Nitric Oxide Reacts Very Fast with Hydrogen Sulfide, Alcohols, and Thiols to Produce HNO: Revised Rate Constants.

The chemical reactivity of NO and its role in several biological processes seem well established. Despite this, the chemical reduction of •NO toward HNO has been historically discarded, mainly because of the negative reduction potential of NO. However, this value and its implications are nowadays under revision. The last reported redox potential, E'(NO,H+/HNO), at micromolar and picomolar concentrations of •NO and HNO, respectively, is between -0.3 and 0 V at pH 7.4. This potential implies that the one-electron-reduction process for NO is feasible under biological conditions and could be promoted by well-known biological reductants with reduction potentials of around -0.3 to -0.5 V. Moreover, the biologically compatible chemical reduction of •NO (nonenzymatic), like direct routes to HNO by alkylamines, aromatic and pseudoaromatic alcohols, thiols, and hydrogen sulfide, has been extensively explored by our group during the past decade. The aim of this work is to use a kinetic modeling approach to analyze electrochemical HNO measurements and to report for the first-time direct reaction rate constants between •NO and moderate reducing agents, producing HNO. These values are between 5 and 30 times higher than the previously reported keff values. On the other hand, we also showed that reaction through successive attack by two NO molecules to biologically compatible compounds could produce HNO. After over 3 decades of intense research, the •NO chemistry is still there, ready to be discovered.

HNO Is Produced by the Reaction of NO with Thiols.

Azanone (nitroxyl, HNO) is a highly reactive compound whose biological role is still a matter of debate. One possible route for its formation is NO reduction by biological reductants. These reactions have been historically discarded due to the negative redox potential for the NO,H+/HNO couple. However, the NO to HNO conversion mediated by vitamins C, E, and aromatic alcohols has been recently shown to be feasible from a chemical standpoint. Based on these precedents, we decided to study the reaction of NO with thiols as potential sources of HNO. Using two complementary approaches, trapping by a Mn porphyrin and an HNO electrochemical sensor, we found that under anaerobic conditions aliphatic and aromatic thiols (as well as selenols) are able to convert NO to HNO, albeit at different rates. Further mechanistic analysis using ab initio methods shows that the reaction between NO and the thiol produces a free radical adduct RSNOH•, which reacts with a second NO molecule to produce HNO and a nitrosothiol. The nitrosothiol intermediate reacts further with RSH to produce a second molecule of HNO and RSSR, as previously reported.

Nitric oxide is reduced to HNO by proton-coupled nucleophilic attack by ascorbate, tyrosine, and other alcohols. A new route to HNO in biological media?

The role of NO in biology is well established. However, an increasing body of evidence suggests that azanone (HNO), could also be involved in biological processes, some of which are attributed to NO. In this context, one of the most important and yet unanswered questions is whether and how HNO is produced in vivo. A possible route concerns the chemical or enzymatic reduction of NO. In the present work, we have taken advantage of a selective HNO sensing method, to show that NO is reduced to HNO by biologically relevant alcohols with moderate reducing capacity, such as ascorbate or tyrosine. The proposed mechanism involves a nucleophilic attack to NO by the alcohol, coupled to a proton transfer (PCNA: proton-coupled nucleophilic attack) and a subsequent decomposition of the so-produced radical to yield HNO and an alkoxyl radical.

Reactions of HNO with metal porphyrins: underscoring the biological relevance of HNO.

Azanone ((1)HNO, nitroxyl) shows interesting yet poorly understood chemical and biological effects. HNO has some overlapping properties with nitric oxide (NO), sharing its biological reactivity toward heme proteins, thiols, and oxygen. Despite this similarity, HNO and NO show significantly different pharmacological effects. The high reactivity of HNO means that studies must rely on the use of donor molecules such as trioxodinitrate (Angeli's salt). It has been suggested that azanone could be an intermediate in several reactions and that it may be an enzymatically produced signaling molecule. The inherent difficulty in detecting its presence unequivocally prevents evidence from yielding definite answers. On the other hand, metalloporphyrins are widely used as chemical models of heme proteins, providing us with invaluable tools for the study of the coordination chemistry of small molecules, like NO, CO, and O2. Studies with transition metal porphyrins have shown diverse mechanistic, kinetic, structural, and reactive aspects related to the formation of nitrosyl complexes. Porphyrins are also widely used in technical applications, especially when coupled to a surface, where they can be used as electrochemical gas sensors. Given their versatility, they have not escaped their role as key players in chemical studies involving HNO. This Account presents the research performed during the last 10 years in our group concerning azanone reactions with iron, manganese, and cobalt porphyrins. We begin by describing their HNO trapping capabilities, which result in formation of the corresponding nitrosyl complexes. Kinetic and mechanistic studies of these reactions show two alternative operating mechanisms: reaction of the metal center with HNO or with the donor. Moreover, we have also shown that azanone can be stabilized by coordination to iron porphyrins using electron-attracting substituents attached to the porphyrin ring, which balance the negatively charged NO¯. Second, we describe an electrochemical HNO sensing device based on the covalent attachment of a cobalt porphyrin to gold. A surface effect affects the redox potentials and allows discrimination between HNO and NO. The reaction with the former is fast, efficient, and selective, lacking spurious signals due to the presence of reactive nitrogen and oxygen species. The sensor is both biologically compatible and highly sensitive (nanomolar). This time-resolved detection allows kinetic analysis of reactions producing HNO. The sensor thus offers excellent opportunities to be used in experiments looking for HNO. As examples, we present studies concerning (a) HNO donation capabilities of new HNO donors as assessed by the sensor, (b) HNO detection as an intermediate in O atom abstraction to nitrite by phosphines, and (c) NO to HNO interconversion mediated by alcohols and thiols. Finally, we briefly discuss the key experiments required to demonstrate endogenous HNO formation to be done in the near future, involving the in vivo use of the HNO sensing device.

Redox potential determines the reaction mechanism of HNO donors with Mn and Fe porphyrins: defining the better traps.

Azanone ((1)HNO, nitroxyl) is a highly reactive molecule with interesting chemical and biological properties. Like nitric oxide (NO), its main biologically related targets are oxygen, thiols, and metalloproteins, particularly heme proteins. As HNO dimerizes with a rate constant between 10(6) and 10(7) M(-1) s(-1), reactive studies are performed using donors, which are compounds that spontaneously release HNO in solution. In the present work, we studied the reaction mechanism and kinetics of two azanone donors Angelís Salt and toluene sulfohydroxamic acid (TSHA) with eight different Mn porphyrins as trapping agents. These porphyrins differ in their total peripheral charge (positively or negatively charged) and in their Mn(III)/Mn(II) reduction potential, showing for each case positive (oxidizing) and negative (reducing) values. Our results show that the reduction potential determines the azanone donor reaction mechanism. While oxidizing porphyrins accelerate decomposition of the donor, reducing porphyrins react with free HNO. Our results also shed light into the donor decomposition mechanism using ab initio methods and provide a thorough analysis of which MnP are the best candidates for azanone trapping and quantification experiments.

Time-resolved electrochemical quantification of azanone (HNO) at low nanomolar level.

Azanone (HNO, nitroxyl) is a highly reactive and short-lived compound with intriguing and highly relevant properties. It has been proposed to be a reaction intermediate in several chemical reactions and an in vivo, endogenously produced key metabolite and/or signaling molecule. In addition, its donors have important pharmacological properties. Therefore, given its relevance and elusive nature (it reacts with itself very quickly), the development of reliable analytical methods for quantitative HNO detection is in high demand for the advancement of future research in this area. During the past few years, several methods were developed that rely on chemical reactions followed by mass spectrometry, high-performance liquid chromatography, UV-vis, or fluorescence-trapping-based methodologies. In this work, our recently developed HNO-sensing electrode, based on the covalent attachment of cobalt(II) 5,10,15,20-tetrakis[3-(p-acetylthiopropoxy)phenyl] porphyrin [Co(P)] to a gold electrode, has been thoroughly characterized in terms of sensibility, accuracy, time-resolved detection, and compatibility with complex biologically compatible media. Our results show that the Co(P) electrode: (i) allows time-resolved detection and kinetic analysis of the electrode response (the underlying HNO-producing reactions can be characterized) (ii) is able to selectively detect and reliably quantify HNO in the 1-1000 nM range, and (iii) has good biological media compatibility (including cell culture), displaying a lack of spurious signals due to the presence of O2, NO, and other reactive nitrogen and oxygen species. In summary, the Co(P) electrode is to our knowledge the best prospect for use in studies investigating HNO-related chemical and biological reactions.

Small ligand-globin interactions: reviewing lessons derived from computer simulation.

In this work we review the application of classical and quantum-mechanical atomistic computer simulation tools to the investigation of small ligand interaction with globins. In the first part, studies of ligand migration, with its connection to kinetic association rate constants (kon), are presented. In the second part, we review studies for a variety of ligands such as O2, NO, CO, HS(-), F(-), and NO2(-) showing how the heme structure, proximal effects, and the interactions with the distal amino acids can modulate protein ligand binding. The review presents mainly results derived from our previous works on the subject, in the context of other theoretical and experimental studies performed by others. The variety and extent of the presented data yield a clear example of how computer simulation tools have, in the last decade, contributed to our deeper understanding of small ligand interactions with globins. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.

The pH of HNO donation is modulated by ring substituents in Piloty's acid derivatives: azanone donors at biological pH.

A group of Piloty's acid (N-hydroxybenzenesulfonamide) derivatives were synthesized and fully characterized in order to assess the rates and pH of HNO (azanone, nitroxyl) donation in aqueous media. The derivatives, with electron-withdrawing and -donating substituents include methyl, nitro, fluoro, tri-isopropyl, trifluoromethyl and methoxy groups. The most interesting modulation observed is the change in pH range in which the compounds are able to donate HNO. UV-visible kinetic measurements at different pH values were used to evaluate the decomposition rate of the donors. A novel technique based on electrochemical measurements using a Co-porphyrin sensor was used to assess the release of HNO as a function of pH, by direct measurement of [HNO]. The results were contrasted with DFT calculations in order to understand the electronic effects exerted by the ring substituents, which drastically modify the pH range of donation. For example, while Piloty's acid donates HNO from pH 9.3, the corresponding fluoro derivative starts donating at pH 4.0.

Tetrachlorocarbonyliridates: water-soluble carbon monoxide releasing molecules rate-modulated by the sixth ligand.

A new family of compounds is presented as potential carbon monoxide releasing molecules (CORMs). These compounds, based on tetrachlorocarbonyliridate(III) derivatives, were synthesized and fully characterized by X-ray diffraction, electrospray mass spectrometry, IR, NMR, and density functional theory calculations. The rate of CO release was studied via the myoglobin assay. The results showed that the rate depends on the nature of the sixth ligand, trans to CO, and that a significant modulation on the release rate can be produced by changing the ligand. The reported compounds are soluble in aqueous media, and the rates of CO release are comparable with those for known CORMs, releasing CO at a rate of 0.03-0.58 µM min(-1) in a 10 µM solution of myoglobin and 10 µM of the complexes.

Protein dynamics and ligand migration interplay as studied by computer simulation.

Since proteins are dynamic systems in living organisms, the employment of methodologies contemplating this crucial characteristic results fundamental to allow revealing several aspects of their function. In this work, we present results obtained using classical mechanical atomistic simulation tools applied to understand the connection between protein dynamics and ligand migration. Firstly, we will present a review of the different sampling schemes used in the last years to obtain both ligand migration pathways and the thermodynamic information associated with the process. Secondly, we will focus on representative examples in which the schemes previously presented are employed, concerning the following: i) ligand migration, tunnels, and cavities in myoglobin and neuroglobin; ii) ligand migration in truncated hemoglobin members; iii) NO escape and conformational changes in nitrophorins; iv) ligand selectivity in catalase and hydrogenase; and v) larger ligand migration: the P450 and haloalkane dehalogenase cases. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.

Role of heme distortion on oxygen affinity in heme proteins: the protoglobin case.

The chemical properties of heme proteins largely reflect the electronic properties of their heme group. Often, the porphyrin ring of the heme exhibits significant distortions from its isolated structure, but the impact of these distortions on the chemical properties of the heme is yet uncertain. A systematic study focused on the effects of the distortion of the macrocycle on the binding affinity for oxygen is presented. The results show that out-of-plane distortions decrease the binding affinity, while in-plane distortions can increase or decrease it. Among in-plane distortions, only the breathing mode, which involves the symmetric compression-expansion of the porphyrin ring, strongly modulates the binding affinity. These findings shed light into the peculiar binding affinity of Methanosarcina acetivorans protoglobin, a protein that contains a highly distorted heme. Overall, the results highlight that in-plane distortions might be exploited by certain classes of heme proteins to modulate the ligand affinity.

Successful stabilization of the elusive species {FeNO}8 in a heme model.

Nitroxyl (HNO/NO(-)) heme-adducts have been postulated as intermediates in a variety of catalytic processes carried out by different metalloenzymes. Hence, there is growing interest in obtaining and characterizing heme model nitroxyl complexes. The one-electron chemical reduction of the {FeNO}(7) nitrosyl derivative of Fe(III)(TFPPBr(8))Cl, Fe(II)(TFPPBr(8))NO (1) (TFPPBr(8) = 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-[Tetrakis-(pentafluorophenyl)]porphyrin) with cobaltocene yields the significantly stable {FeNO}(8) complex, [Co(C(5)H(5))(2)](+)[Fe(TFPPBr(8))NO](-) (2). Complex 2 was isolated and characterized by UV-vis, FTIR, (1)H and (15)N NMR spectroscopies. In addition, DFT calculations were performed to get more insight into the structure of 2. According to the spectroscopic and DFT results, we can state unequivocally that the surprisingly stable complex 2 is the elusive {FeNO}(8) species. Both experimental and computational data allow to assign the electronic structure of 2 as intermediate between Fe(II)NO(-) and Fe(I)NO, which is contrasted with the predominant Fe(II)NO(-) character of known nonheme {FeNO}(8) complexes. The enhanced stability achieved for a heme model {FeNO}(8) is expected to allow further studies related to the reactivity of this elusive species.

DFT study on the reactivity of iron porphyrins tuned by ring substitution.

The effect of beta-substituents (-NO2, -Br, -OCH3) in the reactivity of Fe(II) and Fe(III) porphyrins is studied by means of density functional theory (DFT) calculations. The binding of nitric oxide, carbon monoxide and dioxygen (NO, CO, O2) was explored due to the relevance of their interactions in the chemistry of heme proteins and in biomimetic catalysis. The binding capability (BC) of the porphyrins was found to be strongly modulated both by the donor and attractor substituents used in the work. Unexpectedly, we found that the BC of Fe(II) porphyrins is mainly decreased for the diatomic ligands, when both donor or withdrawing substituents were considered. This effect was particularly significant when the ligand was oxygen. The correlation of Fe-X and X-O (X=N, C, O) bond distances is explained in terms of backdonation effects.

Oxygen affinity controlled by dynamical distal conformations: the soybean leghemoglobin and the Paramecium caudatum hemoglobin cases.

The binding of diatomic ligands, such as O(2), NO, and CO, to heme proteins is a process intimately related with their function. In this work, we analyzed by means of a combination of classical Molecular Dynamics (MD) and Hybrid Quantum-Classical (QM/MM) techniques the existence of multiple conformations in the distal site of heme proteins and their influence on oxygen affinity regulation. We considered two representative examples: soybean leghemoglobin (Lba) and Paramecium caudatum truncated hemoglobin (PcHb). The results presented in this work provide a molecular interpretation for the kinetic, structural, and mutational data that cannot be obtained by assuming a single distal conformation.

Modeling heme proteins using atomistic simulations.

Heme proteins are found in all living organisms, and perform a wide variety of tasks ranging from electron transport, to the oxidation of organic compounds, to the sensing and transport of small molecules. In this work we review the application of classical and quantum-mechanical atomistic simulation tools to the investigation of several relevant issues in heme proteins chemistry: (i) conformational analysis, ligand migration, and solvation effects studied using classical molecular dynamics simulations; (ii) electronic structure and spin state energetics of the active sites explored using quantum-mechanics (QM) methods; (iii) the interaction of heme proteins with small ligands studied through hybrid quantum mechanics-molecular mechanics (QM-MM) techniques; (iv) and finally chemical reactivity and catalysis tackled by a combination of quantum and classical tools.

Two distinct heme distal site states define Cerebratulus lacteus mini-hemoglobin oxygen affinity.

The nerve tissue hemoglobin of Cerebratulus lacteus (CerHb) is the smallest naturally occurring known hemoglobin. Stabilization of the diatomic bound species (e.g., O(2)) is achieved through a network of hydrogen bonds based on three key residues TyrB10, GlnE7, and ThrE11. The first two residues are typically associated in hemoglobins with enhanced O(2) affinity, related to hydrogen bond stabilization of the heme-bound O(2) resulting in a decrease of the ligand dissociation rates. In contrast to the above observations, the affinity of CerHb for O(2) is only moderate, and the rate of O(2) dissociation is unexpectedly high. To gain insight on the diverse molecular mechanisms controlling ligand affinities, we have analyzed w.t. CerHb and its ThrE11-->Val mutant by means of joint molecular dynamics and quantum mechanics simulation techniques, complementing recent site-directed mutagenesis experiments. Our results suggest that the observed O(2) dissociation rates can only be explained through a dynamic equilibrium between high and low affinity states of the w.t. CerHb heme distal site.

Dioxygen affinity in heme proteins investigated by computer simulation.

We present an investigation of the molecular basis of the modulation of oxygen affinity in heme proteins using computer simulation. QM-MM calculations are applied to explore distal and proximal effects on O(2) binding to the heme, while classical molecular dynamics simulations are employed to investigate ligand migration across the polypeptide to the active site. Trends in binding energies and in the kinetic constants are illustrated through a number of selected examples highlighting the virtues and the limitations of the applied methodologies. These examples cover a wide range of O(2)-affinities, and include: the truncated-N and truncated-O hemoglobins from Mycobacterium tuberculosis, the mammalian muscular O(2) storage protein: myoglobin, the hemoglobin from the parasitic nematode Ascaris lumbricoides, the oxygen transporter in the root of leguminous plants: leghemoglobin, the Cerebratulus lacteus nerve tissue hemoglobin, and the Alcaligenes xyloxidans cytochrome c'.

Solvation and structure of LiAlH(4) in ethereal solvents.

The nature of the solute species present in ethereal solutions of LiAlH(4) is of crucial importance for understanding the mechanisms for the reduction of ketones and other functional groups by LiAlH(4). We have employed a combination of theoretical and experimental techniques to investigate the structure of LiAlH(4) in ethereal solutions. Using complexation agents, we measured the IR spectra of LiAlH(4) and AlH(4)(-) in tetrahydrofuran (THF). Hybrid quantum-classical (QM-MM) simulations have also been carried out to compute the IR spectra of associated and dissociated LiAlH(4) species and the free-energy profile for the dissociation process in solution. Our experimental estimate of the dissociation constant in THF is 0.021 +/- 0.002, while the predicted computational value corresponding to a model dimethyl ether solvent is 0.001. The free-energy profile shows only one minimum corresponding to a contact ion pair at a Li-Al separation distance of 3.0 A. These results are consistent with the fact that LiAlH(4) is essentially associated in ethereal solutions forming contact ion pairs.