HNO Production Research Articles

Theoretical insight into the NO* Coverage-Dependent selectivity of Pd and Cu electrocatalysts for nitrate reduction

Nitrate (NO3−) is a concerning contaminant in groundwater that is harmful to human health. Electrocatalytic reduction of NO3− can be achieved with transition metals such as Pd and Cu. In this work, we apply density functional theory (DFT) to discern how the electronic properties of the metal catalyst affect the selectivity of the nitrate reduction mechanism toward either N2 products (e.g., N2 and N2O) or N1 products (e.g., NH3 and NH4+). We find that the greater d-band filling of Cu results in more electron accumulation on the N atom of adsorbed NO*, which drives N–H bond formation favoring the production of HNO* and eventually N1 products (NH3). Conversely, the more delocalized d orbitals of Pd lead to a strong adsorbate–adsorbate coverage effect that lowers key N–N coupling barriers at high NO* coverage favoring selectivity toward N2 products (N2 and N2O). This work elucidates why, at the electronic-structure level, nitrate reduction is highly sensitive to NO* coverage on some metals (such as Pd) and less so on others (such as Cu).

Front Cover: Update on Endogenous HNO Production: An Exploration of Unanswered Questions and Challenges (Eur. J. Inorg. Chem. 21/2024)

Front Cover: Update on Endogenous HNO Production: An Exploration of Unanswered Questions and Challenges (Eur. J. Inorg. Chem. 21/2024)

A dual-response fluorescent probe to reveal the role of ferroptosis in drug-induced liver injury

Drug-induced liver injury (DILI) has already been recognized as a formidable obstacle in global health issues. Despite the proposed common cellular death types in DILI, exploration of alternative mechanisms such as ferroptosis for prevention and therapy remains limited. Visualization of the role of ferroptosis in DILI holds profound promise in advancing diagnostic and therapeutic strategies for DILI. To address this, herein, a unique multi-functional fluorescent probe, VIS-HNO, was elaborately built up and endowed with the extraordinary performance of excellent selectivity and high sensitivity toward HNO and viscosity. Employing the probe VIS-HNO with the confocal fluorescence imaging, the up-regulations of HNO and viscosity in the process of DILI and ferroptosis were disclosed. Then the remediation of DILI was achieved by VIS-HNO under the treatment of ferrostatin1 (Fer-1), indicating its therapeutic value for DILI. In addition, taking into account the significance of HNO in determining the role of ferroptosis in DILI, the pathway of HNO production in DILI was further explored, which presented essential insights into the role of ferroptosis in DILI. Overall, these findings demonstrated that VIS-HNO may be a potent tool to aid in diagnosing ferroptosis and DILI, more importantly, to understand the role of ferroptosis in DILI by evaluating HNO and viscosity level, which will facilitate the exploration of unique strategies for DILI treatment.

Update on Endogenous HNO Production:An Exploration of Unanswered Questions and Challenges

This review consolidates the evidence supporting endogenous HNO generation through enzymatic and non‐enzymatic pathways, emphasizing advancements in real‐time HNO sensing within living systems. Azanone (nitroxyl, HNO) is the one‐electron‐reduced congener of nitric oxide (NO•) and shares various biological actions. HNO exhibits unique properties, including positive inotropic and lusitropic effects, along with resistance to scavenging by reactive oxygen species (ROS), particularly superoxide. Research on HNO has intensified over the last two decades, focusing on its reactivity and the prospect of endogenous formation due to its potential significance. The high reactivity of HNO arises from reactions with biologically relevant species such as oxygen, NO•, and thiols, as well as self‐dimerization. Detecting and quantifying HNO has posed challenges, initially relying on nitrous oxide (N2O) detection, a byproduct of dimerization. Recent advancements, including fluorescent probes and a specific electrochemical nanomolar‐level sensor, have facilitated direct detection, enhancing understanding of HNO reactivity and formation.

Decoding the enigmatic last gasotransmitter. The intriguing journey of HNO: From hidden origins to clues on reactivity and detection

This review focuses on HNO, a molecule of immense chemical and biological importance that has intrigued scientists for decades. Despite its elusive and transient nature, HNO may play an important role in various physiological processes, particularly in cardiovascular regulation. This review thoroughly examines the formation, chemical properties, and biological significance of HNO and highlights ongoing research efforts to unravel its mysteries. Challenges in studying HNO arise from its high reactivity, short half-life, and complex interactions with other nitrogen oxides, particularly nitric oxide. Detection and quantification of HNO in biological systems pose difficulties, prompting the development of advanced techniques. Active research into endogenous HNO formation is revealing intricate pathways within biological systems, the elucidation of which is crucial for exploiting its therapeutic potential. The multifaceted role of HNO in cardiovascular regulation, influencing vasorelaxation, blood pressure reduction, and enhanced cardiac contractility, underscores its profound impact on the circulatory system. Ongoing research holds promise for treating conditions such as hypertension and heart failure. As clinical applications expand, HNO research may unlock treatments for cardiovascular disease, inflammatory disorders, and cancer. The recent discovery of endogenous HNO production in plants adds a new dimension. While numerous clues have emerged, the scientific saga underscores that mysteries persist, evolve, and beckon to perpetual exploration in the realm of science.

Pyruvate dehydrogenase operates as an intramolecular nitroxyl generator during macrophage metabolic reprogramming

M1 macrophages enter a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase 2 and pyruvate dehydrogenase (PDH) activity. Here, we provide evidence that NO targets the PDH complex by using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 enzyme, also known as dihydrolipoamide dehydrogenase (DLD), is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated production of HNO, which interacts with thiols forming irreversible modifications including sulfinamide. In addition, we reveal a macrophage signature of proteins with reduction-resistant modifications, including in DLD, and identify potential HNO targets. Consistently, DLD enzyme is modified in an HNO-dependent manner at Cys477 and Cys484, and molecular modeling and mutagenesis show these modifications impair the formation of DLD homodimers. In conclusion, our work demonstrates that HNO is produced physiologically. Moreover, the production of HNO is dependent on the lipoate-rich PDH complex facilitating irreversible modifications that are critical to NO-dependent metabolic rewiring.

Pyruvate Dehydrogenase Operates as an Intramolecular Nitroxyl Generator During Macrophage Metabolic Reprogramming

Abstract M1 macrophages “commit” to a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase (Aco)-2 and pyruvate dehydrogenase (PDH). Here we provide evidence that NOdirectly targets the PDH complex using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 subunit is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated HNO production that interacts with thiols forming modifications resistant to reduction. Consequently, we reveal a macrophage signature of proteins with reduction resistant modifications, including E3, and identify a subset of potential HNO targets. Consistently, recombinant E3 is modified in HNO-dependent manner at Cys 477and Cys 484, and molecular modeling shows these cause impairment of functional E3 homodimer formation. Our work demonstrates that HNO is produced physiologically, and its presence is peculiar to the lipoate rich PDH complex facilitating irreversible modifications critical in NO-dependent metabolic rewiring.

Light‐induced NO release from iron‐nitrosyl‐thiolato complex: The role of noncovalent thiol/thioether

AbstractFour low‐spin {FeNO}6 complexes, [Fe(NO)(PS2)(PS2H)] (1, PS2H2 = bis(2‐dimercaptophenyl)phenylphosphine) with a pendant thiol, [Fe(NO)(PS2)(PS2CH3)] (2) bearing a pendant thioether, and [Fe(NO)(PS2)(RPS)] (RH, 4a; RTMS, 4b) without the noncovalent thiol/thioether group are spectroscopically and structurally characterized. In comparisons of the νNO, absorption energy in UV/vis spectra and structural parameters from single X‐ray diffraction studies, the four iron‐nitrosyl‐thiolato compounds share similarity in electronic structure. Complex 1 with a pendant thiol leads to NO and HNO production upon exposure to the light. Photolysis of 2 bearing a pendant thioether only affords NO. Effective detection of HNO or NO from 1 or 2 is achieved by the employment of [MnIII(TMSPS3)(DABCO)]. In contrast, 4a and 4b show inertness toward visible‐light stimulus. Photolysis and having pendant thiol/thioether group play key roles in NO production from these iron‐nitrosyl‐thiolato complexes, that is, the Fe‐NO bond is weakened by exposure to light and the noncovalent SH of 1 or SCH3 of 2 can serve as an incoming ligand to interact with Fe atom, resulting in a transient with intramolecular [RS⋅⋅⋅Fe⋅⋅⋅NO] interaction (RH and CH3) which could facilitate NO dissociation.

A modular scaffold for triggerable and tunable nitroxyl (HNO) generation with a fluorescence reporter.

Nitroxyl (HNO) is a short-lived mediator of cell signalling and can enhance the sulfane sulfur pool, a cellular antioxidant reservoir, by reacting with hydrogen sulfide (H2S). Here, we report esterase-activated HNO-generators that are suitable for tunable HNO release and the design of these donors allows for real-time monitoring of HNO release. These tools will help gain a better understanding of the cross-talk among short-lived gaseous signalling molecules that have emerged as major players in health and disease.

A bifunctional fluorescent probe based on “AND logic” for the simultaneous recognition of H2S/HNO and its bioimaging applications

The reaction of NO and H2S to form HNO is a classical pathway in physiological conditions. The reported single recognition-type fluorescent probes are difficult to track precisely the relationships of H2S and HNO. It is necessary to develop a bifunctional fluorescence probe (NJA) for monitoring simultaneously the production of endogenous HNO and H2S. Using 7-Nitrobenzofurazan (NBD) and 2-(diphenylphosphine) benzoate as recognition sites, the obatined NJA can detect specifically HS− and HNO. The detection limit of HS− and HNO are 0.46 μM and 1.42 μM, respectively. Based on the dual recognition sites and input signals of the probe, a molecular “AND” logic gate was established to detect successfully H2S and HNO in MCF-7 cells. NJA based on “AND logic” provided a simple and robust tool for monitoring the production of endogenous HNO correlative with H2S and NO in living cells.

The Underlying Mechanism of HNO Production by the Myoglobin-Mediated Oxidation of Hydroxylamine.

Azanone (HNO, nitroxyl) is a highly reactive molecule that, in the past few years, has drawn significant interest because of its pharmacological properties. However, the understanding of how, when, and where endogenous HNO is produced remains a matter of discussion. In this study, we examined the ability of myoglobin to produce HNO via the peroxidation of hydroxylamine with H2O2 using both experimental and computational approaches. The production of HNO was confirmed using an azanone selective electrochemical method and by the detection of N2O using FTIR. The catalytic capacity of myoglobin was characterized by the determination of the turnover number. The reaction kinetics of the hydroxylamine peroxidation were studied by both electrochemical and UV-vis methods. Further evidence about the reaction mechanism was obtained by EPR spectroscopy. Additionally, quantum mechanical/molecular mechanics experiments were performed to calculate the energy barrier for HNO production and to gain insight into the reaction mechanism. Our results confirm that myoglobin produces HNO via the peroxidation of hydroxylamine with a great catalytic capacity. In addition, our mechanistic study allows us to state that the Mb ferryl state is the most likely intermediate that reacts with hydroxylamine, yielding important evidence for endogenous HNO generation.

Photoinduced NO and HNO Production from Mononuclear {FeNO}6 Complex Bearing a Pendant Thiol.

Light triggers the formation of HNO from a metal-nitrosyl species, facilitated by an intramolecular pendant thiol proton. Two {FeNO}6 complexes (the Enemark-Felthan notation), [Fe(NO)(TMSPS2)(TMSPS2H)] (1, TMSPS2H2 = 2,2'-dimercapto-3,3'-bis(trimethylsilyl)diphenyl)phenylphosphine; H is a dissociable proton) with a pendant thiol and [Fe(NO)(TMSPS2)(TMSPS2CH3)] (2) bearing a pendant thioether, are spectroscopically and structurally characterized. Both complexes are highly sensitive to visible light. Upon photolysis, complex 2 undergoes NO dissociation to yield a mononuclear Fe(III) complex, [Fe(TMSPS2)(TMSPS2CH3)] (3). In contrast, the pendant SH of 1 can act as a trap for the departing NO radical upon irradiation, resulting in the formation of an intermediate A with an intramolecular [SH···ON-Fe] interaction. As suggested by computational results (density functional theory), the NO stretching frequency (νNO) is sensitive to the intramolecular interaction between the pendant ligand and the iron-bound NO, and a shift of νNO from 1833 (1) to 1823 cm-1 (A) is observed experimentally. Subsequent photolysis of the intermediate A results in HNO production and a thiyl group that then coordinates to the Fe center for the formation of [Fe(TMSPS2)2] (4). In contrast with the common acid-base coupling pathway, the HNO is not voluntarily yielded from 1 but rather is generated by the photopromoted pathway. The photogenerated HNO can further react with [MnIII(TMSPS3)(DABCO)] (TMSPS3H3 = (2,2'2''-trimercapto-3,3',3''-tris(trimethylsilyl)triphenylphosphine; DABCO = 1,4-diazabicyclo[2.2.2]octane) in organic media to yield anionic [Mn(NO)(TMSPS3)]- (5-) with a {MnNO}6 electronic configuration, whereas [MnIII(TMSPS3)(DABCO)] reacts with NO gas for the formation of a {MnNO}5 species, [Mn(NO)(TMSPS3)] (6). Effective differentiation of the formation of HNO from complex 1 with the pendant SH versus NO from 2 with the pendant SMe is achieved by the employment of [MnIII(TMSPS3)(DABCO)].

Using reduced density matrix techniques to capture static and dynamic correlation in the energy landscape for the decomposition of the CH2CH2ONO radical and support a non-IRC pathway.

The unexpected abundance of HNO in the photodecomposition of the radical 2-nitrosooxy ethyl (CH2CH2ONO) is investigated through calculations of the potential energy surface by the anti-Hermitian contracted Schrödinger equation (ACSE) method, which directly generates the 2-electron reduced density matrix. The ACSE, which is able to balance single-reference (dynamic) and multi-reference (static) correlation effects, reveals some subtle correlation effects along the intrinsic reaction coordinate (IRC) en route to NO + oxirane, an IRC which offers a potential bifurcation to the HNO + vinoxy product channel. These effects were not fully captured by either single-reference techniques, such as coupled cluster, or multi-reference techniques, such as second-order multi-reference perturbation theory. These correlation effects reveal small to moderate energy changes in key transition states, which have implications for the reaction mechanism as related to the production of HNO.

Covalent attachment of the heme to Synechococcus hemoglobin alters its reactivity toward nitric oxide

The cyanobacterium Synechococcus sp. PCC 7002 produces a monomeric hemoglobin (GlbN) implicated in the detoxification of reactive nitrogen and oxygen species. GlbN contains a b heme, which can be modified under certain reducing conditions. The modified protein (GlbN-A) has one heme–histidine C–N linkage similar to the C–S linkage of cytochrome c. No clear functional role has been assigned to this modification. Here, optical absorbance and NMR spectroscopies were used to compare the reactivity of GlbN and GlbN-A toward nitric oxide (NO). Both forms of the protein are capable of NO dioxygenase activity and both undergo heme bleaching after multiple NO challenges. GlbN and GlbN-A bind NO in the ferric state and form diamagnetic complexes (FeIII–NO) that resist reductive nitrosylation to the paramagnetic FeII–NO forms. Dithionite reduction of FeIII–NO GlbN and GlbN-A, however, resulted in distinct outcomes. Whereas GlbN-A rapidly formed the expected FeII–NO complex, NO binding to FeII GlbN caused immediate heme loss and, remarkably, was followed by slow heme rebinding and HNO (nitrosyl hydride) production. Additionally, combining FeIII GlbN, 15N-labeled nitrite, and excess dithionite resulted in the formation of FeII–H15NO GlbN. Dithionite-mediated HNO production was also observed for the related GlbN from Synechocystis sp. PCC 6803. Although ferrous GlbN-A appeared capable of trapping preformed HNO, the histidine–heme post-translational modification extinguished the NO reduction chemistry associated with GlbN. Overall, the results suggest a role for the covalent modification in FeII GlbNs: protection from NO-mediated heme loss and prevention of HNO formation.

Solvation and Proton-Coupled Electron Transfer Reduction Potential of 2NO• to 1HNO in Aqueous Solution: A Theoretical Investigation.

In this work, quantum mechanical calculations and Monte Carlo statistical mechanical simulations were carried out to investigate the solvation properties of HNO in aqueous solution and to evaluate the proton-coupled one electron reduction potential of 2NO to 1HNO, which is essential missing information to understand the fate of 2NO in the biological medium. Our results showed that the 1HNO molecule acts mainly as a hydrogen bond donor in aqueous solution with an average energy of -5.5 ± 1.3 kcal/mol. The solvation free energy of 1HNO in aqueous solution, computed using three approaches based on the linear response theory, revealed that the current prediction of the hydration free energy of HNO is, at least, 2 times underestimated. We proposed two pathways for the production of HNO through reduction of NO. The first pathway is the direct reduction of NO through proton-coupled electron transfer to produce HNO, and the second path is the reduction of the radical anion HONO•-, which is involved in equilibrium with NO in aqueous solution. We have shown that both pathways are viable processes under physiological conditions, having reduction potentials of E°' = -0.161 V and E°' ≈ 1 V for the first and second pathways, respectively. The results shows that both processes can be promoted by well-known biological reductants such as NADH, ascorbate, vitamin E (tocopherol), cysteine, and glutathione, for which the reduction potential at physiological pH is around -0.3 to -0.5 V. The computed reduction potential of NO through the radical anion HONO•- can also explain the recent experimental findings on the formation of HNO through the reduction of NO, promoted by H2S, vitamin C, and aromatic alcohols. Therefore, these results contribute to shed some light into the question of whether and how HNO is produced in vivo and also for the understanding of the biochemical and physiological effects of NO.

Modeling of S-Nitrosothiol-Thiol Reactions of Biological Significance: HNO Production by S-Thiolation Requires a Proton Shuttle and Stabilization of Polar Intermediates.

Nitroxyl (HNO), a reduced form of the important gasotransmitter nitric oxide, exhibits its own unique biological activity. A possible biological pathway of HNO formation is the S-thiolation reaction between thiols and S-nitrosothiols (RSNOs). Our density functional theory (DFT) calculations suggested that S-thiolation proceeds through a proton transfer from the thiol to the RSNO nitrogen atom, which increases electrophilicity of the RSNO sulfur, followed by nucleophilic attack by thiol, yielding a charge-separated zwitterionic intermediate structure RSS+ (R)N(H)O- (Zi), which decomposes to yield HNO and disulfide RSSR. In the gas phase, the proton transfer and the S-S bond formation are asynchronous, resulting in a high activation barrier (>40 kcal mol-1 ), making the reaction infeasible. However, the barrier can decrease below the S-N bond dissociation energy in RSNOs (≈30 kcal mol-1 ) upon transition into an aqueous environment that stabilizes Zi and provides a proton shuttle to synchronize the proton transfer and the S-S bond formation. These mechanistic features suggest that S-thiolation can easily lend itself to enzymatic catalysis and thus can be a possible route of endogenous HNO production.

Cover Picture: Modeling of S‐Nitrosothiol–Thiol Reactions of Biological Significance: HNO Production by S‐Thiolation Requires a Proton Shuttle and Stabilization of Polar Intermediates (ChemBioChem 8/2017)

Cover Picture: Modeling of <i>S</i>‐Nitrosothiol–Thiol Reactions of Biological Significance: HNO Production by S‐Thiolation Requires a Proton Shuttle and Stabilization of Polar Intermediates (ChemBioChem 8/2017)

Amphiphilic Copolymers Capable of Concomitant Release of HNO and Small Molecule Organics.

We demonstrate concomitant release of HNO and small molecule organics from amphiphilic poly(norbornene)-based copolymers. This key function was achieved by incorporation of thermally labile oxazine units within random and block copolymer architectures. Upon thermolysis, we observed generation of HNO and release of a small molecule conjugate. Importantly, the release kinetics of HNO and a UV-active small molecule (4-nitroaniline) were found to be 1:1, signifying an ability to monitor HNO production indirectly, or to simultaneously release organic therapeutics (e.g., nonsteroidal anti-inflammatory drugs) along with HNO. To our knowledge, these are the first reported polymeric materials demonstrating HNO release from covalently attached HNO donors.

Thiol-Activated HNO Release from a Ruthenium Antiangiogenesis Complex and HIF-1α Inhibition for Cancer Therapy.

Metallonitrosyl complexes are promising as nitric oxide (NO) donors for the treatment of cardiovascular, endothelial, and pathogenic diseases, as well as cancer. Recently, the reduced form of NO– (protonated as HNO, nitroxyl, azanone, isoelectronic with O2) has also emerged as a candidate for therapeutic applications including treatment of acute heart failure and alcoholism. Here, we show that HNO is a product of the reaction of the RuII complex [Ru(bpy)2(SO3)(NO)]+ (1) with glutathione or N-acetyl-L-cysteine, using met-myoglobin and carboxy-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) as trapping agents. Characteristic absorption spectroscopic profiles for HNO reactions with met-myoglobin were obtained, as well as EPR evidence from carboxy-PTIO experiments. Importantly, the product HNO counteracted NO-induced as well as hypoxia-induced stabilization of the tumor-suppressor HIF-1α in cancer cells. The functional disruption of neovascularization by HNO produced by this metallonitrosyl complex was demonstrated in an in vitro angiogenesis model. This behavior is consistent with HNO biochemistry and contrasts with NO-mediated stabilization of HIF-1α. Together, these results demonstrate for the first time thiol-dependent production of HNO by a ruthenium complex and subsequent destabilization of HIF-1α. This work suggests that the complex warrants further investigation as a promising antiangiogenesis agent for the treatment of cancer.

Metal-based optical probes for live cell imaging of nitroxyl (HNO).

Nitroxyl (HNO) is a biological signaling agent that displays distinctive reactivity compared to nitric oxide (NO). As a consequence, these two reactive nitrogen species trigger different physiological responses. Selective detection of HNO over NO has been a challenge for chemists, and several fluorogenic molecular probes have been recently developed with that goal in mind. Common constructs take advantage of the HNO-induced reduction of Cu(II) to Cu(I). The sensing mechanism of such probes relies on the ability of the unpaired electron in a d orbital of the Cu(II) center to quench the fluorescence of a photoemissive ligand by either an electron or energy transfer mechanism. Experimental and theoretical mechanistic studies suggest that proton-coupled electron transfer mediates this process, and careful tuning of the copper coordination environment has led to sensors with optimized selectivity and kinetics. The current optical probes cover the visible and near-infrared regions of the spectrum. This palette of sensors comprises structurally and functionally diverse fluorophores such as coumarin (blue/green emission), boron dipyrromethane (BODIPY, green emission), benzoresorufin (red emission), and dihydroxanthenes (near-infrared emission). Many of these sensors have been successfully applied to detect HNO production in live cells. For example, copper-based optical probes have been used to detect HNO production in live mammalian cells that have been treated with H2S and various nitrosating agents. These studies have established a link between HSNO, the smallest S-nitrosothiol, and HNO. In addition, a near-infrared HNO sensor has been used to perform multicolor/multianalyte microscopy, revealing that exogenously applied HNO elevates the concentration of intracellular mobile zinc. This mobilization of zinc ions is presumably a consequence of nitrosation of cysteine residues in zinc-chelating proteins such as metallothionein. Future challenges for the optical imaging of HNO include devising probes that can detect HNO reversibly, especially because ratiometric imaging can only report equilibrium concentrations when the sensing event is reversible. Another important aspect that needs to be addressed is the creation of probes that can sense HNO in specific subcellular locations. These tools would be useful to identify the organelles in which HNO is produced in mammalian cells and probe the intracellular signaling networks in which this reactive nitrogen species is involved. In addition, near-infrared emitting probes might be applied to detect HNO in thicker specimens, such as acute tissue slices and even live animals, enabling the investigation of the roles of HNO in physiological or pathological conditions in multicellular systems.