Heme Nitric Oxide/oxygen Research Articles

HNOXPred: a web tool for the prediction of gas-sensing H-NOX proteins from amino acid sequence.

HNOXPred is a webserver for the prediction of gas-sensing heme-nitric oxide/oxygen (H-NOX) proteins from amino acid sequence. H-NOX proteins are gas-sensing hemoproteins found in diverse organisms ranging from bacteria to eukaryotes. Recently, gas-sensing complex multi-functional proteins containing only the conserved amino acids at the heme centers of H-NOX proteins, have been identified through a motif-based approach. Based on experimental data and H-NOX candidates reported in the literature, HNOXPred is created to automate and facilitate the identification of similar H-NOX centers across systems. The server features HNOXSCORES scaled from 0 to 1 that consider in its calculation, the physicochemical properties of amino acids constituting the heme center in H-NOX in addition to the conserved amino acids within the center. From user input amino acid sequence, the server returns positive hits and their calculated HNOXSCORES ordered from high to low confidence which are accompanied by interpretation guides and recommendations. The utility of this server is demonstrated using the human proteome as an example. The HNOXPred server is available at https://www.hnoxpred.com. Supplementary data are available at Bioinformatics online.

Nitric oxide sensing revisited.

Nitric oxide (NO) sensing is an ancient trait enabled by hemoproteins harboring a highly conserved Heme-Nitric oxide/OXygen (H-NOX) domain that operates throughout bacteria, fungi, and animal kingdoms including in humans, but that has long thought to be absent in plants. Recently, H-NOX-containing plant hemoproteins mediating crucial NO-dependent responses such as stomatal closure and pollen tube guidance have been reported. There are indications that the detection method that led to these discoveries will uncover many more heme-based NO sensors that operate as regulatory sites in complex proteins. Their characterizations will in turn offer a much more complete picture of plant NO responses at both the molecular and systems level.

Replacement of heme by soluble guanylate cyclase (sGC) activators abolishes heme-nitric oxide/oxygen (H-NOX) domain structural plasticity.

The gasotransmitter nitric oxide (NO) is a critical endogenous regulator of homeostasis, in major part via the generation of cGMP (cyclic guanosine monophosphate) from GTP (guanosine triphosphate) by NO's main physiological receptor, the soluble guanylate cyclase (sGC). sGC is a heterodimer, composed of an α1 and a β1 subunit, of which the latter contains the heme-nitric oxide/oxygen (H-NOX) domain, responsible for NO recognition, binding and signal initiation. The NO/sGC/cGMP axis is dysfunctional in a variety of diseases, including hypertension and heart failure, especially since oxidative stress results in heme oxidation, sGC unresponsiveness to NO and subsequent degradation. As a central player in this axis, sGC is the focus of intense research efforts aiming to develop therapeutic molecules that enhance its activity. A class of drugs named sGC “activators” aim to replace the oxidized heme of the H-NOX domain, thus stabilizing the enzyme and restoring its activity. Although numerous studies outline the pharmacology and binding behavior of these compounds, the static 3D models available so far do not allow a satisfactory understanding of the structural basis of sGC's activation mechanism by these drugs. Herein, application NMR describes different conformational states during the replacement of the heme by a sGC activators. We show that the two sGC activators (BAY 58-2667 and BAY 60-2770) significantly decrease the conformational plasticity of the recombinant H-NOX protein domain of Nostoc sp. cyanobacterium, rendering it a lot more rigid compared to the heme-occupied H-NOX. NMR methodology also reveals, for the first time, a surprising bi-directional competition between reduced heme and these compounds, pointing to a highly dynamic regulation of the H-NOX domain. This competitive, bi-directional mode of interaction is also confirmed by monitoring cGMP generation in A7r5 vascular smooth muscle cells by these activators. We show that, surprisingly, heme's redox state impacts differently the bioactivity of these two structurally similar compounds. In all, by NMR-based and functional approaches we contribute unique experimental insight into the dynamic interaction of sGC activators with the H-NOX domain and its dependence on the heme redox status, with the ultimate goal to permit a better design of such therapeutically important molecules.

Structural insights into the mechanism of oxidative activation of heme-free H-NOX from Vibrio cholerae.

Bacterial heme nitric oxide/oxygen (H-NOX) domains are nitric oxide (NO) or oxygen sensors. This activity is mediated through binding of the ligand to a heme cofactor. However, H-NOX from Vibrio cholerae (Vc H-NOX) can be easily purified in a heme-free state that is capable of reversibly responding to oxidation, suggesting a heme-independent function as a redox sensor. This occurs by oxidation of Cys residues at a zinc-binding site conserved in a subset of H-NOX homologs. Remarkably, zinc is not lost from the protein upon oxidation, although its ligation environment is significantly altered. Using a combination of computational and experimental approaches, we have characterized localized structural changes that accompany the formation of specific disulfide bonds between Cys residues upon oxidation. Furthermore, the larger-scale structural changes accompanying oxidation appear to mimic those changes observed upon NO binding to the heme-bound form. Thus, Vc H-NOX and its homologs may act as both redox and NO sensors by completely separate mechanisms.

Discovery of a Nitric Oxide-Responsive Protein in Arabidopsis thaliana.

In plants, much like in animals, nitric oxide (NO) has been established as an important gaseous signaling molecule. However, contrary to animal systems, NO-sensitive or NO-responsive proteins that bind NO in the form of a sensor or participating in redox reactions have remained elusive. Here, we applied a search term constructed based on conserved and functionally annotated amino acids at the centers of Heme Nitric Oxide/Oxygen (H-NOX) domains in annotated and experimentally-tested gas-binding proteins from lower and higher eukaryotes, in order to identify candidate NO-binding proteins in Arabidopsis thaliana. The selection of candidate NO-binding proteins identified from the motif search was supported by structural modeling. This approach identified AtLRB3 (At4g01160), a member of the Light Response Bric-a-Brac/Tramtrack/Broad Complex (BTB) family, as a candidate NO-binding protein. AtLRB3 was heterologously expressed and purified, and then tested for NO-response. Spectroscopic data confirmed that AtLRB3 contains a histidine-ligated heme cofactor and importantly, the addition of NO to AtLRB3 yielded absorption characteristics reminiscent of canonical H-NOX proteins. Furthermore, substitution of the heme iron-coordinating histidine at the H-NOX center with a leucine strongly impaired the NO-response. Our finding therefore established AtLRB3 as a NO-interacting protein and future characterizations will focus on resolving the nature of this response.

Comparative Studies of the Dynamics Effects of BAY60-2770 and BAY58-2667 Binding with Human and Bacterial H-NOX Domains.

Soluble guanylate cyclase (sGC) is a key enzyme implicated in various physiological processes such as vasodilation, thrombosis and platelet aggregation. The enzyme’s Heme-Nitric oxide/Oxygen (H-NOX) binding domain is the only sensor of nitric oxide (NO) in humans, which on binding with NO activates sGC to produce the second messenger cGMP. H-NOX is thus a hot target for drug design programs. BAY60-2770 and BAY58-2667 are two widely studied activators of sGC. Here we present comparative molecular dynamics studies to understand the molecular details characterizing the binding of BAY60-2770 and BAY58-2667 with the human H-NOX (hH-NOX) and bacterial H-NOX (bH-NOX) domains. HartreeFock method was used for parametrization of both the activators. A 50 ns molecular dynamics (MD) simulation was run to identify the functionally critical regions of the H-NOX domains. The CPPTRAJ module was used for analysis. BAY60-2770 on binding with bH-NOX, triggered rotational movement in signaling helix F and significant dynamicity in loops α and β, but in hH-NOX domain the compound showed relatively lesser aforementioned structural fluctuations. Conversely, hH-NOX ligated BAY58-2667 experienced highest transitions in its helix F due to electrostatic interactions with D84, T85 and R88 residues which are not conserved in bH-NOX. These conformational transformations might be essential to communicate with downstream PAS, CC and cyclase domains of sGC. Comparative MD studies revealed that BAY bound bHNOX dynamics varied from that of hH-NOX, plausibly due to some key residues such as R40, F74 and Y112 which are not conserved in bacteria. These findings will help to the design of novel drug leads to cure diseases associated to human sGC.

Native Alanine Substitution in the Glycine Hinge Modulates Conformational Flexibility of Heme Nitric Oxide/Oxygen (H-NOX) Sensing Proteins.

Heme nitric oxide/oxygen sensing (H-NOX) domains are direct NO sensors that regulate a variety of biological functions in both bacteria and eukaryotes. Previous work on H-NOX proteins has shown that upon NO binding, a conformational change occurs along two glycine residues on adjacent helices (termed the glycine hinge). Despite the apparent importance of the glycine hinge, it is not fully conserved in all H-NOX domains. Several H-NOX sensors from the family Flavobacteriaceae contain a native alanine substitution in one of the hinge residues. In this work, the effect of the increased steric bulk within the Ala-Gly hinge on H-NOX function was investigated. The hinge in Kordia algicida OT-1 ( Ka H-NOX) is composed of A71 and G145. Ligand-binding properties and signaling function for this H-NOX were characterized. The variant A71G was designed to convert the hinge region of Ka H-NOX to the typical Gly-Gly motif. In activity assays with its cognate histidine kinase (HnoK), the wild type displayed increased signal specificity compared to A71G. Increasing titrations of unliganded A71G gradually inhibits HnoK autophosphorylation, while increasing titrations of unliganded wild type H-NOX does not inhibit HnoK. Crystal structures of both wild type and A71G Ka H-NOX were solved to 1.9 and 1.6 Å, respectively. Regions of H-NOX domains previously identified as involved in protein-protein interactions with HnoK display significantly higher b-factors in A71G compared to wild-type H-NOX. Both biochemical and structural data indicate that the hinge region controls overall conformational flexibility of the H-NOX, affecting NO complex formation and regulation of its HnoK.

Soluble guanylate cyclase activation by nitric oxide

Since the discovery of nitric oxide (NO) formation in animals, it has become clear that this toxic, free radical, diatomic gas plays a central role in cellular function in eukaryotes. NO acts as a signaling agent in the cardiovascular system and other tissues. Using a low concentration of NO mitigates the toxicity problem but places a difficult chemical requirement on the NO receptor, the soluble isoform of guanylate cyclase (sGC). sGC contains a heme cofactor that binds and traps NO, thereby activating the enzyme to convert GTP to cGMP. The heme in sGC has unique ligand binding properties enabling sGC to act as a selective receptor for NO. The heme domain of sGC was found to be part of the H-NOX (Heme-Nitric oxide/Oxygen) family of proteins with homologues in both aerobic and anaerobic prokaryotes. Studies with H-NOX proteins has provided a molecular explanation for selective NO trapping at low concentrations. The structure/function picture that is emerging provides new clues to the activation mechanism. It is also become clear that the activation and deactivation of sGC is more complicated than the simple model of NO binding to the heme. As a physiological model is developed it must also account for activity properties of stimulators such as the FDA-approved Adempas®.

Heme Sensor Proteins for Nitric Oxide: A Versatile Use of Captive Iron

Since the discovery of nitric oxide (NO) formation in animals, it has become clear that this toxic, free radical, diatomic gas plays a central role in cellular function. NO is a signaling agent in the cardiovascular system and other tissues where the NO receptor is the soluble isoform of guanylate cyclase (sGC). The immune system also utilizes NO in the host response to infection. NO in signaling is synthesized by constitutive isoforms of enzyme nitric oxide synthase (NOS) that tightly regulates NO formation; thereby avoiding toxicity. sGC contains a heme cofactor that traps NO, thereby activating the enzyme to convert GTP to cGMP. The heme domain of sGC was found to be part of the H‐NOX (Heme‐Nitric oxide OXygen) family of proteins with homologues in aerobic and anaerobic prokaryotes. Some H‐NOX domains form stable and tight complexes with oxygen while others are selective for NO. Structural and biochemical studies have provided a molecular explanation for selective NO trapping at low concentrations. Study of H‐NOX sensors has uncovered novel functions in biology such as oxygen‐regulated sGCs and NO sensors in pathogens. The function of NO in biology is even more pervasive and more complicated than originally described. The structure/function picture that is emerging points to some common themes though many questions remain.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Activation of the H‐NOX redox sensor in Vibrio cholerae by a zinc ligand switch mechanism

Heme Nitric Oxide/Oxygen (H-NOX) binding proteins are bacterial heme binding proteins that interact with nitric oxide (NO) or oxygen (O2). In some gram-negative bacteria, such as Shewanella oneidensis, H-NOX influences the activity of downstream protein histidine kinases in an NO-dependent manner, ultimately impacting biofilm formation and dispersal. Vibrio cholerae has a homologous H-NOX to Shewanella oneidensis, and the histidine kinase (HnoK) and downstream proteins mediating the signaling cascade responsible for biofilm formation are conserved in this organism. We have expressed and purified the H-NOX and HnoK from Vibrio cholera and demonstrated that HnoK kinase activity is inhibited by both the holo and apo protein. Interestingly, the inhibitory function of H-NOX on HnoK is lost when the protein is reduced with DTT. Further investigation showed that cysteine residues in H-NOX are reversibly oxidized to form disulfide bonds, mediating HnoK binding and inhibitory activity and implicating this protein as a redox sensor. Currently our focus is on the structural details of reduced and oxidized H-NOX. There is a ligand switch mechanism at a conserved zinc site whereby cysteine ligands to the zinc are replaced by other protein side chains in the oxidized state. Detailed structural information on the interaction of HNOX with HnoK may be important for V. cholerae pathogenicity and environmental persistence. Support or Funding InformationNational Science Foundation (IIA-1301346) Apo Vc H-NOX signaling mediated by zinc ligand switch

Heme-independent Redox Sensing by the Heme-Nitric Oxide/Oxygen-binding Protein (H-NOX) from Vibrio cholerae

Heme nitric oxide/oxygen (H-NOX)-binding proteins act as nitric oxide (NO) sensors among various bacterial species. In several cases, they act to mediate communal behavior such as biofilm formation, quorum sensing, and motility by influencing the activity of downstream signaling proteins such as histidine kinases (HisKa) in a NO-dependent manner. An H-NOX/HisKa regulatory circuit was recently identified in Vibrio cholerae, and the H-NOX protein has been spectroscopically characterized. However, the influence of the H-NOX protein on HisKa autophosphorylation has not been evaluated. This process may be important for persistence and pathogenicity in this organism. Here, we have expressed and purified the V. cholerae HisKa (HnoK) and H-NOX in its heme-bound (holo) and heme-free (apo) forms. Autophosphorylation assays of HnoK in the presence of H-NOX show that the holoprotein in the Fe(II)-NO and Fe(III) forms is a potent inhibitor of HnoK. Activity of the Fe(III) form and aerobic instability of the Fe(II) form suggested that Vibrio cholerae H-NOX may act as a sensor of the redox state as well as NO. Remarkably, the apoprotein also showed robust HnoK inhibition that was dependent on the oxidation of cysteine residues to form disulfide bonds at a highly conserved zinc site. The importance of cysteine in this process was confirmed by mutagenesis, which also showed that holo Fe(III), but not Fe(II)-NO, H-NOX relied heavily upon cysteine for activation. These results highlight a heme-independent mechanism for activation of V. cholerae H-NOX that implicates this protein as a dual redox/NO sensor.

Structural insights into the role of iron-histidine bond cleavage in nitric oxide-induced activation of H-NOX gas sensor proteins.

Heme-nitric oxide/oxygen (H-NOX) binding domains are a recently discovered family of heme-based gas sensor proteins that are conserved across eukaryotes and bacteria. Nitric oxide (NO) binding to the heme cofactor of H-NOX proteins has been implicated as a regulatory mechanism for processes ranging from vasodilation in mammals to communal behavior in bacteria. A key molecular event during NO-dependent activation of H-NOX proteins is rupture of the heme-histidine bond and formation of a five-coordinate nitrosyl complex. Although extensive biochemical studies have provided insight into the NO activation mechanism, precise molecular-level details have remained elusive. In the present study, high-resolution crystal structures of the H-NOX protein from Shewanella oneidensis in the unligated, intermediate six-coordinate and activated five-coordinate, NO-bound states are reported. From these structures, it is evident that several structural features in the heme pocket of the unligated protein function to maintain the heme distorted from planarity. NO-induced scission of the iron-histidine bond triggers structural rearrangements in the heme pocket that permit the heme to relax toward planarity, yielding the signaling-competent NO-bound conformation. Here, we also provide characterization of a nonheme metal coordination site occupied by zinc in an H-NOX protein.

Crystal structure of the Alpha subunit PAS domain from soluble guanylyl cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric heme protein of ≈ 150 kDa and the primary nitric oxide receptor. Binding of NO stimulates cyclase activity, leading to regulation of cardiovascular physiology and providing attractive opportunities for drug discovery. How sGC is stimulated and where candidate drugs bind remains unknown. The α and β sGC chains are each composed of Heme-Nitric Oxide Oxygen (H-NOX), Per-ARNT-Sim (PAS), coiled-coil and cyclase domains. Here, we present the crystal structure of the α1 PAS domain to 1.8 Å resolution. The structure reveals the binding surfaces of importance to heterodimer function, particularly with respect to regulating NO binding to heme in the β1 H-NOX domain. It also reveals a small internal cavity that may serve to bind ligands or participate in signal transduction.

Soluble guanylate cyclase crystal clear: 1stcrystal structure of the wild-type human heterodimeric sGC catalytic domains and implications for activity

Background Soluble guanylate cyclase (sGC) is the key enzyme in the NO-sGC-cGMP signaling cascade crucial in regulating the cardiovascular system. Low output of this system causes hypertension and acute heart failure, which are the leading causes of death globally. Mammalian sGC is a heterodimer of two homologous subunits (a and b), which contain four domains: an Nterminal regulatory domain (HNOX: Heme Nitric oxide OXygen), an HNOX associated (HNOXA) domain and a coiled-coil (CC) domain important for dimerization, and a C-terminal catalytic domain (GC) (Figure 1). The enzyme is basally active, but NO binding to the heme group in the b subunit regulatory domain enhances sGC catalytic output several hundred fold. The molecular mechanism by which the regulatory domain relays the activation signal to the catalytic domain remains elusive. Several studies have highlighted the crucial role of the HNOXA and CC domains for sGC dimerization necessary for catalytic activity [1-9]. Others have shown that the

Insights into the distal heme pocket of H-NOX using fluoride as a probe for H-bonding interactions

Nitric oxide (NO) and dioxygen (O2) are gases of similar size, shape, and electrostatic potential, but different physiological function. In aerobic organisms, the cellular concentration of O2 far exceeds that of NO; instead NO relies heavily on the ability of its receptor to discriminate against O2. In mammals, soluble guanylate cyclase (sGC) serves this role, binding NO with picomolar sensitivity and excluding O2 binding. Interestingly, some bacterial homologs of sGC, including the H-NOX (heme-nitric oxide/oxygen) domain from Thermoanaerobacter tengcongensis, tightly bind O2. Three distal pocket residues (Trp9, Asn74, and Tyr140) form a hydrogen-bonding network that stabilizes O2 binding to TtH-NOX. Therefore, a current hypothesis to explain sGC ligand specificity is that sGC lacks H-bond donors that preferentially stabilize O2 binding. The wavelength maximum of the charge-transfer band (CT1) in the electronic spectrum of the fluoride complex of ferric hemoproteins is a sensitive probe of H-bonding. Here, in order to gain further understanding of the distal pocket H-bonding network in TtH-NOX, we employ fluoride as a spectroscopic probe. As expected, our results indicate that Y140 donates a strong H-bond to the heme-bound ligand. We find that an H-bond from Asn74 as well as distal pocket crowding contributes to positioning Tyr140 for a strong and directed H-bond to iron-bound ligands; indeed crowding may be the primary role for Trp9. We clarify the role of H-bonding in sGC ligand discrimination and suggest that sterics also regulate ligand binding in the H-NOX family.

P64: Biochemical and structural characterization of the soluble guanylate cyclase catalytic domain

Background Soluble guanylate cyclase (sGC) is a key enzyme in the NO–sGC–cGMP signaling cascade and is crucial to cardiovascular system regulation. NO binding to the sGC regulatory domain enhances its basal catalytic activity to convert GTP to cGMP The second messenger cGMP modulates downstream targets leading to vasodilation. Low output of the NO–sGC–cGMP system results in hypertension and acute heart failure. Nitrite delivery has been shown to enhance hypoxic NO signaling via cGMP production. sGC is a heterodimer of two homologous subunits, α and β, which both contain three domains: an N-terminal regulatory domain (HNOX: Heme Nitric oxide OXygen), a central dimerization HNOX associated (HNOXA) and coiled-coil (CC) domain, and a C-terminal catalytic domain (GC). The sGC enzyme is basally active in the absence of NO, but NO binding to the heme group of the β-subunit HNOX domain enhances catalytic output several hundred fold. The mechanism by which the HNOX domain inhibits activity in the basal state and relays the NO activation signal to the catalytic domain remains elusive. Methods and results Here, we report the first crystal structure of wild type human heterodimeric αβGC at 1.9 A resolution as well as activity data comparing full-length sGC to our truncated catalytic constructs. The cGMP activity measurements show that αβCC–GC exhibits higher levels of catalytic activity than both abGC and basally active full-length sGC. Conclusions Activity levels of αβCC–GC compared to basal and activated full-length sGC and abGC hint at structural differences between the two constructs and full-length sGC. Our results also suggest that additional sGC domains are necessary for full enzymatic activity. These differences will be further characterized by structural studies using protein X-ray crystallography. Disclosure Supported by American Heart Association Scientist Development Grant.

Understanding How H‐NOX (Heme Nitric Oxide/Oxygen) Domain Works Needs First Clarifying How Diatomic Gases Are Relocated Inside This Sensing Protein. A Molecular‐Mechanics Approach

H-NOX (Heme Nitric Oxide/Oxygen) domain has widespread occurrence, either standalone or associated with functional proteins, sending signals for functions that span from modulating vasodilation and neurotransmission with humans to competition and symbiosis with bacteria. Understanding how H-NOX works, and possibly intervening on degeneration for health purposes, needs first clarifying how diatomic gases are relocated through this protein in relation to the deeply buried heme. To this end, a biased form of molecular dynamics, i.e., Random Accelaration Molecular Dynamics (RAMD), is used by applying a randomly oriented tiny force to heme-dissociated CO of Nostoc sp. H-NOX, while changing randomly the direction of the force, if CO travels less than specified for the evaluated block. The result is that a large area of the protein, comprising amino acids from serine 44 to leucine 67 along two adjacent helices, offers a broad portal to CO from the surrounding medium to the deeply buried heme. Most traffic is concentrated through a channel lined by tyrosine 49, valine 52, and leucine 67. This modifies the picture drawn from mapping Xe cavities on pressurizing Nostoc sp. H-NOX with Xe gas. What is the main pathway with Xe-cavity mapping becomes a minor pathway with RAMD, and vice versa. The reason is that the fluctuating protein under MD creates clefts for CO slipping through, as it is expected to occur in nature.

Biochemical and structural characterization of C-terminal constructs of bovine soluble guanylate cyclase

Background Soluble guanylate cyclase (sGC) is the key enzyme in the NO-sGC-cGMP signaling cascade crucial in regulating the cardiovascular system. Low output of this system causes hypertension and acute heart failure, which are the leading causes of death globally. Mammalian sGC is a heterodimer. Each of the two homologous subunits (a and b) contains three domains: an N-terminal regulatory domain (HNOX: Heme Nitric oxide OXygen), a central dimerization HNOX associated (HNOXA) and coiled-coil (CC) domain, and a C-terminal catalytic domain (GC). The enzyme is basally active, but NO binding to the heme group in the b subunit’s regulatory domain enhances sGC catalytic output several hundred fold. The exact mechanism by which the regulatory domain relays the NO activation signal to the catalytic domain remains elusive. Furthermore, it has been proposed that the HNOX regulatory domain inhibits the activity of the catalytic GC domain [1]. Winger et al. showed that the GC heterodimer by itself exhibits catalytic activity in the presence of Mg and Mn[1]. On the contrary, Wedel et al. propose that additional amino acids are required for dimerization, folding, and catalytic activity [2]. We aim to test the hypothesis that additional domains are necessary for full activity of the catalytic domain by combining mutagenesis, activity assays, fluorescence spectroscopy, Small-Angle X-ray Scattering (SAXS), and protein crystallography.

Biochemical and structural characterization of the activation of soluble Guanylate Cyclase

Soluble Guanylate Cyclase (sGC) is a heterodimeric enzyme converting GTP to cyclic GMP. Each subunit contains an N‐terminal, heme‐containing regulatory domain (HNOX: Heme Nitric oxide OXygen), a dimerization PAS (Per/Arnt/Sim) and coiled‐coil (CC) domain, and a C‐terminal catalytic domain (GC). Controlling crucial physiological functions such as vasorelaxation, a low turnover of sGC causes cardiovascular pathologies. Binding of nitric oxide (NO) to HNOX maximally activates sGC and alleviates a presumed inhibitory effect of HNOX on GC. However, the isolated GC exhibits low activity excluding HNOX as the only brake on catalytic turnover.To elucidate the activation mechanism and identify the domains required for maximum activity, we are studying truncated bovine sGC constructs. These include GC, CC‐GC, and PAS‐CC‐GC as well as the HNOX‐PAS and HNOX‐PAS‐CC heterodimers.We report preliminary SAXS (Small Angle X‐ray Scattering) ab initio envelopes on HNOX‐containing constructs and present activity assays for the GC‐containing constructs.Combinations of activity measurements, SAXS, and x‐ray crystallography have been used to elucidate the activation mechanism of sGC. These studies will provide a foundation for structure‐based drug design of novel sGC activators.Work supported by American Heart Association Scientist Development Grant (E. Garcin) and NIH CBI Training Grant NIH T32 GM066706‐06.

Structural insights into the molecular mechanism of H‐NOX activation

Nitric oxide (NO) signaling in mammals controls important processes such as smooth muscle relaxation and neurotransmission by the activation of soluble guanylate cyclase (sGC). NO binding to the heme domain of sGC leads to dissociation of the iron-histidine (Fe-His) bond, which is required for enzyme activity. The heme domain of sGC belongs to a larger class of proteins called H-NOX (Heme-Nitric oxide/OXygen) binding domains. Previous crystallographic studies on H-NOX domains demonstrate a correlation between heme bending and protein conformation. It was unclear, however, whether these structural changes were important for signal transduction. Subsequent NMR solution structures of H-NOX proteins show a conformational change upon disconnection of the heme and proximal helix, similar to those observed in the crystallographic studies. The atomic details of these conformational changes, however, are lacking in the NMR structures especially at the heme pocket. Here, a high-resolution crystal structure of an H-NOX mutant mimicking a broken Fe-His bond is reported. This mutant exhibits specific changes in heme conformation and major N-terminal displacements relative to the wild-type H-NOX protein. Fe-His ligation is ubiquitous in all H-NOX domains, and therefore, the heme and protein conformational changes observed in this study are likely to occur throughout the H-NOX family when NO binding leads to rupture of the Fe-His bond.