Hydrogen sulfide production does not affect antibiotic resistance in Pseudomonas aeruginosa.

Hydrogen sulfide (H2S) has been proposed to protect bacteria from antibiotics, pointing to H2S-producing enzymes as possible targets for the development of antibiotic adjuvants. Here, MIC assays performed with Pseudomonas aeruginosa mutants producing altered H2S levels demonstrate that H2S does not affect antibiotic resistance in this bacterium. Moreover, correlation analyses in a large collection of P. aeruginosa cystic fibrosis isolates argue against the protective role of H2S from antibiotic activity during chronic lung infection.

Cyanide Insensitive Oxidase Confers Hydrogen Sulfide and Nitric Oxide Tolerance to Pseudomonas aeruginosa Aerobic Respiration.

Hydrogen sulfide (H2S) and nitric oxide (NO) are long-known inhibitors of terminal oxidases in the respiratory chain. Yet, they exert pivotal signaling roles in physiological processes, and in several bacterial pathogens have been reported to confer resistance against oxidative stress, host immune responses, and antibiotics. Pseudomonas aeruginosa, an opportunistic pathogen causing life-threatening infections that are difficult to eradicate, has a highly branched respiratory chain including four terminal oxidases of the haem-copper type (aa3, cbb3-1, cbb3-2, and bo3) and one oxidase of the bd-type (cyanide-insensitive oxidase, CIO). As Escherichia coli bd-type oxidases have been shown to be H2S-insensitive and to readily recover their activity from NO inhibition, here we tested the effect of H2S and NO on CIO by performing oxygraphic measurements on membrane preparations from P. aeruginosa PAO1 and isogenic mutants depleted of CIO only or all other terminal oxidases except CIO. We show that O2 consumption by CIO is unaltered even in the presence of high levels of H2S, and that CIO expression is enhanced and supports bacterial growth under such stressful conditions. In addition, we report that CIO is reversibly inhibited by NO, while activity recovery after NO exhaustion is full and fast, suggesting a protective role of CIO under NO stress conditions. As P. aeruginosa is exposed to H2S and NO during infection, the tolerance of CIO towards these stressors agrees with the proposed role of CIO in P. aeruginosa virulence.

Human Cystathionine γ-Lyase Is Inhibited by s-Nitrosation: A New Crosstalk Mechanism between NO and H2S.

The 'gasotransmitters' hydrogen sulfide (H2S), nitric oxide (NO), and carbon monoxide (CO) act as second messengers in human physiology, mediating signal transduction via interaction with or chemical modification of protein targets, thereby regulating processes such as neurotransmission, blood flow, immunomodulation, or energy metabolism. Due to their broad reactivity and potential toxicity, the biosynthesis and breakdown of H2S, NO, and CO are tightly regulated. Growing evidence highlights the active role of gasotransmitters in their mutual cross-regulation. In human physiology, the transsulfuration enzymes cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) are prominent H2S enzymatic sources. While CBS is known to be inhibited by NO and CO, little is known about CSE regulation by gasotransmitters. Herein, we investigated the effect of s-nitrosation on CSE catalytic activity. H2S production by recombinant human CSE was found to be inhibited by the physiological nitrosating agent s-nitrosoglutathione (GSNO), while reduced glutathione had no effect. GSNO-induced inhibition was partially reverted by ascorbate and accompanied by the disappearance of one solvent accessible protein thiol. By combining differential derivatization procedures and mass spectrometry-based analysis with functional assays, seven out of the ten protein cysteine residues, namely Cys84, Cys109, Cys137, Cys172, Cys229, Cys307, and Cys310, were identified as targets of s-nitrosation. By generating conservative Cys-to-Ser variants of the identified s-nitrosated cysteines, Cys137 was identified as most significantly contributing to the GSNO-mediated CSE inhibition. These results highlight a new mechanism of crosstalk between gasotransmitters.

Bacterial Oxidases of the Cytochrome bd Family: Redox Enzymes of Unique Structure, Function, and Utility As Drug Targets.

Significance: Cytochrome bd is a ubiquinol:oxygen oxidoreductase of many prokaryotic respiratory chains with a unique structure and functional characteristics. Its primary role is to couple the reduction of molecular oxygen, even at submicromolar concentrations, to water with the generation of a proton motive force used for adenosine triphosphate production. Cytochrome bd is found in many bacterial pathogens and, surprisingly, in bacteria formally denoted as anaerobes. It endows bacteria with resistance to various stressors and is a potential drug target. Recent Advances: We summarize recent advances in the biochemistry, structure, and physiological functions of cytochrome bd in the light of exciting new three-dimensional structures of the oxidase. The newly discovered roles of cytochrome bd in contributing to bacterial protection against hydrogen peroxide, nitric oxide, peroxynitrite, and hydrogen sulfide are assessed. Critical Issues: Fundamental questions remain regarding the precise delineation of electron flow within this multihaem oxidase and how the extraordinarily high affinity for oxygen is accomplished, while endowing bacteria with resistance to other small ligands. Future Directions: It is clear that cytochrome bd is unique in its ability to confer resistance to toxic small molecules, a property that is significant for understanding the propensity of pathogens to possess this oxidase. Since cytochrome bd is a uniquely bacterial enzyme, future research should focus on harnessing fundamental knowledge of its structure and function to the development of novel and effective antibacterial agents.

The multifaceted roles of sulfane sulfur species in cancer-associated processes.

Sulfane sulfur species comprise a variety of biologically relevant hydrogen sulfide (H2S)-derived species, including per- and poly-sulfidated low molecular weight compounds and proteins. A growing body of evidence suggests that H2S, currently recognized as a key signaling molecule in human physiology and pathophysiology, plays an important role in cancer biology by modulating cell bioenergetics and contributing to metabolic reprogramming. This is accomplished through functional modulation of target proteins via H2S binding to heme iron centers or H2S-mediated reversible per- or poly-sulfidation of specific cysteine residues. Since sulfane sulfur species are increasingly viewed not only as a major source of H2S but also as key mediators of some of the biological effects commonly attributed to H2S, the multifaceted role of these species in cancer biology is reviewed here with reference to H2S, focusing on their metabolism, signaling function, impact on cell bioenergetics and anti-tumoral properties.

Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa3 Supercomplex.

Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa3 supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatisaa3-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O2 and reductants is resistant to inhibition by the ligand, being able to metabolize NO at 25 °C with an apparent turnover number as high as ≈303 mol NO (mol enzyme)-1 min-1 at 30 µM NO. The rate of NO consumption proved to be proportional to that of O2 consumption, with 2.65 ± 0.19 molecules of NO being consumed per O2 molecule by the mycobacterial bcc-aa3. The enzyme was found to metabolize the ligand even under anaerobic reducing conditions with a turnover number of 2.8 ± 0.5 mol NO (mol enzyme)-1 min-1 at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial bcc-aa3 supercomplexes against NO stress.

Hydrogen Sulfide Metabolism and Signaling in the Tumor Microenvironment.

Hydrogen sulfide (H2S), while historically perceived merely as a toxicant, has progressively emerged as a key regulator of numerous processes in mammalian physiology, exerting its signaling function essentially through interaction with and/or modification of proteins, targeting mainly cysteine residues and metal centers. As a gaseous signaling molecule that freely diffuses across aqueous and hydrophobic biological milieu, it has been designated the third 'gasotransmitter' in mammalian physiology. H2S is synthesized and detoxified by specialized endogenous enzymes that operate under a tight regulation, ensuring homeostatic levels of this otherwise toxic molecule. Indeed, imbalances in H2S levels associated with dysfunctional H2S metabolism have been growingly correlated with various human pathologies, from cardiovascular and neurodegenerative diseases to cancer. Several cancer cell lines and specimens have been shown to naturally overexpress one or more of the H2S-synthesizing enzymes. The resulting increased H2S levels have been proposed to promote cancer development through the regulation of various cancer-related processes, which led to the interest in pharmacological targeting of H2S metabolism. Herein are summarized some of the key observations that place H2S metabolism and signaling pathways at the forefront of the cellular mechanisms that support the establishment and development of a tumor within its complex and challenging microenvironment. Special emphasis is given to the mechanisms whereby H2S helps shaping cancer cell bioenergetic metabolism and affords resistance and adaptive mechanisms to hypoxia.

In the respiratory chain of Escherichia coli cytochromes bd-I and bd-II are more sensitive to carbon monoxide inhibition than cytochrome bo3.

Bacteria can not only encounter carbon monoxide (CO) in their habitats but also produce the gas endogenously. Bacterial respiratory oxidases, thus, represent possible targets for CO. Accordingly, host macrophages were proposed to produce CO and release it into the surrounding microenvironment to sense viable bacteria through a mechanism that in Escherichia (E.) coli was suggested to involve the targeting of a bd-type respiratory oxidase by CO. The aerobic respiratory chain of E. coli possesses three terminal quinol:O2-oxidoreductases: the heme-copper oxidase bo3 and two copper-lacking bd-type oxidases, bd-I and bd-II. Heme-copper and bd-type oxidases differ in the mechanism and efficiency of proton motive force generation and in resistance to oxidative and nitrosative stress, cyanide and hydrogen sulfide. Here, we investigated at varied O2 concentrations the effect of CO gas on the O2 reductase activity of the purified cytochromes bo3, bd-I and bd-II of E. coli. We found that CO, in competition with O2, reversibly inhibits the three enzymes. The inhibition constants Ki for the bo3, bd-I and bd-II oxidases are 2.4 ±â€¯0.3, 0.04 ±â€¯0.01 and 0.2 ±â€¯0.1 µM CO, respectively. Thus, in E. coli, bd-type oxidases are more sensitive to CO inhibition than the heme-copper cytochrome bo3. The possible physiological consequences of this finding are discussed.

N-Acetylcysteine Serves as Substrate of 3-Mercaptopyruvate Sulfurtransferase and Stimulates Sulfide Metabolism in Colon Cancer Cells.

Hydrogen sulfide (H2S) is an endogenously produced signaling molecule. The enzymes 3-mercaptopyruvate sulfurtransferase (MST), partly localized in mitochondria, and the inner mitochondrial membrane-associated sulfide:quinone oxidoreductase (SQR), besides being respectively involved in the synthesis and catabolism of H2S, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the H2S biological effects. Reprogramming of H2S metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and N-acetylcysteine (NAC) was recently suggested to act as an antioxidant by increasing intracellular levels of sulfane sulfur species, here we evaluated the effect of prolonged exposure to NAC on the H2S metabolism of SW480 colon cancer cells. Cells exposed to NAC for 24 h displayed increased expression and activity of MST and SQR. Furthermore, NAC was shown to: (i) persist at detectable levels inside the cells exposed to the drug for up to 24 h and (ii) sustain H2S synthesis by human MST more effectively than cysteine, as shown working on the isolated recombinant enzyme. We conclude that prolonged exposure of colon cancer cells to NAC stimulates H2S metabolism and that NAC can serve as a substrate for human MST.

Hydrogen Sulfide Oxidation: Adaptive Changes in Mitochondria of SW480 Colorectal Cancer Cells upon Exposure to Hypoxia.

Hydrogen sulfide (H2S), a known inhibitor of cytochrome c oxidase (CcOX), plays a key signaling role in human (patho)physiology. H2S is synthesized endogenously and mainly metabolized by a mitochondrial sulfide-oxidizing pathway including sulfide:quinone oxidoreductase (SQR), whereby H2S-derived electrons are injected into the respiratory chain stimulating O2 consumption and ATP synthesis. Under hypoxic conditions, H2S has higher stability and is synthesized at higher levels with protective effects for the cell. Herein, working on SW480 colon cancer cells, we evaluated the effect of hypoxia on the ability of cells to metabolize H2S. The sulfide-oxidizing activity was assessed by high-resolution respirometry, measuring the stimulatory effect of sulfide on rotenone-inhibited cell respiration in the absence or presence of antimycin A. Compared to cells grown under normoxic conditions (air O2), cells exposed for 24 h to hypoxia (1% O2) displayed a 1.3-fold reduction in maximal sulfide-oxidizing activity and 2.7-fold lower basal O2 respiration. Based on citrate synthase activity assays, mitochondria of hypoxia-treated cells were 1.8-fold less abundant and displayed 1.4-fold higher maximal sulfide-oxidizing activity and 2.6-fold enrichment in SQR as evaluated by immunoblotting. We speculate that under hypoxic conditions mitochondria undergo these adaptive changes to protect cell respiration from H2S poisoning.

Screening Pyridine Derivatives against Human Hydrogen Sulfide-synthesizing Enzymes by Orthogonal Methods.

Biosynthesis of hydrogen sulfide (H2S), a key signalling molecule in human (patho)physiology, is mostly accomplished by the human enzymes cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST). Several lines of evidence have shown a close correlation between increased H2S production and human diseases, such as several cancer types and amyotrophic lateral sclerosis. Identifying compounds selectively and potently inhibiting the human H2S-synthesizing enzymes may therefore prove beneficial for pharmacological applications. Here, the human enzymes CBS, CSE and MST were expressed and purified from Escherichia coli, and thirty-one pyridine derivatives were synthesized and screened for their ability to bind and inhibit these enzymes. Using differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), circular dichroism spectropolarimetry (CD), and activity assays based on fluorimetric and colorimetric H2S detection, two compounds (C30 and C31) sharing structural similarities were found to weakly inhibit both CBS and CSE: 1 mM C30 inhibited these enzymes by approx. 50% and 40%, respectively, while 0.5 mM C31 accounted for CBS and CSE inhibition by approx. 40% and 60%, respectively. This work, while presenting a robust methodological platform for screening putative inhibitors of the human H2S-synthesizing enzymes, highlights the importance of employing complementary methodologies in compound screenings.

Substrate-induced conformational change in cytochrome P450 OleP.

The regulation of cytochrome P450 activity is often achieved by structural transitions induced by substrate binding. We describe the conformational transition experienced upon binding by the P450 OleP, an epoxygenase involved in oleandomycin biosynthesis. OleP bound to the substrate analog 6DEB crystallized in 2 forms: one with an ensemble of open and closed conformations in the asymmetric unit and another with only the closed conformation. Characterization of OleP-6DEB binding kinetics, also using the P450 inhibitor clotrimazole, unveiled a complex binding mechanism that involves slow conformational rearrangement with the accumulation of a spectroscopically detectable intermediate where 6DEB is bound to open OleP. Data reported herein provide structural snapshots of key precatalytic steps in the OleP reaction and explain how structural rearrangements induced by substrate binding regulate activity.-Parisi, G., Montemiglio, L. C., Giuffrè, A., Macone, A., Scaglione, A., Cerutti, G., Exertier, C., Savino, C., Vallone, B. Substrate-induced conformational change in cytochrome P450 OleP.

Hydrogen Sulfide Biochemistry and Interplay with Other Gaseous Mediators in Mammalian Physiology.

Hydrogen sulfide (H2S) has emerged as a relevant signaling molecule in physiology, taking its seat as a bona fide gasotransmitter akin to nitric oxide (NO) and carbon monoxide (CO). After being merely regarded as a toxic poisonous molecule, it is now recognized that mammalian cells are equipped with sophisticated enzymatic systems for H2S production and breakdown. The signaling role of H2S is mainly related to its ability to modify different protein targets, particularly by promoting persulfidation of protein cysteine residues and by interacting with metal centers, mostly hemes. H2S has been shown to regulate a myriad of cellular processes with multiple physiological consequences. As such, dysfunctional H2S metabolism is increasingly implicated in different pathologies, from cardiovascular and neurodegenerative diseases to cancer. As a highly diffusible reactive species, the intra- and extracellular levels of H2S have to be kept under tight control and, accordingly, regulation of H2S metabolism occurs at different levels. Interestingly, even though H2S, NO, and CO have similar modes of action and parallel regulatory targets or precisely because of that, there is increasing evidence of a crosstalk between the three gasotransmitters. Herein are reviewed the biochemistry, metabolism, and signaling function of hydrogen sulfide, as well as its interplay with the other gasotransmitters, NO and CO.

The presence of glutamate residues on the PAS sequence of the stimuli-sensitive nano-ferritin improves in vivo biodistribution and mitoxantrone encapsulation homogeneity.

A genetically engineered human ferritin heavy chain (HFt)-based construct has been recently shown by our group to efficiently entrap and deliver doxorubicin to cancer cells. This construct, named HFt-MP-PAS, contained a tumor-selective sequence (MP) responsive to proteolytic cleavage by tumor proteases (MMPs), located between each HFt subunit and an outer shielding polypeptide sequence rich in proline (P), serine (S) and alanine (A) residues (PAS). HFt-MP-PAS displayed excellent therapeutic efficacy in xenogenic pancreatic and head and neck cancer models in vivo, leading to a significant increase in overall animal survivals. Here we report a new construct obtained by the genetic insertion of two glutamate residues in the PAS sequence of HFt-MP-PAS. Such new construct, named HFt-MP-PASE, is characterized by improved performances as drug biodistribution in a xenogenic pancreatic cancer model in vivo. Moreover, HFt-MP-PASE efficiently encapsulates the anti-cancer drug mitoxantrone (MIT), and the resulting MIT-loaded nanoparticles proved to be more soluble and monodispersed than the HFt-MP-PAS counterparts. Importantly, in vitro MIT-loaded HFt-MP-PASE kills several cancer cell lines of different origin (colon, breast, sarcoma and pancreas) at least as efficiently as the free drug. Finally, our MIT loaded protein nanocages allowed in vivo an impressive incrementing of the drug accumulation in the tumor with respect to the free drug.

Nitrosative stress defences of the enterohepatic pathogenic bacterium Helicobacter pullorum.

Helicobacter pullorum is an avian bacterium that causes gastroenteritis, intestinal bowel and hepatobiliary diseases in humans. Although H. pullorum has been shown to activate the mammalian innate immunity with release of nitric oxide (NO), the proteins that afford protection against NO and reactive nitrogen species (RNS) remain unknown. Here several protein candidates of H. pullorum, namely a truncated (TrHb) and a single domain haemoglobin (SdHb), and three peroxiredoxin-like proteins (Prx1, Prx2 and Prx3) were investigated. We report that the two haemoglobin genes are induced by RNS, and that SdHb confers resistance to nitrosative stress both in vitro and in macrophages. For peroxiredoxins, the prx2 and prx3 expression is enhanced by peroxynitrite and hydrogen peroxide, respectively. Mutation of prx1 does not alter the resistance to these stresses, while the single ∆prx2 and double ∆prx1∆prx2 mutants have decreased viability. To corroborate the physiological data, the biochemical analysis of the five recombinant enzymes was done, namely by stopped-flow spectrophotometry. It is shown that H. pullorum SdHb reacts with NO much more quickly than TrHb, and that the three Prxs react promptly with peroxynitrite, Prx3 displaying the highest reactivity. Altogether, the results unveil SdHb and Prx3 as major protective systems of H. pullorum against nitrosative stress.

Cytochrome bd and Gaseous Ligands in Bacterial Physiology.

Cytochrome bd is a unique prokaryotic respiratory terminal oxidase that does not belong to the extensively investigated family of haem-copper oxidases (HCOs). The enzyme catalyses the four-electron reduction of O2 to 2H2O, using quinols as physiological reducing substrates. The reaction is electrogenic and cytochrome bd therefore sustains bacterial energy metabolism by contributing to maintain the transmembrane proton motive force required for ATP synthesis. As compared to HCOs, cytochrome bd displays several distinctive features in terms of (i) metal composition (it lacks Cu and harbours a d-type haem in addition to two haems b), (ii) overall three-dimensional structure, that only recently has been solved, and arrangement of the redox cofactors, (iii) lesser energetic efficiency (it is not a proton pump), (iv) higher O2 affinity, (v) higher resistance to inhibitors such as cyanide, nitric oxide (NO) and hydrogen sulphide (H2S) and (vi) ability to efficiently metabolize potentially toxic reactive oxygen and nitrogen species like hydrogen peroxide (H2O2) and peroxynitrite (ONOO-). Compelling evidence suggests that, beyond its bioenergetic role, cytochrome bd plays multiple functions in bacterial physiology and affords protection against oxidative and nitrosative stress. Relevant to human pathophysiology, thanks to its peculiar properties, the enzyme has been shown to promote virulence in several bacterial pathogens, being currently recognized as a target for the development of new antibiotics. This review aims to give an update on our current understanding of bd-type oxidases with a focus on their reactivity with gaseous ligands and its potential impact on bacterial physiology and human pathophysiology.

A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionine ß-Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5'-phosphate- (PLP-) dependent cystathionine ß-synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (H2S). CBS activity, contributing to cellular redox homeostasis, is positively regulated by S-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenic CBS mutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired H2S-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria.

The Terminal Oxidase Cytochrome bd Promotes Sulfide-resistant Bacterial Respiration and Growth.

Hydrogen sulfide (H2S) impairs mitochondrial respiration by potently inhibiting the heme-copper cytochrome c oxidase. Since many prokaryotes, including Escherichia (E.) coli, generate H2S and encounter high H2S levels particularly in the human gut, herein we tested whether bacteria can sustain sulfide-resistant O2-dependent respiration. E. coli has three respiratory oxidases, the cyanide-sensitive heme-copper bo3 enzyme and two bd oxidases much less sensitive to cyanide. Working on the isolated enzymes, we found that, whereas the bo3 oxidase is inhibited by sulfide with half-maximal inhibitory concentration IC50 = 1.1 ± 0.1 µM, under identical experimental conditions both bd oxidases are insensitive to sulfide up to 58 µM. In E. coli respiratory mutants, both O2-consumption and aerobic growth proved to be severely impaired by sulfide when respiration was sustained by the bo3 oxidase alone, but unaffected by ≤200 µM sulfide when either bd enzyme acted as the only terminal oxidase. Accordingly, wild-type E. coli showed sulfide-insensitive respiration and growth under conditions favouring the expression of bd oxidases. In all tested conditions, cyanide mimicked the functional effect of sulfide on bacterial respiration. We conclude that bd oxidases promote sulfide-resistant O2-consumption and growth in E. coli and possibly other bacteria. The impact of this discovery is discussed.

Bioenergetic relevance of hydrogen sulfide and the interplay between gasotransmitters at human cystathionine ß-synthase.

Merely considered as a toxic gas in the past, hydrogen sulfide (H2S) is currently viewed as the third 'gasotransmitter' in addition to nitric oxide (NO) and carbon monoxide (CO), playing a key signalling role in human (patho)physiology. H2S can either act as a substrate or, similarly to CO and NO, an inhibitor of mitochondrial respiration, in the latter case by targeting cytochrome c oxidase (CcOX). The impact of H(2)S on mitochondrial energy metabolism crucially depends on the bioavailability of this gaseous molecule and its interplay with the other two gasotransmitters. The H(2)S-producing human enzyme cystathionine ß-synthase (CBS), sustaining cellular bioenergetics in colorectal cancer cells, plays a role in the interplay between gasotransmitters. The enzyme was indeed recently shown to be negatively modulated by physiological concentrations of CO and NO, particularly in the presence of its allosteric activator S-adenosyl-l-methionine (AdoMet). These newly discovered regulatory mechanisms are herein reviewed. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

Evidence for Detrimental Cross Interactions between Reactive Oxygen and Nitrogen Species in Leber's Hereditary Optic Neuropathy Cells.

Here we have collected evidence suggesting that chronic changes in the NO homeostasis and the rise of reactive oxygen species bioavailability can contribute to cell dysfunction in Leber's hereditary optic neuropathy (LHON) patients. We report that peripheral blood mononuclear cells (PBMCs), derived from a female LHON patient with bilateral reduced vision and carrying the pathogenic mutation 11778/ND4, display increased levels of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as revealed by flow cytometry, fluorometric measurements of nitrite/nitrate, and 3-nitrotyrosine immunodetection. Moreover, viability assays with the tetrazolium dye MTT showed that lymphoblasts from the same patient are more sensitive to prolonged NO exposure, leading to cell death. Taken together these findings suggest that oxidative and nitrosative stress cooperatively play an important role in driving LHON pathology when excess NO remains available over time in the cell environment.