Why do bacteria use so many enzymes to scavenge hydrogen peroxide?

Hydrogen peroxide (H(2)O(2)) is continuously formed by the autoxidation of redox enzymes in aerobic cells, and it also enters from the environment, where it can be generated both by chemical processes and by the deliberate actions of competing organisms. Because H(2)O(2) is acutely toxic, bacteria elaborate scavenging enzymes to keep its intracellular concentration at nanomolar levels. Mutants that lack such enzymes grow poorly, suffer from high rates of mutagenesis, or even die. In order to understand how bacteria cope with oxidative stress, it is important to identify the key enzymes involved in H(2)O(2) degradation. Catalases and NADH peroxidase (Ahp) are primary scavengers in many bacteria, and their activities and physiological impacts have been unambiguously demonstrated through phenotypic analysis and through direct measurements of H(2)O(2) clearance in vivo. Yet a wide variety of additional enzymes have been proposed to serve similar roles: thiol peroxidase, bacterioferritin comigratory protein, glutathione peroxidase, cytochrome c peroxidase, and rubrerythrins. Each of these enzymes can degrade H(2)O(2) in vitro, but their contributions in vivo remain unclear. In this review we examine the genetic, genomic, regulatory, and biochemical evidence that each of these is a bonafide scavenger of H(2)O(2) in the cell. We also consider possible reasons that bacteria might require multiple enzymes to catalyze this process, including differences in substrate specificity, compartmentalization, cofactor requirements, kinetic optima, and enzyme stability. It is hoped that the resolution of these issues will lead to an understanding of stress resistance that is more accurate and perceptive.     

Are minidisc recorders adequate for the study of respiratory sounds?

Digital audio tape (DAT) recorders have become the de facto gold standard recording devices for lung sounds. Sound recorded on DAT is compact-disk (CD) quality with adequate sensitivity from below 20 Hz to above 20 KHz. However, DAT recorders have drawbacks. Although small, they are relatively heavy, the recording mechanism is complex and delicate, and finding one desired track out of many is inconvenient. A more recent development in portable recording devices is the minidisc (MD) recorder. These recorders are widely available, inexpensive, small and light, rugged, mechanically simple, and record digital data in tracks that may be named and accessed directly. Minidiscs hold as much recorded sound as a compact disk but in about 1/5 of the recordable area. The data compression is achieved by use of a technique known as adaptive transform acoustic coding for minidisc (ATRAC). This coding technique makes decisions about what components of the sound would not be heard by a human listener and discards the digital information that represents these sounds. Most of this compression takes place on sounds above 5.5 KHz. As the intended use of these recorders is the storage and reproduction of music, it is unknown whether ATRAC will discard or distort significant portions of typical lung sound signals. We determined the suitability of MD recorders for respiratory sound research by comparing a variety of normal and pathologic lung sounds that were digitized directly into a computer and also after recording by a DAT recorder and 2 different MD recorders (Sharp and Sony). We found that the frequency spectra and waveforms of respiratory sounds were not distorted in any important way by recording on the two MD recorders tested.     

Superoxide-dependent and ascorbate-dependent formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Are lactoferrin and transferrin promoters of hydroxyl-radical generation?

Apo-lactoferrin and apo-transferrin protect against iron-ion-dependent hydroxyl-radical (.OH) generation from H2O2 in the presence of superoxide radicals or ascorbic acid at pH 7.4, whether the necessary iron is added as ionic iron or as ferritin. Iron-loaded transferrin and lactoferrin [2 mol of Fe(III)/mol] show no protective ability, but do not themselves accelerate .OH production unless chelating agents are present in the reaction mixture, especially if the proteins are incorrectly loaded with iron. At acidic pH values, the protective ability of the apoproteins is diminished, and the fully iron-loaded proteins can release some iron in a form able to accelerate .OH generation. The physiological significance of these observations is discussed.     

Theoretical study of the hydrogen-bonded complexes serotonin–water/hydrogen peroxide

Eight H-bonded complexes between serotonin (5-hydroxy-tryptamine) and water/hydrogen peroxide were studied at the B3LYP and HF levels of theory, using the 6-31+G(d) basis set. A thermodynamic analysis was performed in order to find the most stable complex. The calculated bonding parameters showed that the most stable H-bonded complex is formed between serotonin and hydrogen peroxide by means of the intermolecular H-bond –H₂N...H–OOH. Fig. a Theoretical study of the hydrogen-bonded supersystems serotonin-water/hydrogen peroxide     

Dexamethasone-mediated regulation of death and differentiation of muscle cells. Is hydrogen peroxide involved in the process?

The hypothetical involvement of H2O2 in dexamethasone-mediated regulation of muscle cell differentiation and elimination was studied. Rat L6 myoblasts and mouse C2C12 satellite cells were chosen for acute (24 h) and chronic (5 or 10 day) experiments. Mitogenicity and anabolism were both affected by H2O2. Micromolar concentrations of H2O2 inhibited DNA while stimulating protein synthesis. At the millimolar level, H2O2 led to cell death by apoptosis.Synthetic glucocorticoi - dexamethasone (Dex) was shown to effect muscle cell fate similarly to H2O2. Chronic treatment with H2O2 or Dex dose-dependently accelerated either the formation of myotubes or cell elimination. Dex-induced cell death slightly differed from classical apoptosis and was featured by the symptoms of cell senescence such as extensive cytoplasm vacuolisation, accumulation of inclusion-bodies and lack of low molecular weight oligonucleosomal DNA fragmentation but chromatin condensation. Antioxidants (sodium ascorbate, N-acetyl-L-cysteine, catalase) abrogated Dex-dependent cell death. We conclude that H2O2 directly influences myogenesis and muscle cell elimination. Moreover, H2O2 can be considered as the potent mediator of glucocorticoid-dependent effects on muscle cells.     

Kinetic advantage of the interaction between the fatty acid β-oxidation enzymes and the complexes of the respiratory chain

Respiration-linked oxidation of 3-hydroxybutyryl-CoA, crotonyl-CoA and saturated fatty acyl (C₄, C₈ and C₁₄)-CoA esters was studied in different mitochondrial preparations. Oxidation of acyl-CoA esters was poor in intact mitochondria; however, it was significant, as well as, NAD⁺ and CoA-dependent in gently and in vigorously sonicated mitochondria. The respiration-linked oxidation of crotonyl-CoA and 3-hydroxybutyryl-CoA proceeded at much higher rates (over 700%) in gently disrupted mitochondria than in completely disrupted mitochondria. The redox dye-linked oxidation of crotonyl-CoA (with inhibited respiratory chain) was also higher in gently disrupted mitochondria (149%) than in disrupted ones. During the respiration-linked oxidation of 3-hydroxybutyryl-CoA the steady-state NADH concentrations in the reaction chamber were determined, and found to be 8 μM in gently sonicated and 15 μM in completely sonicated mitochondria in spite of the observation that the gently sonicated mitochondria oxidized the 3-hydroxybutyryl-CoA much faster than the completely sonicated mitochondria. The NAD⁺-dependence of 3-hydroxybutyryl-CoA oxidation showed that a much smaller NAD⁺ concentration was enough to half-saturate the reaction in gently disrupted mitochondria than in completely disrupted ones. Thus, these observations indicate the positive kinetic consequence of organization of β-oxidation enzyme in situ. Respiration-linked oxidation of bytyryl-, oxtanoyl- and palmitoyl-CoA was also studied and these CoA intermediates were oxidized at approx. 50% of the rate of crotonyl- and 3-hydroxybutyryl-CoA in the gently disrupted mitochondria. In vigorously disrupted mitochondria the oxidation rate of these saturated acyl-CoA intermediates was hardly detectable indicating that the connection between the acyl-CoA dehydrogenase and the respiratory chain had been disrupted.     

Tryptophan or tyrosine? On the nature of the amino acid radical formed following hydrogen peroxide treatment of cytochrome c oxidase

It has been reported that different amino acid radicals are formed following the addition of hydrogen peroxide to cytochrome c oxidase (CcO) from bovine heart or from Paracoccus denitrificans. A broad unresolved signal in the electron paramagnetic resonance (EPR) spectra of bovine CcO has been assigned to a tryptophan radical, probably Trp126 [Rigby et al. Biochemistry 2000, 39, 5921-5928]. In the P. denitrificans enzyme, a similarly broad signal but with a well-resolved hyperfine structure was shown to originate from a tyrosyl radical and was tentatively assigned to the active site Tyr280 [MacMillan et al. Biochemistry 1999, 38, 9179-9184]. We confirm that the EPR signal from P. denitrificans CcO can be simulated using spectral parameters typical for known Tyr radicals in other systems. However, the rotational conformation of the phenolic ring of Tyr280 is inconsistent with our simulation. Instead, the simulation parameters we used correspond to the rotational conformation of ring that matches very accurately the conformation found in Tyr167, a residue that is close enough ( approximately 10 A) to the binuclear centre to readily donate an electron. The broad unresolved EPR signal in the bovine oxidase has been thought previously to be inconsistent with a tyrosyl radical. However, we have simulated a hypothetical EPR spectrum arising from a Tyr129 radical (the equivalent of Tyr167 in P. denitrificans CcO) and showed that it is similar to the observed broad signal. The possibility exists, therefore, that the homological tyrosine amino acid (Tyr167/Tyr129) is responsible for the EPR spectrum in both the Paraccoccus and the bovine enzyme. This correspondence between the two enzymes at least allows the possibility that this radical may have functional importance.     

How relevant is the reoxidation of ferrocytochrome c by hydrogen peroxide when determining superoxide anion production?

In a recent publication [(1987) FEBS Lett. 210, 195-198] the authors claim the use of cytochrome c to detect superoxide anion underestimates the real rate of superoxide anion formation on the basis that: (i) the rate of uric acid formation by xanthine oxidase is about 4-fold faster than the rate of cytochrome c reduction and (ii) hydrogen peroxide formed upon dismutation of the superoxide anion generated by xanthine oxidase is capable of reoxidizing ferrocytochrome c. That paper may have been misleading for readers not very familiar with the field of oxygen radicals, since both assumptions are, in fact, incorrect. In this report we demonstrate that the build up in concentration of H2O2 during most reactions in which superoxide anion is being produced is not enough to affect the rate of cytochrome c reduction. Our results suggest that the authors may have been misled by an artifact due to exposure of the samples containing H2O2 to UV light, which generates hydroxyl radicals by photolysis.     

Are Phragmites australis enzymes involved in the degradation of the textile azo dye acid orange 7?

The role of antioxidant and detoxification enzymes of Phragmites australis, in the degradation of an azo dye, acid orange 7 (AO7), was studied. Activities of several enzymes involved in plant protection against stress were assayed through the activity characterization of superoxide dismutase (SOD), peroxidases (POD), catalase (CAT), ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR) and glutathione S-transferase (GST), obtained from P. australis crude extracts of leaves, stems and roots. A sub-surface vertical flow constructed wetland, planted with P. australis was used to test the plants response to the AO7 exposure at two different concentrations (130 and 700 mg l(-1)). An activity increase was detected for an AO7 concentration of 130 mg l(-1) for most enzymes studied (SOD, CAT and APOX), especially in leaves, suggesting a response of the reactive oxygen species scavenging enzymes to the chemical stress imposed. GST activity increase in this situation can also be interpreted as an activation of the detoxification pathway and subsequent AO7 conjugation. A totally different behaviour was observed for AO7 at 700 mg l(-1). An evident decrease in activity was observed for SOD, CAT, APOX and GST, probably due to enzymatic inhibition by AO7. Contrarily, DHAR activity augmented drastically in this situation. POD activity was not greatly affected during trial. Altogether these results suggest that P. australis effectively uses the ascorbate-glutathione pathway for the detoxification of AO7.     

Are primary cultures realistic models of prostate cancer?

Primary cultures fill a unique niche among the repertoire of in vitro model systems available to investigate the biology of the normal and malignant human prostate. This review summarizes some of the properties of primary cultures, with special emphasis on two questions: are primary cultures from adenocarcinomas really comprised of cancer rather than normal cells, and do primary cultures faithfully retain characteristics of cells of origin? © 2003 Wiley‐Liss, Inc.     

Intestinal cholesterol esterase: intracellular enzyme or contamination of cytosol by pancreatic enzymes?

The location of cholesterol esterase in rabbit intestine was re-evaluated. In three different experiments that were designed to eliminate contaminating mucus and pancreatic enzymes from the lumen of the small intestine, it was observed that the activities of cholesterol esterase and amylase in intestinal cytosol and whole homogenate decreased in parallel fashion. After the mucus was carefully wiped from the intestinal mucosa prior to the preparation of cytosol, amylase and cholesterol esterase activities decreased sevenfold. The recovery of the total activity of both enzymes in the cytosol was approximately 15%. When the lumen of the small intestine was filled with phosphate buffer and incubated at 37 degrees C for 20 min, cholesterol esterase and amylase activities in the cytosol prepared from this segment were further decreased. Moreover, the activities of amylase and cholesterol esterase were completely recovered from the lumen. Amylase and cholesterol esterase activities in the cytosol were eliminated if dithiothreitol was used as a mucolytic agent to prepare intestinal mucosa for the isolation of intestinal cells. In whole homogenates prepared from these intestinal segments, approximately 10-15% of the total cholesterol esterase activity remained. This activity, which could not be accounted for by pancreatic contamination, was associated with intestinal nuclei and cellular debris. Progesterone, ethinyl estradiol, and 25-hydroxycholesterol regulated microsomal acyl CoA:cholesterol acyltransferase activity and caused similar directional changes in the rate of cholesteryl ester synthesis in isolated intestinal cells. These same sterols, however, failed to affect cytosolic cholesterol esterase activity in vitro.     

Are sirtuin deacylase enzymes important modulators of mitochondrial energy metabolism?

Background

In recent years, reversible lysine acylation of proteins has emerged as a major post-translational modification across the cell, and importantly has been shown to regulate many proteins in mitochondria. One key family of deacylase enzymes is the sirtuins, of which SIRT3, SIRT4, and SIRT5 are localised to the mitochondria and regulate acyl modifications in this organelle.

Scope of review

In this review we discuss the emerging role of lysine acylation in the mitochondrion and summarise the evidence that proposes mitochondrial sirtuins are important players in the modulation of mitochondrial energy metabolism in response to external nutrient cues, via their action as lysine deacylases. We also highlight some key areas of mitochondrial sirtuin biology where future research efforts are required.

Major conclusions

Lysine deacetylation appears to play some role in regulating mitochondrial metabolism. Recent discoveries of new enzymatic capabilities of mitochondrial sirtuins, including desuccinylation and demalonylation activities, as well as an increasing list of novel protein substrates have identified many new questions regarding the role of mitochondrial sirtuins in the regulation of energy metabolism.

General significance

Dynamic changes in the regulation of mitochondrial metabolism may have far-reaching consequences for many diseases, and despite promising initial findings in knockout animals and cell models, the role of the mitochondrial sirtuins requires further exploration in this context. This article is part of a Special Issue entitled Frontiers of mitochondrial research.     

Formation of hydroxyl radicals from hydrogen peroxide in the presence of iron. Is haemoglobin a biological Fenton reagent?

The ability of oxyhaemoglobin and methaemoglobin to generate hydroxyl radicals (OH.) from H2O2 has been investigated using deoxyribose and phenylalanine as 'detector molecules' for OH.. An excess of H2O2 degrades methaemoglobin, releasing iron ions that react with H2O2 to form a species that appears to be OH.. Oxyhaemoglobin reacts with low concentrations of H2O2 to form a 'reactive species' that degrades deoxyribose but does not hydroxylate phenylalanine. This 'reactive species' is less amenable to scavenging by certain scavengers (salicylate, phenylalanine, arginine) than is OH., but it appears more reactive than OH. is to others (Hepes, urea). The ability of haemoglobin to generate not only this 'reactive species', but also OH. in the presence of H2O2 may account for the damaging effects of free haemoglobin in the brain, the eye, and at sites of inflammation.     

Do nitroxides protect cardiomyocytes from hydrogen peroxide or superoxide?

The aim of the research was to study the role played by extracellular O ₂ ^.- radicals, which are implicated in cardiac cell damage and the protective effect by cell-permeable, nitroxide, superoxide dismutase-mimics. Cardiomyocytes cultures from 1-day-old rats served as the test-system. Experiments were performed since 5th day in culture when >80% of the cells were beating myocardial cells. Oxidative damage was induced by 0.5 mM hypoxanthine and 0.06 U/ml xanthine oxidase or by 10 mM glucose and 0.15 U/ml glucose oxidase. The parameters used to evaluate damages were spontaneous beating, lactate dehydrogenase release and ATP level. The rhythmic pulsation was followed microscopically. To determine the kinetics of cytosolic enzyme release from the cells, media samples were collected at various points of time and assayed for enzyme activity. To determine the cellular ATP, cells were washed with sodium phosphate buffer, scraped off and boiled for 3 min with sodium phosphate buffer. Following centrifugation the supernatant was collected and ATP was determined by the chemiluminogenic assay using firefly tails. The present results indicate that nitroxide stable free radicals, in the millimolar concentration range, provide full protection without toxic side-effect. Unlike exogenously added SOD that failed to protect, exogenous catalase provided almost full protection. In addition, the metal-chelating agent dipyridyl, but not diethylene-triamine-pentaacetate or desferrioxamine, protected the cultured cells. The present results suggest that H₂O₂ is the predominant toxic species mediating the oxidative damage whereas extracellular superoxide radical does not contribute to cultured cardiomyocyte damage. Since nitroxides do not remove H₂O₂ they can protect the cells possibly by oxidizing the metal ions and inhibiting the Fenton reaction. The superoxide dismutase-mimic activity of nitroxides does not seem to underlie their protective effect, however, the involvement of intracellular O ₂ ^.- cannot be excluded.Abbreviations CHDO 2-spirocyclohexane doxyl (2-cyclohexane-5,5-demethyl-3-oxazolidinoxyl) - DF desferrioxamine - DTPA diethylene-triamine-pentaacetate - EPR electron paramagnetic resonance - HX hypoxanthine - LDH lactate dehydrogenase - SOD superoxide dismutase - SEM standard error of mean: TEMPOL, 4-hydroxy-2,2,6,6-tetramethyl-piperidinoxyl - TEMPAMINE 4-amino-2,2,6,6-tetramethyl-piperidinoxyl - XO xanthine oxidase - CAT catalase     

The catalase–hydrogen peroxide system. A theoretical appraisal of the mechanism of catalase action

1. The mechanisms of catalase action advanced by Jones & Wynne-Jones (1962) and by Nicholls (1964) are compared in terms of their relative plausibilities and their utility for extension to accommodate more recent experimental information. 2. A revised formal mechanism is advanced that avoids the less satisfactory features of these mechanisms and attempts to account for the roles of catalase sub-units in both reversible and irreversible deactivation phenomena. 3. Theoretical studies of the redox chemistry of peroxides are used to provide the basis for a discussion of the mechanism of the redox act in catalatic action at the molecular level. It is suggested that an important feature of catalase action may be a mediation of the formation of a reactive intermediate by stereospecifically located acid-base functions in the active site. 4. A more detailed statement of this concept is attempted in terms of a hypothetical partial molecular model for the composition and stereochemistry of the active site of catalase. The utility of this model in describing the catalatic and peroxidatic actions of catalase is assessed.     

Is hydrogen peroxide an EDHF in human radial arteries?

In human radial arteries, a nitric oxide/prostanoid-independent mechanism that has the pharmacological characteristics of an EDHF contributes to endothelium-dependent relaxation. H2O2 can act as an EDHF in some vascular beds. We examined the hypothesis that endogenously produced H2O2 mediated the nitric oxide/prostanoid-independent relaxation to carbachol in radial arteries obtained from patients undergoing coronary artery bypass surgery. Superoxide levels, measured by chemiluminescence, were similar in radial and internal mammary arteries, but immunohistochemical staining for Cu/Zn superoxide dismutase (SOD) was lower in endothelium from radial arteries. In organ chamber studies, neither addition of catalase nor addition of SOD to the bathing fluid modified nitric oxide/prostanoid-independent relaxations to carbachol in radial arteries. However, nitric oxide-dependent vasorelaxation was enhanced in the presence of SOD. Thus the nitric oxide/prostanoid-independent relaxation to carbachol is not due to H2O2 and, unlike nitric oxide-mediated vasorelaxation, is not attenuated by superoxide. Blood vessels showing EDHF-mediated relaxations resistant to oxidative stress may provide favorable outcomes in revascularization surgery.     

Is hydrogen peroxide a second messenger of salicylic acid in systemic acquired resistance?

Elevated levels of salicylic acid (SA) are required for the induction of systemic acquired resistance (SAR) in plants. Recently, a salicylic acid-binding protein (SABP) isolated from tobacco was shown to have catalase activity. Based on this finding elevated levels of hydrogen peroxide (H₂O₂) were postulated to act as a second messenger of SA in the SAR signal transduction pathway. A series of experiments have been carried out to clarify the role of H₂O₂ in SAR-signaling. No increase of H₂O₂ was found during the onset of SAR. Induction of the SAR gene, PR-1, by H₂O₂ and H₂O₂-inducing chemicals is strongly suppressed in transgenic tobacco plants that express the bacterial salicylate hydroxylase gene, indicating that H₂O₂ induction of SAR genes is dependent on SA accumulation. Following treatment of plants with increasing concentrations of H₂O₂, a dose-dependent accumulation of total SA species was found, suggesting that H₂O₂ may induce PR-1 gene expression through SA accumulation. While the results do not support a role for H₂O₂ in SAR signaling, it is suggested that SA inhibition of catalase activity may be important in tissues undergoing a hypersensitive response.     

Development of a new hydrogen peroxide–based vaccine platform

Safe and effective vaccines are crucial for maintaining public health and reducing the global burden of infectious disease. Here we introduce a new vaccine platform that uses hydrogen peroxide (H(2)O(2)) to inactivate viruses for vaccine production. H(2)O(2) rapidly inactivates both RNA and DNA viruses with minimal damage to antigenic structure or immunogenicity and is a highly effective method when compared with conventional vaccine inactivation approaches such as formaldehyde or β-propiolactone. Mice immunized with H(2)O(2)-inactivated lymphocytic choriomeningitis virus (LCMV) generated cytolytic, multifunctional virus-specific CD8(+) T cells that conferred protection against chronic LCMV infection. Likewise, mice vaccinated with H(2)O(2)-inactivated vaccinia virus or H(2)O(2)-inactivated West Nile virus showed high virus-specific neutralizing antibody titers and were fully protected against lethal challenge. Together, these studies demonstrate that H(2)O(2)-based vaccines are highly immunogenic, provide protection against a range of viral pathogens in mice and represent a promising new approach to future vaccine development.     

Are intracellular bacteria hallmarks for human tumors?

正There are ten major characteristics that are used to differentiate cancer from normal cells and tissue (Hanahan and Weinberg, 2011); detection of bacterial species associated with cancer cells is not included among these characteristics,and at first glance this might seem to be irrelevant. The