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.

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration.

The terminal oxidases of bacterial aerobic respiratory chains are redox-active electrogenic enzymes that catalyze the four-electron reduction of O2 to 2H2O taking out electrons from quinol or cytochrome c. Living bacteria often deal with carbon monoxide (CO) which can act as both a signaling molecule and a poison. Bacterial terminal oxidases contain hemes; therefore, they are potential targets for CO. However, our knowledge of this issue is limited and contradictory. Here, we investigated the effect of CO on the cell growth and aerobic respiration of three different Escherichia coli mutants, each expressing only one terminal quinol oxidase: cytochrome bd-I, cytochrome bd-II, or cytochrome bo3. We found that following the addition of CO to bd-I-only cells, a minimal effect on growth was observed, whereas the growth of both bd-II-only and bo3-only strains was severely impaired. Consistently, the degree of resistance of aerobic respiration of bd-I-only cells to CO is high, as opposed to high CO sensitivity displayed by bd-II-only and bo3-only cells consuming O2. Such a difference between the oxidases in sensitivity to CO was also observed with isolated membranes of the mutants. Accordingly, O2 consumption of wild-type cells showed relatively low CO sensitivity under conditions favoring the expression of a bd-type oxidase.

Preparations of Terminal Oxidase Cytochrome bd-II Isolated from Escherichia coli Reveal Significant Hydrogen Peroxide Scavenging Activity.

Cytochrome bd-II is one of the three terminal quinol oxidases of the aerobic respiratory chain of Escherichia coli. Preparations of the detergent-solubilized untagged bd-II oxidase isolated from the bacterium were shown to scavenge hydrogen peroxide (H2O2) with high rate producing molecular oxygen (O2). Addition of H2O2 to the same buffer that does not contain enzyme or contains thermally denatured cytochrome bd-II does not lead to any O2 production. The latter observation rules out involvement of adventitious transition metals bound to the protein. The H2O2-induced O2 production is not susceptible to inhibition by N-ethylmaleimide (the sulfhydryl binding compound), antimycin A (the compound that binds specifically to a quinol binding site), and CO (diatomic gas that binds specifically to the reduced heme d). However, O2 formation is inhibited by cyanide (IC50 = 4.5 ± 0.5 µM) and azide. Addition of H2O2 in the presence of dithiothreitol and ubiquinone-1 does not inactivate cytochrome bd-II and apparently does not affect the O2 reductase activity of the enzyme. The ability of cytochrome bd-II to detoxify H2O2 could play a role in bacterial physiology by conferring resistance to the peroxide-mediated stress.

Bioenergetics and Reactive Nitrogen Species in Bacteria.

The production of reactive nitrogen species (RNS) by the innate immune system is part of the host's defense against invading pathogenic bacteria. In this review, we summarize recent studies on the molecular basis of the effects of nitric oxide and peroxynitrite on microbial respiration and energy conservation. We discuss possible molecular mechanisms underlying RNS resistance in bacteria mediated by unique respiratory oxygen reductases, the mycobacterial bcc-aa3 supercomplex, and bd-type cytochromes. A complete picture of the impact of RNS on microbial bioenergetics is not yet available. However, this research area is developing very rapidly, and the knowledge gained should help us develop new methods of treating infectious diseases.

Terminal Oxidase Cytochrome bd Protects Bacteria Against Hydrogen Sulfide Toxicity.

Hydrogen sulfide (H2S) is often called the third gasotransmitter (after nitric oxide and carbon monoxide), or endogenous gaseous signaling molecule. This compound plays important roles in organisms from different taxonomic groups, from bacteria to animals and humans. In mammalian cells, H2S has a cytoprotective effect at nanomolar concentrations, but becomes cytotoxic at higher concentrations. The primary target of H2S is mitochondria. At submicromolar concentrations, H2S inhibits mitochondrial heme-copper cytochrome c oxidase, thereby blocking aerobic respiration and oxidative phosphorylation and eventually leading to cell death. Since the concentration of H2S in the gut is extremely high, the question arises - how can gut bacteria maintain the functioning of their oxygen-dependent respiratory electron transport chains under such conditions? This review provides an answer to this question and discusses the key role of non-canonical bd-type terminal oxidases of the enterobacterium Escherichia coli, a component of the gut microbiota, in maintaining aerobic respiration and growth in the presence of toxic concentrations of H2S in the light of recent experimental data.

Impact of Hydrogen Sulfide on Mitochondrial and Bacterial Bioenergetics.

This review focuses on the effects of hydrogen sulfide (H2S) on the unique bioenergetic molecular machines in mitochondria and bacteria-the protein complexes of electron transport chains and associated enzymes. H2S, along with nitric oxide and carbon monoxide, belongs to the class of endogenous gaseous signaling molecules. This compound plays critical roles in physiology and pathophysiology. Enzymes implicated in H2S metabolism and physiological actions are promising targets for novel pharmaceutical agents. The biological effects of H2S are biphasic, changing from cytoprotection to cytotoxicity through increasing the compound concentration. In mammals, H2S enhances the activity of FoF1-ATP (adenosine triphosphate) synthase and lactate dehydrogenase via their S-sulfhydration, thereby stimulating mitochondrial electron transport. H2S serves as an electron donor for the mitochondrial respiratory chain via sulfide quinone oxidoreductase and cytochrome c oxidase at low H2S levels. The latter enzyme is inhibited by high H2S concentrations, resulting in the reversible inhibition of electron transport and ATP production in mitochondria. In the branched respiratory chain of Escherichia coli, H2S inhibits the bo3 terminal oxidase but does not affect the alternative bd-type oxidases. Thus, in E. coli and presumably other bacteria, cytochrome bd permits respiration and cell growth in H2S-rich environments. A complete picture of the impact of H2S on bioenergetics is lacking, but this field is fast-moving, and active ongoing research on this topic will likely shed light on additional, yet unknown biological effects.

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.

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.

Cytochrome bd from Escherichia coli catalyzes peroxynitrite decomposition.

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.

Leishmania infantum trypanothione reductase is a promiscuous enzyme carrying an NADPH:O2 oxidoreductase activity shared by glutathione reductase.

BACKGROUND: Leishmania infantum is a protozoan of the trypanosomatid family causing visceral leishmaniasis. Leishmania parasites are transmitted by the bite of phlebotomine sand flies to the human host and are phagocyted by macrophages. The parasites synthesize N1-N8-bis(glutationyl)-spermidine (trypanothione, TS2), which furnishes electrons to the tryparedoxin-tryparedoxin peroxidase couple to reduce the reactive oxygen species produced by macrophages. Trypanothione is kept reduced by trypanothione reductase (TR), a FAD-containing enzyme essential for parasite survival. METHODS: The enzymatic activity has been studied by stopped-flow, absorption spectroscopy, and amperometric measurements. RESULTS: The study reported here demonstrates that the steady-state parameters change as a function of the order of substrates addition to the TR-containing solution. In particular, when the reaction is carried out by adding NADPH to a solution containing the enzyme and trypanothione, the KM for NADPH decreases six times compared to the value obtained by adding TS2 as last reagent to start the reaction (1.9 vs. 12µM). More importantly, we demonstrate that TR is able to catalyze the oxidation of NADPH also in the absence of trypanothione. Thus, TR catalyzes the reduction of O2 to water through the sequential formation of C(4a)-(hydro)peroxyflavin and sulfenic acid intermediates. This NADPH:O2 oxidoreductase activity is shared by Saccharomyces cerevisiae glutathione reductase (GR). CONCLUSIONS: TR and GR, in the absence of their physiological substrates, may catalyze the electron transfer reaction from NADPH to molecular oxygen to yield water. GENERAL SIGNIFICANCE: TR and GR are promiscuous enzymes.

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

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.

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.

Biliverdin Reductase-A integrates insulin signaling with mitochondrial metabolism through phosphorylation of GSK3ß.

Brain insulin resistance links the failure of energy metabolism with cognitive decline in both type 2 Diabetes Mellitus (T2D) and Alzheimer's disease (AD), although the molecular changes preceding overt brain insulin resistance remain unexplored. Abnormal biliverdin reductase-A (BVR-A) levels were observed in both T2D and AD and were associated with insulin resistance. Here, we demonstrate that reduced BVR-A levels alter insulin signaling and mitochondrial bioenergetics in the brain. Loss of BVR-A leads to IRS1 hyper-activation but dysregulates Akt-GSK3ß complex in response to insulin, hindering the accumulation of pGSK3ßS9 into the mitochondria. This event impairs oxidative phosphorylation and fosters the activation of the mitochondrial Unfolded Protein Response (UPRmt). Remarkably, we unveil that BVR-A is required to shuttle pGSK3ßS9 into the mitochondria. Our data sheds light on the intricate interplay between insulin signaling and mitochondrial metabolism in the brain unraveling potential targets for mitigating the development of brain insulin resistance and neurodegeneration.

Cytochrome c oxidase and nitric oxide in action: molecular mechanisms and pathophysiological implications.

BACKGROUND: The reactions between Complex IV (cytochrome c oxidase, CcOX) and nitric oxide (NO) were described in the early 60's. The perception, however, that NO could be responsible for physiological or pathological effects, including those on mitochondria, lags behind the 80's, when the identity of the endothelial derived relaxing factor (EDRF) and NO synthesis by the NO synthases were discovered. NO controls mitochondrial respiration, and cytotoxic as well as cytoprotective effects have been described. The depression of OXPHOS ATP synthesis has been observed, attributed to the inhibition of mitochondrial Complex I and IV particularly, found responsible of major effects. SCOPE OF REVIEW: The review is focused on CcOX and NO with some hints about pathophysiological implications. The reactions of interest are reviewed, with special attention to the molecular mechanisms underlying the effects of NO observed on cytochrome c oxidase, particularly during turnover with oxygen and reductants. MAJOR CONCLUSIONS AND GENERAL SIGNIFICANCE: The NO inhibition of CcOX is rapid and reversible and may occur in competition with oxygen. Inhibition takes place following two pathways leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) of the enzyme reduced, or a more labile nitrite-derivative (CcOX-NO(2)(-)) of the enzyme oxidized, and during turnover. The pathway that prevails depends on the turnover conditions and concentration of NO and physiological substrates, cytochrome c and O(2). All evidence suggests that these parameters are crucial in determining the CcOX vs NO reaction pathway prevailing in vivo, with interesting physiological and pathological consequences for cells.

The terminal oxidase cytochrome bd-I confers carbon monoxide resistance to Escherichia coli cells.

Carbon monoxide (CO) plays a multifaceted role in the physiology of organisms, from poison to signaling molecule. Heme proteins, including terminal oxidases, are plausible CO targets. Three quinol oxidases terminate the branched aerobic respiratory chain of Escherichia coli. These are the heme­copper cytochrome bo3 and two copper-lacking bd-type cytochromes, bd-I and bd-II. All three enzymes generate a proton motive force during the four-electron oxygen reduction reaction that is used for ATP production. The bd-type oxidases also contribute to mechanisms of bacterial defense against various types of stresses. Here we report that in E. coli cells, at the enzyme concentrations tested, cytochrome bd-I is much more resistant to inhibition by CO than cytochrome bd-II and cytochrome bo3. The apparent half-maximal inhibitory concentration values, IC50, for inhibition of O2 consumption of the membrane-bound bd-II and bo3 oxidases by CO at ~150 µM O2 were estimated to be 187.1 ± 11.1 and 183.3 ± 13.5 µM CO, respectively. Under the same conditions, the maximum inhibition observed with the membrane-bound cytochrome bd-I was 20 ± 10% at ~200 µM CO.

Catalytic intermediates of cytochrome bd terminal oxidase at steady-state: ferryl and oxy-ferrous species dominate.

The cytochrome bd ubiquinol oxidase from Escherichia coli couples the exergonic two-electron oxidation of ubiquinol and four-electron reduction of O(2) to 2H(2)O to proton motive force generation by transmembrane charge separation. The oxidase contains two b-type hemes (b(558) and b(595)) and one heme d, where O(2) is captured and converted to water through sequential formation of a few intermediates. The spectral features of the isolated cytochrome bd at steady-state have been examined by stopped-flow multiwavelength absorption spectroscopy. Under turnover conditions, sustained by O(2) and dithiothreitol (DTT)-reduced ubiquinone, the ferryl and oxy-ferrous species are the mostly populated catalytic intermediates, with a residual minor fraction of the enzyme containing ferric heme d and possibly one electron on heme b(558). These findings are unprecedented and differ from those obtained with mammalian cytochrome c oxidase, in which the oxygen intermediates were not found to be populated at detectable levels under similar conditions [M.G. Mason, P. Nicholls, C.E. Cooper, The steady-state mechanism of cytochrome c oxidase: redox interactions between metal centres, Biochem. J. 422 (2009) 237-246]. The data on cytochrome bd are consistent with the observation that the purified enzyme has the heme d mainly in stable oxy-ferrous and ferryl states. The results are here discussed in the light of previously proposed models of the catalytic cycle of cytochrome bd.

Mitochondria and nitric oxide: chemistry and pathophysiology.

Cell respiration is controlled by nitric oxide (NO) reacting with respiratory chain complexes, particularly with Complex I and IV. The functional implication of these reactions is different owing to involvement of different mechanisms. Inhibition of complex IV is rapid (milliseconds) and reversible, and occurs at nanomolar NO concentrations, whereas inhibition of complex I occurs after a prolonged exposure to higher NO concentrations. The inhibition of Complex I involves the reversible S-nitrosation of a key cysteine residue on the ND3 subunit. The reaction of NO with cytochrome c oxidase (CcOX) directly involves the active site of the enzyme: two mechanisms have been described leading to formation of either a relatively stable nitrosyl-derivative (CcOX-NO) or a more labile nitrite-derivative (CcOX-NO (2) (-) ). Both adducts are inhibited, though with different K(I); one mechanism prevails on the other depending on the turnover conditions and availability of substrates, cytochrome c and O(2). SH-SY5Y neuroblastoma cells or lymphoid cells, cultured under standard O(2) tension, proved to follow the mechanism leading to degradation of NO to nitrite. Formation of CcOX-NO occurred upon rising the electron flux level at this site, artificially or in the presence of higher amounts of endogenous reduced cytochrome c. Taken together, the observations suggest that the expression level of mitochondrial cytochrome c may be crucial to determine the respiratory chain NO inhibition pathway prevailing in vivo under nitrosative stress conditions. The putative patho-physiological relevance of the interaction between NO and the respiratory complexes is addressed.

A sulfite respiration pathway from Thermus thermophilus and the key role of newly identified cytochrome c550.

Sulfite, produced for instance during amino acid metabolism, is a very reactive and toxic compound. Various detoxification mechanisms exist, but sulfite oxidoreductases (SORs) are one of the major actors in sulfite remediation in bacteria and animals. Here we describe the existence of an operon in the extreme thermophilic bacterium Thermus thermophilus HB8 encoding both a SOR and a diheme c-type cytochrome. The in vitro analysis clearly showed that the newly identified cytochrome c550 acts as an acceptor of the electrons generated by the SOR enzyme during the oxidation of sulfite. The electrons are then rapidly shuttled via cytochrome c552 to the terminal ba3- and caa3-type oxidases, thereby unveiling a novel electron transfer pathway, linking sulfite oxidation to oxygen reduction in T. thermophilus: sulfite → SOR(HB8) → cytochrome c550 → cytochrome c552 → ba3 oxidase/caa3 oxidase → O2. The description of the complete pathway reveals that electrons generated during sulfite oxidation by the SOR are funneled into the respiratory chain, participating in the energy production of T. thermophilus.

Giardia intestinalis escapes oxidative stress by colonizing the small intestine: A molecular hypothesis.

Giardia intestinalis is the microaerophilic protozoon causing giardiasis, a common infectious intestinal disease. Giardia possesses an O(2) -scavenging activity likely essential for survival in the host. We report that Giardia trophozoites express the O(2) -detoxifying flavodiiron protein (FDP), detected by immunoblotting, and are able to reduce O(2) to H(2) O rapidly (∼3 µM O(2) × min × 10(6) cells at 37 °C) and with high affinity (C(50) = 3.4 ± 0.7 µM O(2)). Following a short-term (minutes) exposure to H(2) O(2) ≥ 100 µM, the O(2) consumption by the parasites is irreversibly impaired, and the FDP undergoes a degradation, prevented by the proteasome-inhibitor MG132. Instead, H(2) O(2) does not cause degradation or inactivation of the isolated FDP. On the basis of the elevated susceptibility of Giardia to oxidative stress, we hypothesize that the parasite preferentially colonizes the small intestine since, compared with colon, it is characterized by a greater capacity for redox buffering and a lower propensity to oxidative stress.

The characterization of Thermotoga maritima ferritin reveals an unusual subunit dissociation behavior and efficient DNA protection from iron-mediated oxidative stress.

Ferritin from the hyperthermophilic anaerobe Thermotoga maritima, a bacterium of ancient phylogenetic origin, is structurally similar to known bacterial and eukaryotic ferritins: 24 identical subunits assemble into a shell having octahedral symmetry and a Mr of about 460 kDa. T. maritima ferritin (TmFtn), purified to homogeneity as a recombinant protein, contains approximately 2-3 iron atoms and can incorporate efficiently up to 3,500 atoms in the form of a ferric oxy-hydroxide mineral at 80°C, the optimal growth temperature of the bacterium. The 24-mer unexpectedly dissociates reversibly into dimers at low ionic strengths. In turn, dimers re-associate into the native 24-mer assembly at high protein concentrations and upon incorporation of iron micelles containing at least 500 Fe(III). TmFtn uses O(2) as efficient iron oxidant. The reaction stoichiometry is 3-4 O(2):Fe(II) as in all bacterial ferritins. Accordingly no H(2)O(2) is released into solution, a feature reflected in the in vitro ability of TmFtn to reduce significantly iron-mediated oxidative damage to DNA at 80°C. A similar TmFtn-mediated ROS detoxifying role likely occurs in the bacterium which lacks the SOD/catalase defense systems of the aerobic world.