Fabio Doctorovich

H2S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO-TRPA1-CGRP signalling pathway.

Nitroxyl (HNO) is a redox sibling of nitric oxide (NO) that targets distinct signalling pathways with pharmacological endpoints of high significance in the treatment of heart failure. Beneficial HNO effects depend, in part, on its ability to release calcitonin gene-related peptide (CGRP) through an unidentified mechanism. Here we propose that HNO is generated as a result of the reaction of the two gasotransmitters NO and H2S. We show that H2S and NO production colocalizes with transient receptor potential channel A1 (TRPA1), and that HNO activates the sensory chemoreceptor channel TRPA1 via formation of amino-terminal disulphide bonds, which results in sustained calcium influx. As a consequence, CGRP is released, which induces local and systemic vasodilation. H2S-evoked vasodilatatory effects largely depend on NO production and activation of HNO–TRPA1–CGRP pathway. We propose that this neuroendocrine HNO–TRPA1–CGRP signalling pathway constitutes an essential element for the control of vascular tone throughout the cardiovascular system.

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.

Physiological concentrations of melatonin inhibit the nitridergic pathway in the Syrian hamster retina.

In the present work, the effect of melatonin on the hamster retinal nitridergic pathway was examined. When the retinas were incubated in the presence of low concentrations (1 pM–10 nM) of melatonin for 15 min, a significant decrease of nitric oxide synthase (NOS) activity was observed. However, when crude retinal homogenates were preincubated with melatonin for 15 min, no changes in NOS activity were detected, despite the fact that under the same conditions trifluoperazine, a calmodulin inhibitor, significantly decreased enzymatic activity. Kinetic analysis showed that melatonin decreased the Vmax of retinal NOS without changes in the Km. On the other hand, low concentrations (100 pM) of melatonin significantly reduced retinal L‐arginine influx. A decrease in the Vmax of L‐arginine uptake was observed in the presence of melatonin, whereas the Km remained unchanged. Melatonin significantly inhibited the accumulation of cyclic guanosine monophosphate (cGMP) levels induced by both L‐arginine and sodium nitroprusside (SNP). In summary, the present results indicate that melatonin could be a potent inhibitor of the retinal nitridergic pathway.

Nitrite Reduction Mediated by Heme Models. Routes to NO and HNO

The water-soluble ferriheme model Fe(III)(TPPS) mediates oxygen atom transfer from inorganic nitrite to a water-soluble phosphine (tppts), dimethyl sulfide, and the biological thiols cysteine (CysSH) and glutathione (GSH). The products with the latter reductant are the respective sulfenic acids CysS(O)H and GS(O)H, although these reactive intermediates are rapidly trapped by reaction with excess thiol. The nitrosyl complex Fe(II)(TPPS)(NO) is the dominant iron species while excess substrate is present. However, in slightly acidic media (pH ≈ 6), the system does not terminate at this very stable ferrous nitrosyl. Instead, it displays a matrix of redox transformations linking spontaneous regeneration of Fe(III)(TPPS) to the formation of both N2O and NO. Electrochemical sensor and trapping experiments demonstrate that HNO (nitroxyl) is formed, at least when tppts is the reductant. HNO is the likely predecessor of the N2O. A key pathway to NO formation is nitrite reduction by Fe(II)(TPPS), and the kinetics of this iron-mediated transformation are described. Given that inorganic nitrite has protective roles during ischemia/reperfusion (I/R) injury to organs, attributed in part to NO formation, and that HNO may also reduce net damage from I/R, the present studies are relevant to potential mechanisms of such nitrite protection.

A Surface Effect Allows HNO/NO Discrimination by a Cobalt Porphyrin Bound to Gold

Nitroxyl (HNO) is a small short-lived molecule for which it has been suggested that it could be produced, under certain cofactors conditions, by nitric oxide (NO) synthases. Biologically relevant targets of HNO are heme proteins, thiols, molecular oxygen, NO, and HNO itself. Given the overlap of the targets and reactivity between NO and HNO, it is very difficult to discriminate their physiopathological role conclusively, and accurate discrimination between them still remains critical for interpretation of the ongoing research in this field. The high reactivity and stability of cobalt(II) porphyrins toward NO and the easy and efficient way of covalently joining porphyrins to electrodes through S-Au bonds prompted us to test cobalt(II) 5,10,15,20-tetrakis[3-(p-acetylthiopropoxy)phenyl]porphyrin [Co(P)], as a possible candidate for the electrochemical discrimination of both species. For this purpose, first, we studied the reaction between NO, NO donors, and commonly used HNO donors, with Co(II)(P) and Co(III)(P). Second, we covalently attached Co(II)(P) to gold electrodes and characterized its redox and structural properties by electrochemical techniques as well as scanning tunneling microscopy, X-ray photoelectron spectroscopy, and solid-state density functional theory calculations. Finally, we studied electrochemically the NO and HNO donor reactions with the electrode-bound Co(P). Our results show that Co(P) is positioned over the gold surface in a lying-down configuration, and a surface effect is observed that decreases the Co(III)(P) (but not Co(III)(P)NO(-)) redox potential by 0.4 V. Using this information and when the potential is fixed to values that oxidize Co(III)(P)NO(-) (0.8 V vs SCE), HNO can be detected by amperometric techniques. Under these conditions, Co(P) is able to discriminate between HNO and NO donors, reacting with the former in a fast, efficient, and selective manner with concomitant formation of the Co(III)(P)NO(-) complex, while it is inert or reacts very slowly with NO donors.

A Microscopic Study of the Deoxyhemoglobin-Catalyzed Generation of Nitric Oxide from Nitrite Anion†

There is recent evidence suggesting that nitrite anion (NO 2 (-)) represents the major intravascular NO storage molecule whose transduction to NO is facilitated by a reduction mechanism catalyzed by deoxygenated hemoglobin (deoxy-Hb). In this work, we provide a detailed microscopic study of deoxy-Hb nitrite reductase (NIR) activity by combining classical molecular dynamics and hybrid quantum mechanical-molecular mechanical simulations. Our results point out that two alternative mechanisms could be operative and suggest that the most energetic barriers should stem from either reprotonation of the distal histidine or NO dissociation from the ferric heme. In the first proposed mechanism, which is similar to that proposed for bacterial NIRs, nitrite anion or nitrous acid coordinates to the heme through the N atom. This pathway involves HisE7 in a one or two proton transfer process, depending on whether the active species is nitrite anion or nitrous acid, to yield an intermediate Fe(III)NO species which eventually dissociates leading to NO and methemoglobin. In the second mechanism, the nitrite anion coordinates to the heme through the O atom. This pathway requires only one proton transfer from HisE7 and leads directly to the formation of a hydroxo Fe(III) complex and NO.

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.

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.

Nitrosation of melatonin by nitric oxide: a computational study

Melatonin is being increasingly promoted as a therapeutic agent for the treatment of jet lag and insomnia, and is an efficient free radical scavenger. We have recently characterized a product for the reaction of melatonin with nitric oxide (NO), N‐nitrosomelatonin. In the present work, reaction pathways with N1, C2, C4, C6 and C7 as possible targets for its reaction with NO that yield the respective nitroso derivatives have been investigated using semiempirical AM1 computational tools, both in vacuo and aqueous solution. Specifically, two different pathways were studied: a radical mechanism involving the hydrogen atom abstraction to yield a neutral radical followed by NO addition, and an ionic mechanism involving addition of nitrosonium ion to the indolic moiety. Our results show that the indolic nitrogen is the most probable site for nitrosation by the radical mechanism, whereas different targets are probable considering the ionic pathway. These results are in good agreement with previous experimental findings and provide a coherent picture for the interaction of melatonin with NO.

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.