Orthogonal properties of the redox siblings nitroxyl and nitric oxide in the cardiovascular system: a novel redox paradigm

Endogenous formation of nitric oxide (NO) and related nitrogen oxides in the vascular system is critical to regulation of multiple physiological functions. An imbalance in the production or availability of these species can result in progression of disease. Nitrogen oxide research in the cardiovascular system has primarily focused on the effects of NO and higher oxidation products. However, nitroxyl (HNO), the one-electron-reduction product of NO, has recently been shown to have unique and potentially beneficial pharmacological properties. HNO and NO often induce discrete biological responses, providing an interesting redox system. This article discusses the emerging aspects of HNO chemistry and attempts to provide a framework for the distinct effects of NO and HNO in vivo.     

The potential of Angeli's salt to decrease nitric oxide scavenging by plasma hemoglobin

Release of hemoglobin from the erythrocyte during intravascular hemolysis contributes to the pathology of a variety of diseased states. This effect is partially due to the enhanced ability of cell-free plasma hemoglobin, which is primarily found in the ferrous, oxygenated state, to scavenge nitric oxide. Oxidation of the cell-free hemoglobin to methemoglobin, which does not effectively scavenge nitric oxide, using inhaled nitric oxide has been shown to be effective in limiting pulmonary and systemic vasoconstriction. However, the ferric heme species may be reduced back to ferrous hemoglobin in plasma and has the potential to drive injurious redox chemistry. We propose that compounds that selectively convert cell-free hemoglobin to ferric, and ideally iron-nitrosylated heme species that do not actively scavenge nitric oxide, would effectively treat intravascular hemolysis. We show here that nitroxyl generated by Angeli's salt (sodium alpha-oxyhyponitrite, Na2N2O3) preferentially reacts with cell-free hemoglobin compared to that encapsulated in the red blood cell under physiologically relevant conditions. Nitroxyl oxidizes oxygenated ferrous hemoglobin to methemoglobin and can convert the methemoglobin to a more stable, less toxic species, iron-nitrosyl hemoglobin. These results support the notion that Angeli's salt or a similar compound could be used to effectively treat conditions associated with intravascular hemolysis.     

Mechanism and biological role of nitric oxide binding to cytochrome c'

The binding of nitric oxide to ferric and ferrous Chromatium vinosum cytochrome c' was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c' data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c' was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c' was found to be in the range typical of receptors and carriers. In addition, cytochrome c' was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c' functioning as a nitric oxide carrier was proposed.     

Differential biological effects of products of nitric oxide (NO) synthase: it is not enough to say NO

The radical gas nitric oxide (NO) is implicated in an enormous number of biological function both in physiological and pathological conditions. Often it is not clear if it plays a deleterious or beneficial role. Here briefly, are analyzed some of the reasons of this multitude of effects. Emphasis is given to factors influencing NO formation and to the type and quantity of radicals formed by nitric oxide synthase. In particular, a comparison between the biological effects of nitroxyl anion (HNO/NO(-)) and nitric oxide NO(.) is considered. These redox siblings often exhibit orthogonal behavior in physiological and pathological conditions. In the light of the multitude of effects of NO, the role of this gas, their siblings and their derivatives in cardiac ischemic preconditioning scenario is more extensively analyzed.     

Direct evidence for nitric oxide production by a nitric-oxide synthase-like protein from Bacillus subtilis

Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have diverse functions in physiology. Recent genome sequencing revealed NOS-like protein in bacteria, but whether these proteins generate nitric oxide is unknown. We therefore cloned, expressed, and purified a NOS-like protein from Bacillus subtilis (bsNOS) and characterized its catalytic parameters in both multiple and single turnover reactions. bsNOS was dimeric, bound l-Arg and 6R-tetrahydrobiopterin with similar affinity as mammalian NOS, and generated nitrite from l-Arg when incubated with NADPH and a mammalian NOS reductase domain. Stopped-flow analysis showed that ferrous bsNOS reacted with O(2) to form a transient heme Fe(II)O(2) species in the presence of either Arg or the reaction intermediate N-hydroxy-l-arginine. In the latter case, disappearance of the Fe(II)O(2) species was kinetically and quantitatively coupled to formation of a transient heme Fe(III)NO product, which then dissociated to form ferric bsNOS. This behavior mirrors mammalian NOS enzymes and unambiguously shows that bsNOS can generate NO. NO formation required a bound tetrahydropteridine, and the kinetic effects of this cofactor were consistent with it donating an electron to the Fe(II)O(2) intermediate during the reaction. Dissociation of the heme Fe(III)NO product was much slower in bsNOS than in mammalian NOS. This constrains allowable rates of ferric heme reduction by a protein redox partner and underscores the utility of using a tetrahydropteridine electron donor in bsNOS.     

Formation and reactions of the heme-dioxygen intermediate in the first and second steps of nitric oxide synthesis as studied by stopped-flow spectroscopy under single-turnover conditions

To better understand the mechanism of nitric oxide (NO) synthesis, we studied conversion of N-hydroxy-L-arginine (NOHA) or L-arginine (Arg) to citrulline and NO under single-turnover conditions using the oxygenase domain of neuronal nitric oxide synthase (nNOSoxy) and rapid scanning stopped-flow spectroscopy. When anaerobic nNOSoxy saturated with H(4)B and NOHA was provided with 0.5 or 1 electron per heme and then exposed to air at 25 degrees C, it formed 0.5 or 1 mol of citrulline/mol of heme, respectively, indicating that NOHA conversion had 1:1 stoichiometry with respect to electrons added. Identical experiments with Arg produced substoichiometric amounts of NOHA or citrulline even when up to 3 electrons were provided per heme. Transient spectral intermediates were investigated at 10 degrees C. For NOHA, four species were observed in the following sequence: starting ferrous nNOSoxy, a transient ferrous-dioxygen complex, a transient ferric-NO complex, and ferric nNOSoxy. For Arg, transient intermediates other than the ferrous-dioxygen species were not apparent during the reaction. Our results provide a kinetic framework for formation and reactions of the ferrous-dioxygen complex in each step of NO synthesis and establish that (1) the ferrous-dioxy enzyme reacts quantitatively with NOHA but not with Arg and (2) its reaction with NOHA forms 1 NO/heme, which immediately binds to form a ferric heme-NO complex.     

Carbon monoxide -- a "new" gaseous modulator of gene expression

Carbon monoxide (CO) is an odorless, tasteless and colorless gas which is generated by heme oxygenase enzymes (HOs). HOs degrade heme releasing equimolar amounts of CO, iron and biliverdin, which is subsequently reduced to bilirubin. CO shares many properties with nitric oxide (NO), an established cellular messenger. Both CO and NO are involved in neural transmission and modulation of blood vessel function, including their relaxation and inhibition of platelet aggregation. CO, like NO, binds to heme proteins, although CO binds only ferrous (FeII) heme, whereas NO binds both ferrous and ferric (FeIII). CO enhances the activity of guanylate cyclase although it is less potent than NO. In contrast, CO inhibits other heme proteins, such as catalase or cytochrome p450. The effects of CO on gene expression can be thus varied, depending on the cellular microenvironment and the metabolic pathway being influenced. In this review the regulation of gene expression by HO/CO in the cardiovascular system is discussed. Recent data, derived also from our studies, indicate that HO/CO are significant modulators of inflammatory reactions, influencing the underlying processes such as cell proliferation and production of cytokines and growth factors.     

Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions

Recent evidence suggests that the reaction of nitrite with deoxygenated hemoglobin and myoglobin contributes to the generation of nitric oxide and S-nitrosothiols in vivo under conditions of low oxygen availability. We have investigated whether ferrous neuroglobin and cytoglobin, the two hexacoordinate globins from vertebrates expressed in brain and in a variety of tissues, respectively, also react with nitrite under anaerobic conditions. Using absorption spectroscopy, we find that ferrous neuroglobin and nitrite react with a second-order rate constant similar to that of myoglobin, whereas the ferrous heme of cytoglobin does not react with nitrite. Deconvolution of absorbance spectra shows that, in the course of the reaction of neuroglobin with nitrite, ferric Fe(III) heme is generated in excess of nitrosyl Fe(II)-NO heme as due to the low affinity of ferrous neuroglobin for nitric oxide. By using ferrous myoglobin as scavenger for nitric oxide, we find that nitric oxide dissociates from ferrous neuroglobin much faster than previously appreciated, consistently with the decay of the Fe(II)-NO product during the reaction. Both neuroglobin and cytoglobin are S-nitrosated when reacting with nitrite, with neuroglobin showing higher levels of S-nitrosation. The possible biological significance of the reaction between nitrite and neuroglobin in vivo under brain hypoxia is discussed.     

A conserved Val to Ile switch near the heme pocket of animal and bacterial nitric-oxide synthases helps determine their distinct catalytic profiles

Nitric oxide (NO) release from nitric oxide synthases (NOSs) is largely dependent on the dissociation of an enzyme ferric heme-NO product complex (Fe(III)NO). Although the NOS-like protein from Bacillus subtilis (bsNOS) generates Fe(III)NO from the reaction intermediate N-hydroxy-l-arginine (NOHA), its NO dissociation is about 20-fold slower than in mammalian NOSs. Crystal structures suggest that a conserved Val to Ile switch near the heme pocket of bsNOS might determine its kinetic profile. To test this we generated complementary mutations in the mouse inducible NOS oxygenase domain (iNOSoxy, V346I) and in bsNOS (I224V) and characterized the kinetics and extent of their NO synthesis from NOHA and their NO-binding kinetics. The mutations did not greatly alter binding of Arg, (6R)-tetrahydrobiopterin, or alter the electronic properties of the heme or various heme-ligand complexes. Stopped-flow spectroscopy was used to study heme transitions during single turnover NOHA reactions. I224V bsNOS displayed three heme transitions involving four species as typically occurs in wild-type NOS, the beginning ferrous enzyme, a ferrous-dioxy (Fe(II)O(2)) intermediate, Fe(III)NO, and an ending ferric enzyme. The rate of each transition was increased relative to wild-type bsNOS, with Fe(III)NO dissociation being 3.6 times faster. In V346I iNOSoxy we consecutively observed the beginning ferrous, Fe(II)O(2), a mixture of Fe(III)NO and ferric heme species, and ending ferric enzyme. The rate of each transition was decreased relative to wild-type iNOSoxy, with the Fe(III)NO dissociation being 3 times slower. An independent measure of NO binding kinetics confirmed that V346I iNOSoxy has slower NO binding and dissociation than wild-type. Citrulline production by both mutants was only slightly lower than wild-type enzymes, indicating good coupling. Our data suggest that a greater shielding of the heme pocket caused by the Val/Ile switch slows down NO synthesis and NO release in NOS, and thus identifies a structural basis for regulating these kinetic variables.     

EPR studies on the photo-induced intermediates of ferric NO complexes of rat neuronal nitric oxide synthase trapped at low temperature.

Rat neuronal nitric oxide synthase (nNOS) was expressed in Escherichia coli and purified. Although the nitric oxide (NO) complex of the ferric heme was EPR-silent, photo-illumination at 5 K to the NO complex of the ferric nNOS in the substrate-free form produced a new high spin EPR signal similar to that of the ferric heme of N(omega)-nitro-L-arginine-bound nNOS, suggesting that the photo-dissociated NO might move away from the heme. Low photo-dissociability of NO in this complex indicated less restricted movement of the dissociated NO in the distal region of the heme, which might result in the rapid rebinding of the NO to the ferric heme at 5 K. In the presence of substrate L-arginine, derivatives, or product L-citrulline, the photo-products from the ferric NO complexes exhibited large novel EPR signals with a spin-coupled interaction between the ferric heme (S = 5/2) and the photolyzed NO (S = 1/2), suggesting a stereochemically restricted interaction between the photo-dissociated NO and the guanidino- or the ureido-group of the substrate analogues at the distal heme region of nNOS. The photo-product from the NO complex produced from citrulline-bound nNOS might be the same intermediate species as that formed in the last step of the catalytic cycle.     

The Effects of Nitroxyl (HNO) on Soluble Guanylate Cyclase Activity: INTERACTIONS AT FERROUS HEME AND CYSTEINE THIOLS

It has been previously proposed that nitric oxide (NO) is the only biologically relevant nitrogen oxide capable of activating the enzyme soluble guanylate cyclase (sGC). However, recent reports implicate HNO as another possible activator of sGC. Herein, we examine the affect of HNO donors on the activity of purified bovine lung sGC and find that, indeed, HNO is capable of activating this enzyme. Like NO, HNO activation appears to occur via interaction with the regulatory ferrous heme on sGC. Somewhat unexpectedly, HNO does not activate the ferric form of the enzyme. Finally, HNO-mediated cysteine thiol modification appears to also affect enzyme activity leading to inhibition. Thus, sGC activity can be regulated by HNO via interactions at both the regulatory heme and cysteine thiols.Nitric oxide (NO)² is the most studied of the endogenously generated nitrogen oxides and is well known to mediate many aspects of cardiovascular function including the regulation of vascular tone and platelet aggregation (for example, see Ref. 1). These responses are in large part due to the interaction of NO with its most established endogenous receptor, soluble guanylate cyclase (sGC) (2). This 150-kDa heterodimeric heme protein catalyzes the production of the second messenger molecule cyclic guanosine monophosphate (cGMP) from guanosine triphosphate (GTP) (3). The basal activity of sGC is enhanced several hundred fold upon binding of NO to the single regulatory heme site. This stimulation of activity is a result of a conformational change induced by cleavage of the proximal histidine heme ligand upon formation of the ferrous nitrosyl complex, which is preferentially pentacoordinate (4). In addition to heme site regulation of sGC, there are numerous reports indicating that oxidation of cysteine thiol residues on this protein can also alter/regulate both the basal activity and the degree of NO-mediated activation (5–10).Recently, the one-electron reduced and protonated congener of NO, nitroxyl (HNO) has received significant interest as a cardiovascular agent whose actions are independent of NO formation (11). For example, a study by Ellis and co-workers (12) suggests that HNO is a vital component of endothelium-derived relaxing factor along with NO in rat aorta. HNO is also able to mediate murine aorta vasorelaxation even in the presence of NO scavengers (13). Furthermore, the vasodilation produced by HNO was inhibited by the sGC heme site inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxalin-1-one implicating sGC activation in this HNO-mediated effect. In addition to its effects on large conduit vessels like the aorta, HNO also dilates rat small mesenteric resistance-like arteries through sGC-dependent and voltage-dependent K⁺ channel-dependent mechanisms (14). Nitroxyl (derived from the HNO-donor Angeli''s salt) is also a potent dilator of feline pulmonary vasculature equal to that of the NO donors SPER/NO, DETA/NO, and SULFI/NO (15). Most recently, HNO was found to be a potent dilator of rat coronary arteries through an sGC-mediated mechanism (16). The evidence presented in these studies suggests that HNO is able to modulate cGMP levels through an interaction with sGC, an idea in conflict with a previous report showing that NO is the only nitrogen oxide capable of directly activating sGC (17).HNO forms a stable adduct with the ferrous heme of deoxymyoglobin (18, 19) providing precedence for a possible interaction between HNO and sGC that is akin to the interaction of NO with ferrous sGC. In light of all the reports indicating possible HNO-mediated activation of sGC, an examination of the direct interaction of HNO with purified sGC was carried out to evaluate the possibility that HNO may be capable of directly interacting with sGC to elicit activation. Moreover, due to the previously reported thiol redox regulation of sGC (see above) and the known thiophilicity of HNO (20), we also examined the effects of HNO-mediated thiol modification on enzyme activity.     

O2- and NO-sensing mechanism through the DevSR two-component system in Mycobacterium smegmatis

The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O(2). The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis. The binding of O(2) to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O(2) and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.     

The interplay between heme iron and protein sulfhydryls in the reaction of dimeric Scapharca inaequivalvis hemoglobin with nitric oxide

The homodimeric hemoglobin from the mollusk Scapharca inaequivalvis possesses a single reactive cysteine residue per monomer, Cys92, which is located in the subunit interface in the vicinity of the heme group. The interplay between the heme iron and Cys92 towards the reaction with NO has been investigated by the combined use of electrospray mass spectrometry, FTIR and UV-Visible spectroscopy. When the ferrous liganded or unliganded protein reacts with free NO in solution Cys92 is not modified, but undergoes nitrosation when the hemoglobin is exposed to the nitric oxide releaser S-nitrosocysteine. When the ferric protein reacts with free NO under anaerobic conditions the heme iron is reduced and Cys92 is nitrosated. At variance with other hemeproteins investigated to date, in Scapharca HbI the heme-iron NO driven reduction is not accompanied by the formation of a ferric iron nitrosyl intermediate in detectable amounts. The results are consistent with the hypothesis that the nitrosating agent is the NO(+) species, which is generated during the NO driven reduction of the ferric heme iron. The possible reaction mechanism is discussed in comparison with recent findings on human hemoglobin and myoglobin.     

The redox behavior of the heme in cystathionine beta-synthase is sensitive to pH

Human cystathionine beta-synthase (CBS) is a unique pyridoxal-5'-phosphate-dependent enzyme in which heme is also present as a cofactor. Because the function of heme in this enzyme has yet to be elucidated, the study presented herein investigated possible relationships between the chemistry of the heme and the strong pH dependence of CBS activity. This study revealed, via study of a truncation variant, that the catalytic core of the enzyme governs the pH dependence of the activity. The heme moiety was found to play no discernible role in regulating CBS enzyme activity by sensing changes in pH, because the coordination sphere of the heme is not altered by changes in pH over a range of pH 6-9. Instead, pH was found to control the equilibrium amount of ferric and ferrous heme present after reaction of CBS with one-electron reducing agents. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, demonstrated that at pH 9 Fe(II) CBS is dominant while at pH 6 Fe(III) CBS is favored. At low pH, Fe(II) CBS forms transiently but reoxidizes by an apparent proton-gated electron-transfer mechanism. Regulation of CBS activity by the iron redox state has been proposed as the role of the heme moiety in this enzyme. Given that the redox behavior of the CBS heme appears to be controlled by pH, interplay of pH and oxidation state effects must occur if CBS activity is redox regulated.     

Proximal effects in the modulation of nitric oxide synthase reactivity: a QM-MM study

Nitric oxide synthases (NOS) are heme proteins that have a cysteine residue as axial ligand, which generates nitric oxide (NO). The proximal environment, specifically H-bonding between tryptophan (Trp) 178 and thiolate, has been proposed to play a fundamental role in the modulation of NOS activity. We analyzed the molecular basis of this modulation by performing electronic structure calculations on isolated model systems and hybrid quantum-classical computations of the active sites in the protein environment for wild-type and mutant (Trp 178 × Gly) proteins. Our results show that in the ferrous proteins NO exhibits a considerable trans effect. We also showed that in the ferrous (Fe⁺²) mutant NOS the absence of Trp, experimentally associated to a protonated cysteine, weakens the Fe–S bond and yields five coordinate complexes. In the ferric (Fe⁺³) state, the NO dissociation energy is shown to be slightly smaller in the mutant NOS, implying that the Fe⁺³–NO complex has a shorter half-life. We found computational evidence suggesting that ferrous NOS is favored in wild-type NOS when compared to the Trp mutant, consistently with the fact that Trp mutants have been shown to accumulate less Fe⁺²–NO dead end species. We also found that the heme macrocycle showed a significant distortion in the wild-type protein, due to the presence of the nearby Trp 178. This may also play a role in the subtle tuning of the electronic structure of the heme moiety.     

Ultrahigh resolution structures of nitrophorin 4: heme distortion in ferrous CO and NO complexes

Nitrophorin 4 (NP4), a nitric oxide (NO)-transport protein from the blood-sucking insect Rhodnius prolixus, uses a ferric (Fe3+) heme to deliver NO to its victims. NO binding to NP4 induces a large conformational change and complete desolvation of the distal pocket. The heme is markedly nonplanar, displaying a ruffling distortion postulated to contribute to stabilization of the ferric iron. Here, we report the ferrous (Fe2+) complexes of NP4 with NO, CO, and H2O formed after chemical reduction of the protein and the characterization of these complexes by absorption spectroscopy, flash photolysis, and ultrahigh-resolution crystallography (resolutions vary from 0.9 to 1.08 A). The absorption spectra, both in solution and in the crystal, are typical for six-coordinated ferrous complexes. Closure and desolvation of the distal pocket occurs upon binding CO or NO to the iron regardless of the heme oxidation state, confirming that the conformational change is driven by distal ligand polarity. The degree of heme ruffling is coupled to the nature of the ligand and the iron oxidation state in the following order: (Fe3+)-NO > (Fe2+)-NO > (Fe2+)-CO > (Fe3+)-H2O > (Fe2+)-H2O. The ferrous coordination geometry is as expected, except for the proximal histidine bond, which is shorter than typically found in model compounds. These data are consistent with heme ruffling and coordination geometry serving to stabilize the ferric state of the nitrophorins, a requirement for their physiological function. Possible roles for heme distortion and NO bending in heme protein function are discussed.     

Why iron–dithiocarbamates ensure detection of nitric oxide in cells and tissues

The in vivo mechanism of NO trapping by iron-dithiocarbamate complexes is considered. Contrary to common belief, we find that in biological systems the NO radicals are predominantly trapped by ferric iron-dithiocarbamates. Therefore, the trapping leads to ferric mononitrosyl complexes which are diamagnetic and cannot be directly detected with Electron Paramagnetic Resonance spectroscopy. The ferric mononitrosyl complexes are far easily reduced to ferrous state with L-cysteine, glutathione, ascorbate or dithiocarbamate ligands than their non-nitrosyl counterpart. When trapping NO in oxygenated biological systems, the majority of trapped nitric oxide is found in diamagnetic ferric mononitrosyl iron complexes. Only a minority fraction of NO is trapped in the form of paramagnetic ferrous mononitrosyl iron complexes with dithiocarbamate ligands. Subsequent ex vivo reduction of biological samples sharply increases the total yield of the paramagnetic mononitrosyl iron complexes. Reduction also eliminates the overlapping EPR spectrum from Cu(2+)-dithiocarbamate complexes. This facilitates the quantification of yields from NO trapping.     

Molecular basis for nitric oxide dynamics and affinity with Alcaligenes xylosoxidans cytochrome c

The bacterial heme protein cytochrome ? from Alcaligenes xylosoxidans (AXCP) reacts with nitric oxide (NO) to form a 5-coordinate ferrous nitrosyl heme complex. The crystal structure of ferrous nitrosyl AXCP has previously revealed that NO is bound in an unprecedented manner on the proximal side of the heme. To understand how the protein structure of AXCP controls NO dynamics, we performed absorption and Raman time-resolved studies at the heme level as well as a molecular computational dynamics study at the entire protein structure level. We found that after NO dissociation from the heme iron, the structure of the proximal heme pocket of AXCP confines NO close to the iron so that an ultrafast (7 ps) and complete (99 +/- 1%) geminate rebinding occurs, whereas the proximal histidine does not rebind to the heme iron on the timescale of NO geminate rebinding. The distal side controls the initial NO binding, whereas the proximal heme pocket controls its release. These dynamic properties allow the trapping of NO within the protein core and represent an extreme behavior observed among heme proteins.     

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.