Hydrogen sulfide (H 2 S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H 2 S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H 2 S and NO exert their effects via separate pathways, the results of the current study show that H 2 S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H 2 S increases intracellular cyclic guanosine 5′-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H 2 S-stimulated angiogenic response, and attenuated H 2 S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H 2 S signaling. Conversely, silencing of the H 2 S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H 2 S in the vascular activity of NO. The actions of H 2 S and NO converge at cGMP; though H 2 S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H 2 S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H 2 S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H 2 S are mutually required for the physiological control of vascular function. N itric oxide (NO) and hydrogen sulfide (H 2 S) are two endogenous gasotransmitters whose regulatory roles in the cardiovascular system include vasorelaxation and stimulation of angiogenesis (1, 2). In endothelial cells, NO is synthesized by the endothelial isoform of NO synthase (eNOS). The principal pathway of NO signaling involves binding to the heme moiety of the soluble guanylyl cyclase (sGC) and production of the second messenger cyclic guanosine 5′-monophosphate (cGMP), followed by the activation of protein kinase G (PKG) (3, 4). However, vascular H 2 S is generated from L-cysteine by two pyridoxal 5′-phosphate-dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), and by the combined action of cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulfurtransferase (3-MST); activation of the ATP-dependent potassium channel (K ATP channel), modulation of cell metabolism, and posttranslational protein modifications via sulfhydration have been identified as some of its key signaling pathways (5-7).It is generally assumed that the signaling pathways of NO and H 2 S are independent. In the ...

Nitric oxide (NO), a simple free radical gas, elicits a surprisingly wide range of physiological and pathophysiological effects. NO interacts with soluble guanylate cyclase to evoke many of these effects. However, NO can also interact with molecular oxygen and superoxide radicals to produce reactive nitrogen species that can modify a number of macromolecules including proteins, lipids, and nucleic acids. NO can also interact directly with transition metals. Here, we have reviewed the non--3',5'-cyclic-guanosine-monophosphate-mediated effects of NO including modifications of proteins, lipids, and nucleic acids.

Selectivity between NO, CO, and O2 is crucial for the physiological function of most heme proteins. Although there is a million-fold variation in equilibrium dissociation constants (KDs), the ratios for NO:CO:O2 binding stay roughly the same, 1:~103:~106 when the proximal ligand is a histidine and the distal site is apolar. For these proteins, there is a “sliding scale rule” for plots of log KD versus ligand type that allows predictions of KD values if one or two are missing. The predicted KD for O2 binding to Ns H-NOX coincides with the value determined experimentally at high pressures. Active site hydrogen bond donors break the rule and selectively increase O2 affinity with little effect on CO and NO binding. Strong field proximal ligands such as thiolate, tyrosinate and imidazolate exert a “leveling” effect on ligand binding affinity. The reported picomolar KD for NO binding to sGC deviates even more dramatically from the sliding scale rule, showing a NO:CO KD ratio of 1: ~108. This deviation is explained by a complex, multi-step process, in which an initial low affinity hexacoordinate NO complex with a measured KD ≈ 54 nM, matching that predicted from the sliding scale rule, is formed initially and then converts to a high affinity pentacoordinate complex. This multi-step 6c to 5c mechanism appears common to all NO sensors that exclude O2 binding in order to capture lower level of cellular NO and prevent its consumption by dioxygenation.

Heme is a vital molecule for all life forms with heme being capable of assisting in catalysis, binding ligands, and undergoing redox changes. Heme-related dysfunction can lead to cardiovascular diseases with the oxidation of the heme of soluble guanylyl cyclase (sGC) critically implicated in some of these cardiovascular diseases. sGC, the main nitric oxide (NO) receptor, stimulates second messenger cGMP production, whereas reactive oxygen species are known to scavenge NO and oxidize/inactivate the heme leading to sGC degradation. This vulnerability of NO-heme signaling to oxidative stress led to the discovery of an NO-independent activator of sGC, cinaciguat (BAY 58 -2667), which is a candidate drug in clinical trials to treat acute decompensated heart failure. Here, we present crystallographic and mutagenesis data that reveal the mode of action of BAY 58 -2667. The 2.3-Å resolution structure of BAY 58 -2667 bound to a heme NO and oxygen binding domain (H-NOX) from Nostoc homologous to that of sGC reveals that the trifurcated BAY 58 -2667 molecule has displaced the heme and acts as a heme mimetic. Carboxylate groups of BAY 58 -2667 make interactions similar to the heme-propionate groups, whereas its hydrophobic phenyl ring linker folds up within the heme cavity in a planar-like fashion. BAY 58 -2667 binding causes a rotation of the ␣F helix away from the heme pocket, as this helix is normally held in place via the inhibitory His 105 -heme covalent bond. The structure provides insights into how BAY 58 -2667 binds and activates sGC to rescue heme-NO dysfunction in cardiovascular diseases.

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