Publications by authors named "Alessandro Giuffrè"

67 Publications

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

Biochim Biophys Acta Bioenerg 2021 02 17;1862(2):148338. Epub 2020 Nov 17.

CNR Institute of Molecular Biology and Pathology, Piazzale Aldo Moro 5, I-00185 Rome, Italy. Electronic address:

Sulfane sulfur species comprise a variety of biologically relevant hydrogen sulfide (HS)-derived species, including per- and poly-sulfidated low molecular weight compounds and proteins. A growing body of evidence suggests that HS, 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 HS binding to heme iron centers or HS-mediated reversible per- or poly-sulfidation of specific cysteine residues. Since sulfane sulfur species are increasingly viewed not only as a major source of HS but also as key mediators of some of the biological effects commonly attributed to HS, the multifaceted role of these species in cancer biology is reviewed here with reference to HS, focusing on their metabolism, signaling function, impact on cell bioenergetics and anti-tumoral properties.
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http://dx.doi.org/10.1016/j.bbabio.2020.148338DOI Listing
February 2021

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

Int J Mol Sci 2020 Nov 12;21(22). Epub 2020 Nov 12.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, 119991 Moscow, Russia.

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 - supercomplex composed of cytochrome and -type terminal oxidase. Strikingly, we found that the enzyme in turnover with O 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) min at 30 µM NO. The rate of NO consumption proved to be proportional to that of O consumption, with 2.65 ± 0.19 molecules of NO being consumed per O molecule by the mycobacterial -. 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) min at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial supercomplexes against NO stress.
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http://dx.doi.org/10.3390/ijms21228521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697965PMC
November 2020

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

Antioxid Redox Signal 2020 Nov 9. Epub 2020 Nov 9.

Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, United Kingdom.

Cytochrome 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 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. We summarize recent advances in the biochemistry, structure, and physiological functions of cytochrome in the light of exciting new three-dimensional structures of the oxidase. The newly discovered roles of cytochrome in contributing to bacterial protection against hydrogen peroxide, nitric oxide, peroxynitrite, and hydrogen sulfide are assessed. 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. It is clear that cytochrome 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 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.
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http://dx.doi.org/10.1089/ars.2020.8039DOI Listing
November 2020

Hydrogen Sulfide Metabolism and Signaling in the Tumor Microenvironment.

Adv Exp Med Biol 2020 ;1219:335-353

Instituto de Tecnologia Química e Biológica António Xavier, NOVA University of Lisbon, Oeiras, Portugal.

Hydrogen sulfide (HS), 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. HS 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 HS levels associated with dysfunctional HS 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 HS-synthesizing enzymes. The resulting increased HS 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 HS metabolism. Herein are summarized some of the key observations that place HS 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 HS helps shaping cancer cell bioenergetic metabolism and affords resistance and adaptive mechanisms to hypoxia.
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http://dx.doi.org/10.1007/978-3-030-34025-4_17DOI Listing
April 2020

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

Biochim Biophys Acta Bioenerg 2019 12 4;1860(12):148088. Epub 2019 Nov 4.

CNR Institute of Molecular Biology and Pathology, Rome, Italy. Electronic address:

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:O-oxidoreductases: the heme-copper oxidase bo 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 O concentrations the effect of CO gas on the O reductase activity of the purified cytochromes bo, bd-I and bd-II of E. coli. We found that CO, in competition with O, reversibly inhibits the three enzymes. The inhibition constants K for the bo, bd-I and bd-II oxidases are 2.4 ± 0.3, 0.04 ± 0.01 and 0.2 ± 0.1 μM CO, respectively. Thus, in E. coli, bd-type oxidases are more sensitive to CO inhibition than the heme-copper cytochrome bo. The possible physiological consequences of this finding are discussed.
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http://dx.doi.org/10.1016/j.bbabio.2019.148088DOI Listing
December 2019

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

Cells 2019 08 4;8(8). Epub 2019 Aug 4.

CNR Institute of Molecular Biology and Pathology, Piazzale Aldo Moro 5, I-00185 Rome, Italy.

Hydrogen sulfide (HS) 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 HS, generate sulfane sulfur species such as persulfides and polysulfides, currently recognized as mediating some of the HS biological effects. Reprogramming of HS metabolism was reported to support cellular proliferation and energy metabolism in cancer cells. As oxidative stress is a cancer hallmark and -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 HS 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 HS 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 HS metabolism and that NAC can serve as a substrate for human MST.
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http://dx.doi.org/10.3390/cells8080828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6721681PMC
August 2019

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

Oxid Med Cell Longev 2019 29;2019:8102936. Epub 2019 Jan 29.

CNR Institute of Molecular Biology and Pathology, Rome, Italy.

Hydrogen sulfide (HS), a known inhibitor of cytochrome oxidase (CcOX), plays a key signaling role in human (patho)physiology. HS is synthesized endogenously and mainly metabolized by a mitochondrial sulfide-oxidizing pathway including sulfide:quinone oxidoreductase (SQR), whereby HS-derived electrons are injected into the respiratory chain stimulating O consumption and ATP synthesis. Under hypoxic conditions, HS 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 HS. 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 O), cells exposed for 24 h to hypoxia (1% O) displayed a 1.3-fold reduction in maximal sulfide-oxidizing activity and 2.7-fold lower basal O 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 HS poisoning.
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http://dx.doi.org/10.1155/2019/8102936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374825PMC
March 2019

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

Sci Rep 2019 01 24;9(1):684. Epub 2019 Jan 24.

Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal.

Biosynthesis of hydrogen sulfide (HS), a key signalling molecule in human (patho)physiology, is mostly accomplished by the human enzymes cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST). Several lines of evidence have shown a close correlation between increased HS production and human diseases, such as several cancer types and amyotrophic lateral sclerosis. Identifying compounds selectively and potently inhibiting the human HS-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 HS 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 HS-synthesizing enzymes, highlights the importance of employing complementary methodologies in compound screenings.
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http://dx.doi.org/10.1038/s41598-018-36994-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6346012PMC
January 2019

Substrate-induced conformational change in cytochrome P450 OleP.

FASEB J 2019 02 12;33(2):1787-1800. Epub 2018 Sep 12.

Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche A. Rossi Fanelli, Sapienza Università di Roma, Rome, Italy.

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.
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http://dx.doi.org/10.1096/fj.201800450RRDOI Listing
February 2019

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

Oxid Med Cell Longev 2018 27;2018:6290931. Epub 2018 Jun 27.

Instituto de Tecnologia Química e Biológica António Xavier, NOVA University of Lisbon, Av. da República (EAN), 2780-157 Oeiras, Portugal.

Hydrogen sulfide (HS) 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 HS production and breakdown. The signaling role of HS 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. HS has been shown to regulate a myriad of cellular processes with multiple physiological consequences. As such, dysfunctional HS 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 HS have to be kept under tight control and, accordingly, regulation of HS metabolism occurs at different levels. Interestingly, even though HS, 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.
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http://dx.doi.org/10.1155/2018/6290931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6040266PMC
October 2018

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

J Control Release 2018 04 20;275:177-185. Epub 2018 Feb 20.

Institute of Molecular Biology and Pathology, CNR - National Research Council of Italy, 00185 Rome, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jconrel.2018.02.025DOI Listing
April 2018

Nitrosative stress defences of the enterohepatic pathogenic bacterium Helicobacter pullorum.

Sci Rep 2017 08 30;7(1):9909. Epub 2017 Aug 30.

Instituto de Tecnologia Química e Biológica António Xavier NOVA, Av. da República, 2780-157, Oeiras, Portugal.

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.
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http://dx.doi.org/10.1038/s41598-017-10375-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577044PMC
August 2017

Cytochrome bd and Gaseous Ligands in Bacterial Physiology.

Adv Microb Physiol 2017 10;71:171-234. Epub 2017 Jul 10.

CNR Institute of Molecular Biology and Pathology, Rome, Italy. Electronic address:

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 O to 2HO, 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 O affinity, (v) higher resistance to inhibitors such as cyanide, nitric oxide (NO) and hydrogen sulphide (HS) and (vi) ability to efficiently metabolize potentially toxic reactive oxygen and nitrogen species like hydrogen peroxide (HO) 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.
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http://dx.doi.org/10.1016/bs.ampbs.2017.05.002DOI Listing
February 2018

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

Oxid Med Cell Longev 2017 22;2017:8940321. Epub 2017 Mar 22.

CNR Institute of Molecular Biology and Pathology, Rome, Italy.

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5'-phosphate- (PLP-) dependent cystathionine -synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (HS). 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 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 HS-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.
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http://dx.doi.org/10.1155/2017/8940321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381205PMC
May 2017

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

Biochim Biophys Acta 2016 Aug 31;1857(8):1127-1138. Epub 2016 Mar 31.

CNR Institute of Molecular Biology and Pathology, Piazzale Aldo Moro 5, I-00185 Rome, Italy. Electronic address:

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 β-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.
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http://dx.doi.org/10.1016/j.bbabio.2016.03.030DOI Listing
August 2016

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

Sci Rep 2016 Mar 31;6:23788. Epub 2016 Mar 31.

CNR Institute of Molecular Biology and Pathology, Rome, Italy.

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.
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http://dx.doi.org/10.1038/srep23788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815019PMC
March 2016

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

Oxid Med Cell Longev 2016 31;2016:3187560. Epub 2015 Dec 31.

Department of Biochemical Sciences and Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy.

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.
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http://dx.doi.org/10.1155/2016/3187560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4736215PMC
December 2016

Antioxidant defence systems in the protozoan pathogen Giardia intestinalis.

Mol Biochem Parasitol 2016 Mar-Apr;206(1-2):56-66. Epub 2015 Dec 7.

CNR Institute of Molecular Biology and Pathology, Rome, Italy. Electronic address:

The microaerophilic protist Giardia intestinalis is the causative agent of giardiasis, one of the most common intestinal infectious diseases worldwide. The pathogen lacks not only respiratory terminal oxidases (being amitochondriate), but also several conventional antioxidant enzymes, including catalase, superoxide dismutase and glutathione peroxidase. In spite of this, since living attached to the mucosa of the proximal small intestine, the parasite should rely on an efficient antioxidant system to survive the oxidative and nitrosative stress conditions found in this tract of the human gut. Here, we review current knowledge on the antioxidant defence systems in G. intestinalis, focusing on the progress made over the last decade in the field. The relevance of this research and future perspectives are discussed.
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http://dx.doi.org/10.1016/j.molbiopara.2015.12.002DOI Listing
July 2017

S-Adenosyl-l-methionine Modulates CO and NO• Binding to the Human H2S-generating Enzyme Cystathionine β-Synthase.

J Biol Chem 2016 Jan 18;291(2):572-81. Epub 2015 Nov 18.

the Institute of Molecular Biology and Pathology, National Research Council of Italy, I-00185 Rome, Italy

Cystathionine β-synthase (CBS) is a key enzyme in human (patho)physiology with a central role in hydrogen sulfide metabolism. The enzyme is composed of a pyridoxal 5'-phosphate-binding catalytic domain, flanked by the following two domains: a heme-binding N-terminal domain and a regulatory C-terminal domain binding S-adenosyl-l-methionine (AdoMet). CO or NO(•) binding at the ferrous heme negatively modulates the enzyme activity. Conversely, AdoMet binding stimulates CBS activity. Here, we provide experimental evidence for a functional communication between the two domains. We report that AdoMet binding significantly enhances CBS inhibition by CO. Consistently, we observed increased affinity (∼5-fold) and faster association (∼10-fold) of CO to the ferrous heme at physiological AdoMet concentrations. NO(•) binding to reduced CBS was also enhanced by AdoMet, although to a lesser extent (∼2-fold higher affinity) as compared with CO. Importantly, CO and NO(•) binding was unchanged by AdoMet in a truncated form of CBS lacking the C-terminal regulatory domain. These unprecedented observations demonstrate that CBS activation by AdoMet puzzlingly sensitizes the enzyme toward inhibition by exogenous ligands, like CO and NO(•). This further supports the notion that CBS regulation is a complex process, involving the concerted action of multiple physiologically relevant effectors.
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http://dx.doi.org/10.1074/jbc.M115.681221DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705378PMC
January 2016

Superoxide reductase from Giardia intestinalis: structural characterization of the first SOR from a eukaryotic organism shows an iron centre that is highly sensitive to photoreduction.

Acta Crystallogr D Biol Crystallogr 2015 Nov 31;71(Pt 11):2236-47. Epub 2015 Oct 31.

Instituto de Tecnologia Química e Biológica, António Xavier Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal.

Superoxide reductase (SOR), which is commonly found in prokaryotic organisms, affords protection from oxidative stress by reducing the superoxide anion to hydrogen peroxide. The reaction is catalyzed at the iron centre, which is highly conserved among the prokaryotic SORs structurally characterized to date. Reported here is the first structure of an SOR from a eukaryotic organism, the protozoan parasite Giardia intestinalis (GiSOR), which was solved at 2.0 Å resolution. By collecting several diffraction data sets at 100 K from the same flash-cooled protein crystal using synchrotron X-ray radiation, photoreduction of the iron centre was observed. Reduction was monitored using an online UV-visible microspectrophotometer, following the decay of the 647 nm absorption band characteristic of the iron site in the glutamate-bound, oxidized state. Similarly to other 1Fe-SORs structurally characterized to date, the enzyme displays a tetrameric quaternary-structure arrangement. As a distinctive feature, the N-terminal loop of the protein, containing the characteristic EKHxP motif, revealed an unusually high flexibility regardless of the iron redox state. At variance with previous evidence collected by X-ray crystallography and Fourier transform infrared spectroscopy of prokaryotic SORs, iron reduction did not lead to dissociation of glutamate from the catalytic metal or other structural changes; however, the glutamate ligand underwent X-ray-induced chemical changes, revealing high sensitivity of the GiSOR active site to X-ray radiation damage.
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http://dx.doi.org/10.1107/S1399004715015825DOI Listing
November 2015

Antigiardial activity of novel triazolyl-quinolone-based chalcone derivatives: when oxygen makes the difference.

Front Microbiol 2015 8;6:256. Epub 2015 Apr 8.

CNR Institute of Molecular Biology and Pathology Rome, Italy.

Giardiasis is a common diarrheal disease worldwide caused by the protozoan parasite Giardia intestinalis. It is urgent to develop novel drugs to treat giardiasis, due to increasing clinical resistance to the gold standard drug metronidazole (MTZ). New potential antiparasitic compounds are usually tested for their killing efficacy against G. intestinalis under anaerobic conditions, in which MTZ is maximally effective. On the other hand, though commonly regarded as an 'anaerobic pathogen,' G. intestinalis is exposed to relatively high O2 levels in vivo, living attached to the mucosa of the proximal small intestine. It is thus important to test the effect of O2 when searching for novel potential antigiardial agents, as outlined in a previous study [Bahadur et al. (2014) Antimicrob. Agents Chemother. 58, 543]. Here, 45 novel chalcone derivatives with triazolyl-quinolone scaffold were synthesized, purified, and characterized by high resolution mass spectrometry, (1)H and (13)C nuclear magnetic resonance and infrared spectroscopy. Efficacy of the compounds against G. intestinalis trophozoites was tested under both anaerobic and microaerobic conditions, and selectivity was assessed in a counter-screen on human epithelial colorectal adenocarcinoma cells. MTZ was used as a positive control in the assays. All the tested compounds proved to be more effective against the parasite in the presence of O2, with the exception of MTZ that was less effective. Under anaerobiosis eighteen compounds were found to be as effective as MTZ or more (up to three to fourfold); the same compounds proved to be up to >100-fold more effective than MTZ under microaerobic conditions. Four of them represent potential candidates for the design of novel antigiardial drugs, being highly selective against Giardia trophozoites. This study further underlines the importance of taking O2 into account when testing novel potential antigiardial compounds.
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http://dx.doi.org/10.3389/fmicb.2015.00256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4389562PMC
April 2015

Cytochrome bd from Escherichia coli catalyzes peroxynitrite decomposition.

Biochim Biophys Acta 2015 Feb 29;1847(2):182-188. Epub 2014 Oct 29.

CNR Institute of Molecular Biology and Pathology, Rome, Italy. Electronic address:

Cytochrome bd is a prokaryotic respiratory quinol oxidase phylogenetically unrelated to heme-copper oxidases, that was found to promote virulence in some bacterial pathogens. Cytochrome bd from Escherichia coli was previously reported to contribute not only to proton motive force generation, but also to bacterial resistance to nitric oxide (NO) and hydrogen peroxide (H2O2). Here, we investigated the interaction of the purified enzyme with peroxynitrite (ONOO(-)), another harmful reactive species produced by the host to kill invading microorganisms. We found that addition of ONOO(-) to cytochrome bd in turnover with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) causes the irreversible inhibition of a small (≤15%) protein fraction, due to the NO generated from ONOO(-) and not to ONOO(-) itself. Consistently, addition of ONOO(-) to cells of the E. coli strain GO105/pTK1, expressing cytochrome bd as the only terminal oxidase, caused only a minor (≤5%) irreversible inhibition of O2 consumption, without measurable release of NO. Furthermore, by directly monitoring the kinetics of ONOO(-) decomposition by stopped-flow absorption spectroscopy, it was found that the purified E. coli cytochrome bd in turnover with O2 is able to metabolize ONOO(-) with an apparent turnover rate as high as ~10 mol ONOO(-) (mol enzyme)(-1) s(-1) at 25°C. To the best of our knowledge, this is the first time that the kinetics of ONOO(-) decomposition by a terminal oxidase has been investigated. These results strongly suggest a protective role of cytochrome bd against ONOO(-) damage.
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http://dx.doi.org/10.1016/j.bbabio.2014.10.006DOI Listing
February 2015

Flavodiiron oxygen reductase from Entamoeba histolytica: modulation of substrate preference by tyrosine 271 and lysine 53.

J Biol Chem 2014 Oct 23;289(41):28260-70. Epub 2014 Aug 23.

From the Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República (EAN), 2781-901 Oeiras, Portugal,

Flavodiiron proteins (FDPs) are a family of enzymes endowed with bona fide oxygen- and/or nitric-oxide reductase activity, although their substrate specificity determinants remain elusive. After a comprehensive comparison of available three-dimensional structures, particularly of FDPs with a clear preference toward either O2 or NO, two main differences were identified near the diiron active site, which led to the construction of site-directed mutants of Tyr(271) and Lys(53) in the oxygen reducing Entamoeba histolytica EhFdp1. The biochemical and biophysical properties of these mutants were studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies coupled to potentiometry. Their reactivity with O2 and NO was analyzed by stopped-flow absorption spectroscopy and amperometric methods. These mutations, whereas keeping the overall properties of the redox cofactors, resulted in increased NO reductase activity and faster inactivation of the enzyme in the reaction with O2, pointing to a role of the mutated residues in substrate selectivity.
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http://dx.doi.org/10.1074/jbc.M114.579086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192481PMC
October 2014

NO* binds human cystathionine β-synthase quickly and tightly.

J Biol Chem 2014 Mar 10;289(12):8579-87. Epub 2014 Feb 10.

From the Metabolism and Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculty of Pharmacy, University of Lisbon, 1649-003 Lisbon, Portugal.

The hexa-coordinate heme in the H2S-generating human enzyme cystathionine β-synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO(•) or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO(•) was reported to bind at the CBS heme with very low affinity (Kd = 30-281 μm). This discrepancy was herein reconciled by investigating the NO(•) reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO(•) binds tightly to the ferrous CBS heme, with an apparent Kd ≤ 0.23 μm. In line with this result, at 25 °C, NO(•) binds quickly to CBS (k on ∼ 8 × 10(3) m(-1) s(-1)) and dissociates slowly from the enzyme (k off ∼ 0.003 s(-1)). The observed rate constants for NO(•) binding were found to be linearly dependent on [NO(•)] up to ∼ 800 μm NO(•), and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO(•) quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO(•). The novel findings reported here shed new light on CBS regulation by NO(•) and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO(•), CO, and H2S in cell signaling.
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http://dx.doi.org/10.1074/jbc.M113.507533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3961681PMC
March 2014

Cytochrome bd oxidase and bacterial tolerance to oxidative and nitrosative stress.

Biochim Biophys Acta 2014 Jul 31;1837(7):1178-87. Epub 2014 Jan 31.

Department of Biochemical Sciences, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy.

Cytochrome bd is a prokaryotic respiratory quinol:O2 oxidoreductase, phylogenetically unrelated to the extensively studied heme-copper oxidases (HCOs). The enzyme contributes to energy conservation by generating a proton motive force, though working with a lower energetic efficiency as compared to HCOs. Relevant to patho-physiology, members of the bd-family were shown to promote virulence in some pathogenic bacteria, which makes these enzymes of interest also as potential drug targets. Beyond its role in cell bioenergetics, cytochrome bd accomplishes several additional physiological functions, being apparently implicated in the response of the bacterial cell to a number of stress conditions. Compelling experimental evidence suggests that the enzyme enhances bacterial tolerance to oxidative and nitrosative stress conditions, owing to its unusually high nitric oxide (NO) dissociation rate and a notable catalase activity; the latter has been recently documented in one of the two bd-type oxidases of Escherichia coli. Current knowledge on cytochrome bd and its reactivity with O2, NO and H2O2 is summarized in this review in the light of the hypothesis that the preferential (over HCOs) expression of cytochrome bd in pathogenic bacteria may represent a strategy to evade the host immune attack based on production of NO and reactive oxygen species (ROS). This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
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http://dx.doi.org/10.1016/j.bbabio.2014.01.016DOI Listing
July 2014

Functional characterization of peroxiredoxins from the human protozoan parasite Giardia intestinalis.

PLoS Negl Trop Dis 2014 9;8(1):e2631. Epub 2014 Jan 9.

CNR Institute of Molecular Biology and Pathology, Rome, Italy.

The microaerophilic protozoan parasite Giardia intestinalis, causative of one of the most common human intestinal diseases worldwide, infects the mucosa of the proximal small intestine, where it has to cope with O2 and nitric oxide (NO). Elucidating the antioxidant defense system of this pathogen lacking catalase and other conventional antioxidant enzymes is thus important to unveil novel potential drug targets. Enzymes metabolizing O2, NO and superoxide anion (O2 (-•)) have been recently reported for Giardia, but it is yet unknown how the parasite copes with H2O2 and peroxynitrite (ONOO(-)). Giardia encodes two yet uncharacterized 2-cys peroxiredoxins (Prxs), GiPrx1a and GiPrx1b. Peroxiredoxins are peroxidases implicated in virulence and drug resistance in several parasitic protozoa, able to protect from nitroxidative stress and repair oxidatively damaged molecules. GiPrx1a and a truncated form of GiPrx1b (deltaGiPrx1b) were expressed in Escherichia coli, purified and functionally characterized. Both Prxs effectively metabolize H2O2 and alkyl-hydroperoxides (cumyl- and tert-butyl-hydroperoxide) in the presence of NADPH and E. coli thioredoxin reductase/thioredoxin as the reducing system. Stopped-flow experiments show that both proteins in the reduced state react with ONOO(-) rapidly (k = 4×10(5) M(-1) s(-1) and 2×10(5) M(-1) s(-1) at 4°C, for GiPrx1a and deltaGiPrx1b, respectively). Consistent with a protective role against oxidative stress, expression of GiPrx1a (but not deltaGiPrx1b) is induced in parasitic cells exposed to air O2 for 24 h. Based on these results, GiPrx1a and deltaGiPrx1b are suggested to play an important role in the antioxidant defense of Giardia, possibly contributing to pathogenesis.
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http://dx.doi.org/10.1371/journal.pntd.0002631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3886907PMC
September 2014

Cytochrome bd oxidase and hydrogen peroxide resistance in Mycobacterium tuberculosis.

mBio 2013 Dec 17;4(6):e01006-13. Epub 2013 Dec 17.

Department of Biochemical Sciences and Istituto Pasteur, Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy.

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http://dx.doi.org/10.1128/mBio.01006-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3870254PMC
December 2013

O(2)-dependent efficacy of novel piperidine- and piperazine-based chalcones against the human parasite Giardia intestinalis.

Antimicrob Agents Chemother 2014 11;58(1):543-9. Epub 2013 Nov 11.

Bio-Organic Laboratory, Department of Chemistry, Delhi University, Delhi, India.

Giardia intestinalis is the most frequent protozoan agent of intestinal diseases worldwide. Though commonly regarded as an anaerobic pathogen, it preferentially colonizes the fairly oxygen-rich mucosa of the proximal small intestine. Therefore, when testing new potential antigiardial drugs, O2 should be taken into account, since it also reduces the efficacy of metronidazole, the gold standard drug against giardiasis. In this study, 46 novel chalcones were synthesized by microwave-assisted Claisen-Schmidt condensation, purified, characterized by high-resolution mass spectrometry, (1)H and (13)C nuclear magnetic resonance, and infrared spectroscopy, and tested for their toxicity against G. intestinalis under standard anaerobic conditions. As a novel approach, compounds showing antigiardial activity under anaerobiosis were also assayed under microaerobic conditions, and their selectivity against parasitic cells was assessed in a counterscreen on human epithelial colorectal adenocarcinoma cells. Among the tested compounds, three [30(a), 31(e), and 33] were more effective in the presence of O2 than under anaerobic conditions and killed the parasite 2 to 4 times more efficiently than metronidazole under anaerobiosis. Two of them [30(a) and 31(e)] proved to be selective against parasitic cells, thus representing potential candidates for the design of novel antigiardial drugs. This study highlights the importance of testing new potential antigiardial agents not only under anaerobic conditions but also at low, more physiological O2 concentrations.
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http://dx.doi.org/10.1128/AAC.00990-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910768PMC
September 2014

Characterization of mitochondrial dysfunction in the 7PA2 cell model of Alzheimer's disease.

J Alzheimers Dis 2013 ;37(4):747-58

Scuola Normale Superiore, Pisa, Italy European Brain Research Institute - "Rita Levi-Montalcini", Rome, Italy.

The 7WD4 and 7PA2 cell lines, widely used as cellular models for Alzheimer's disease (AD), have been used to investigate the effects of amyloid-β protein precursor overexpression and amyloid-β (Aβ) oligomer accumulation on mitochondrial function. Under standard culture conditions, both cell lines, compared to Chinese hamster ovary (CHO) control cells, displayed an ~5% decrease of O2 respiration as sustained by endogenous substrates. Functional impairment of the respiratory chain was found distributed among the protein complexes, though more evident at the level of complex I and complex IV. Measurements of ATP showed that its synthesis by oxidative phosphorylation is decreased in 7WD4 and 7PA2 cells by ~25%, this loss being partly compensated by glycolysis (Warburg effect). Compensation proved to be more efficient in 7WD4 than in 7PA2 cells, the latter cell line displaying the highest reactive oxygen species production. The strongest deficit was observed in mitochondrial membrane potential that is almost 40% and 60% lower in 7WD4 and 7PA2 cells, respectively, in comparison to CHO controls. All functional parameters point to a severe bioenergetic impairment of the AD cells, with the extent of mitochondrial dysfunction being correlated to the accumulation of Aβ peptides and oligomers.
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http://dx.doi.org/10.3233/JAD-130728DOI Listing
July 2014

Cytochrome bd oxidase from Escherichia coli displays high catalase activity: an additional defense against oxidative stress.

FEBS Lett 2013 Jul 30;587(14):2214-8. Epub 2013 May 30.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russian Federation.

Cytochrome bd oxygen reductase from Escherichia coli has three hemes, b558, b595 and d. We found that the enzyme, as-prepared or in turnover with O2, rapidly decomposes H2O2 with formation of approximately half a mole of O2 per mole of H2O2. Such catalase activity vanishes upon cytochrome bd reduction, does not compete with the oxygen-reductase activity, is insensitive to NO, CO, antimycin-A and N-ethylmaleimide (NEM), but is inhibited by cyanide (Ki ~2.5μM) and azide. The activity, possibly associated with heme-b595, was also observed in catalase-deficient E. coli cells following cytochrome bd over-expression suggesting a protective role against oxidative stress in vivo.
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http://dx.doi.org/10.1016/j.febslet.2013.05.047DOI Listing
July 2013