characteristic, the Fe-CO and C-O stretching frequencies were probed in the H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) using resonance Raman (RR) spectroscopy. Four classes of heme pocket mutants were generated to assess the changes in stretching frequency: (i) the distal H-bonding network, (ii) the proximal histidine ligand, (iii) modulation of the heme conformation via Ile-5 and Pro-115, and (iv) the conserved Tyr-Ser-Arg (YxSxR) motif. These mutations revealed important electrostatic interactions that dampen the back-donation of the Fe(II) d(?) electrons into the CO ?* orbitals. The most significant change occurred upon disruption of the H-bonds between the strictly conserved YxSxR motif and the heme propionate groups, producing two dominant CO-bound heme conformations. One conformer was structurally similar to Tt H-NOX WT, whereas the other displayed a decrease in ?(C-O) of up to ?70 cm(-1) relative to the WT protein, with minimal changes in ?(Fe-CO). Taken together, these results show that the electrostatic interactions in the Tt H-NOX binding pocket are primarily responsible for the high ?(C-O) by decreasing the Fe d(?) ? CO ?* back-donation and suggest that the dominant mechanism by which this family modulates the Fe(II)-CO bond likely involves the YxSxR motif.
Project description:Determinants of the Fe-CO and C-O stretching frequencies in (imidazole)heme-CO adducts have been investigated via density functional theory (DFT) analysis, in connection with puzzling characteristics of the heme sensor protein CooA and of the H-NOX (Heme-Nitric Oxide and/or OXygen binding) family of proteins, including soluble guanylate cyclase (sGC). The computations show that two mechanisms of Fe-histidine bond weakening have opposite effects on the nuFeC/nuCO pattern. Mechanical tension is expected to raise nuFeC with little change in nuCO whereas the weakening of H-bond donation from the imidazole ligand has the opposite effect. Data on CooA indicate imidazole H-bond weakening associated with heme displacement, as part of the activation mechanism. The computations also reveal that protein-induced distortion of the porphyrin ring, a prominent structural feature of the H-NOX protein TtTar4H (Thermoanaerobacter tengcongensis Tar4 protein heme domain), has surprisingly little effect on nuFeC or nuCO. However, another structural feature, strong H-bonding to the propionates, is suggested to account for the weakened back bonding that is evident in sGC. TtTar4H-CO itself has an elevated nuFeC, which is successfully modeled as a compression effect, resulting from steric crowding in the distal pocket. nuFeC/nuCO data, in conjunction with modeling, can provide valuable insight into mechanisms for heme-protein modulation.