The Role of Heme Binding by DNA-protective Protein from Starved Cells (Dps) in the Tolerance of Porphyromonas gingivalis to Heme Toxicity

The widely expressed DNA-protective protein from starved-cells (Dps) family proteins are considered major contributors to prokaryotic resistance to stress. We show here that Porphyromonas gingivalis Dps (PgDps), previously described as an iron-storage and DNA-binding protein, also mediates heme sequestration. We determined that heme binds strongly to PgDps with an apparent K_d of 3.7 × 10⁻⁸ m and is coordinated by a single surface-located cysteine at the fifth axial ligand position. Heme and iron sequestered in separate sites by PgDps provide protection of DNA from H₂O₂-mediated free radical damage and were found to be important for growth of P. gingivalis under excess heme as the only iron source. Conservation of the heme-coordinating cysteine among Dps isoforms from the Bacteroidales order suggests that this function may be a common feature within these anaerobic bacteria.     

Role of HemF and HemN in the heme biosynthesis of Vibrio vulnificus under S‐adenosylmethionine‐limiting conditions

Vibrio vulnificus contains two coproporphyrinogen III oxidases (CPOs): O₂‐dependent HemF and O₂‐independent HemN. The growth of the hemF mutant HF1 was similar to wild‐type cells at pH 7.5 under 2% O₂ conditions where HemN was active and had a half‐life of 64 min. However, HF1 did not grow when the medium pH decreased to pH 5.0, where oxidative stress affects endogenous S‐adenosylmethionine (SAM) levels. The growth of HF1 was restored not only by elevating the expression of MnSOD but also through the exogenous addition of SAM. For HF1 to grow under these SAM‐limiting conditions, a mutation arose in hemN, encoding HemN_Y74F. Refolding of the denatured enzymes in vitro revealed that the apparent binding affinity of HemN_Y74F for the cofactor SAM1, which coordinates the 4Fe‐4S cluster, was approximately sixfold higher than that of HemN. The K_m of HemN_Y74F for the co‐substrate SAM2, which provides radicals for CPO reactions, was threefold lower than that of HemN. Thus, affinities for both SAM1 and SAM2 were higher with the Y74F mutation. Taken together, when SAM is limiting, HemN is apparently nonfunctional, and heme synthesis is continued by HemF.     

OxyR介导的细菌氧化胁迫应答调控机制研究进展

OxyR属于LysR型转录因子家族的氧化胁迫调控蛋白,是细菌抵抗氧化胁迫压力的重要调控因子。OxyR能够通过调控过氧化氢酶和过氧化物酶等抗氧化基因的表达清除H₂O₂、参与铁代谢控制胞内过氧化物的产生以及修复生物大分子氧化损伤,从而抵抗氧化胁迫。OxyR的基因表达调控功能依赖于其还原态和氧化态之间的转变,改变调控蛋白对下游基因调控区的亲和能力。氧化态OxyR识别启动子区的结合序列,激活或抑制过氧化氢酶等基因的表达。还原态和氧化态的转换依赖于在氧化状态下分子间二硫键的形成。本文综述了近年来细菌OxyR调控基因表达的最新研究进展,有助于深入理解OxyR在细菌抵抗氧化胁迫的作用方式,为相关致病菌的防治奠定分子基础。     

Mechanisms of neuroprotection by hemopexin: modeling the control of heme and iron homeostasis in brain neurons in inflammatory states

Hemopexin provides neuroprotection in mouse models of stroke and intracerebral hemorrhage and protects neurons in vitro against heme or reactive oxygen species (ROS) toxicity via heme oxygenase‐1 (HO1) activity. To model human brain neurons experiencing hemorrhages and inflammation, we used human neuroblastoma cells, heme–hemopexin complexes, and physiologically relevant ROS, for example, H₂O₂ and HOCl, to provide novel insights into the underlying mechanism whereby hemopexin safely maintains heme and iron homeostasis. Human amyloid precursor protein (hAPP), needed for iron export from neurons, is induced ~twofold after heme–hemopexin endocytosis by iron from heme catabolism via the iron‐regulatory element of hAPP mRNA. Heme–hemopexin is relatively resistant to damage by ROS and retains its ability to induce the cytoprotective HO1 after exposure to tert‐butylhydroperoxide, although induction is impaired, but not eliminated, by exposure to high concentrations of H₂O₂ in vitro. Apo‐hemopexin, which predominates in non‐hemolytic states, resists damage by H₂O₂ and HOCl, except for the highest concentrations likely in vivo. Heme–albumin and albumin are preferential targets for ROS; thus, albumin protects hemopexin in biological fluids like CSF and plasma where it is abundant. These observations provide strong evidence that hemopexin will be neuroprotective after traumatic brain injury, with heme release in the CNS, and during the ensuing inflammation. Hemopexin sequesters heme, thus preventing unregulated heme uptake that leads to toxicity; it safely delivers heme to neuronal cells; and it activates the induction of proteins including HO1 and hAPP that keep heme and iron at safe levels in neurons.     

Structure, function and regulation of the DNA-binding protein Dps and its role in acid and oxidative stress resistance in Escherichia coli: a review

Dps, the DNA‐binding protein from starved cells, is capable of providing protection to cells during exposure to severe environmental assaults; including oxidative stress and nutritional deprivation. The structure and function of Dps have been the subject of numerous studies and have been examined in several bacteria that possess Dps or a structural/functional homologue of the protein. Additionally, the involvement of Dps in stress resistance has been researched extensively as well. The ability of Dps to provide multifaceted protection is based on three intrinsic properties of the protein: DNA binding, iron sequestration, and its ferroxidase activity. These properties also make Dps extremely important in iron and hydrogen peroxide detoxification and acid resistance as well. Regulation of Dps expression in E. coli is complex and partially dependent on the physiological state of the cell. Furthermore, it is proposed that Dps itself plays a role in gene regulation during starvation, ultimately making the cell more resistant to cytotoxic assaults by controlling the expression of genes necessary for (or deleterious to) stress resistance. The current review focuses on the aforementioned properties of Dps in E. coli, its prototypic organism. The consequences of elucidating the protective mechanisms of this protein are far‐reaching, as Dps homologues have been identified in over 1000 distantly related bacteria and Archaea. Moreover, the prevalence of Dps and Dps‐like proteins in bacteria suggests that protection involving DNA and iron sequestration is crucial and widespread in prokaryotes.     

Overlapping and Complementary Oxidative Stress Defense Mechanisms in Nontypeable Haemophilus influenzae

The Gram-negative commensal bacterium nontypeable Haemophilus influenzae (NTHI) can cause respiratory tract diseases that include otitis media, sinusitis, exacerbations of chronic obstructive pulmonary disease, and bronchitis. During colonization and infection, NTHI withstands oxidative stress generated by reactive oxygen species produced endogenously, by the host, and by other copathogens and flora. These reactive oxygen species include superoxide, hydrogen peroxide (H₂O₂), and hydroxyl radicals, whose killing is amplified by iron via the Fenton reaction. We previously identified genes that encode proteins with putative roles in protection of the NTHI isolate strain 86-028NP against oxidative stress. These include catalase (HktE), peroxiredoxin/glutaredoxin (PgdX), and a ferritin-like protein (Dps). Strains were generated with mutations in hktE, pgdX, and dps. The hktE mutant and a pgdX hktE double mutant were more sensitive than the parent to killing by H₂O₂. Conversely, the pgdX mutant was more resistant to H₂O₂ due to increased catalase activity. Supporting the role of killing via the Fenton reaction, binding of iron by Dps significantly mitigated the effect of H₂O₂-mediated killing. NTHI thus utilizes several effectors to resist oxidative stress, and regulation of free iron is critical to this protection. These mechanisms will be important for successful colonization and infection by this opportunistic human pathogen.     

Influence of polyphenols on Escherichia coli resistance to oxidative stress

Among all polyphenols tested (tannic acid and flavonoids belonging to different subclasses) only tannin and quercetin significantly enhanced resistance of Escherichia coli to peroxide stress. Pretreatment of the cells with quercetin and tannin resulted in a decrease in the growth arrest duration under moderate H₂O₂ concentration (2 mM) and an increase in survival under high (10 mM) doses. The shorter growth recovery period in pretreated cells was connected with more rapid H₂O₂ elimination because of induced activity of scavenging enzymes. This effect was absent in the ΔoxyR mutant, which was unable to induce genes responding to peroxide stress. The data obtained suggest that the observed protection was a result of two overlapping effects: induction of OxyR regulon by low concentrations of H₂O₂, accumulated during extracellular autoxidation of quercetin and tannin, and protection of synthesis of OxyR-regulated antioxidant enzymes during H₂O₂ stress because of intracellular binding of iron by quercetin and tannin and suppressing Fenton chemistry.     

Triosephosphate isomerase tyrosine nitration induced by heme–NaNO2–H2O2 or peroxynitrite: Effects of different natural phenolic compounds

Peroxynitrite and heme peroxidases (or heme)–H₂O₂–NaNO₂ system are the two common ways to cause protein tyrosine nitration in vitro, but the effects of antioxidants on reducing these two pathways‐induced protein nitration and oxidation are controversial. Both nitrating systems can dose‐dependently induce triosephosphate isomerase (TIM) nitration, however, heme–H₂O₂–NaNO₂ was less destructive to protein secondary structures and led to more nitrated tyrosine residue than 3‐morpholinosydnonimine hydrochloride (SIN‐1, a peroxynitrite donor). Both of desferrioxamine and catechin could inhibit TIM nitration induced by heme–H₂O₂–NaNO₂ and SIN‐1 and protein oxidation induced by SIN‐1, but promoted heme–H₂O₂–NaNO₂‐induced protein oxidation. Moreover, the antagonism of natural phenolic compounds on SIN‐1‐induced tyrosine nitration was consistent with their radical scavenging ability, but no similar consensus was found in heme–H₂O₂–NaNO₂‐induced nitration. Our results indicated that peroxynitrite and heme–H₂O₂–NaNO₂‐induced protein nitration was different, and the later one could be a better model for anti‐nitration compounds screening.     

An anaerobic bacterium,Bacteroides thetaiotaomicron,uses a consortium of enzymes to scavenge hydrogen peroxide

Obligate anaerobes are periodically exposed to oxygen, and it has been conjectured that on such occasions their low‐potential biochemistry will predispose them to rapid ROS formation. We sought to identify scavenging enzymes that might protect the anaerobe Bacteroides thetaiotaomicron from the H₂O₂ that would be formed. Genetic analysis of eight candidate enzymes revealed that four of these scavenge H₂O₂ in vivo: rubrerythrins 1 and 2, AhpCF, and catalase E. The rubrerythrins served as key peroxidases under anoxic conditions. However, they quickly lost activity upon aeration, and AhpCF and catalase were induced to compensate. The AhpCF is an NADH peroxidase that effectively degraded low micromolar levels of H₂O₂, while the catalytic cycle of catalase enabled it to quickly degrade higher concentrations that might arise from exogenous sources. Using a non‐scavenging mutant we verified that endogenous H₂O₂ formation was much higher in aerated B. thetaiotaomicron than in Escherichia coli. Indeed, the OxyR stress response to H₂O₂ was induced when B. thetaiotaomicron was aerated, and in that circumstance this response was necessary to forestall cell death. Thus aeration is a serious threat for this obligate anaerobe, and to cope it employs a set of defences that includes a repertoire of complementary scavenging enzymes.     

Rapid in vivo screening system for anti‐oxidant activity using bacterial redox sensor strains

Aim: To develop a faster and easier in vivo method to screen compounds for anti‐oxidant activity using a microbial system. Methods and Results: Bacterial redox sensor‐based assay systems were applied. The activities of SoxR and OxyR, the bacterial redox sensors, were monitored to probe the intracellular redox status through two reporter strains, Escherichia coli soxS_p‐lacZ and oxyS_p‐lacZ fusions, which specifically respond to paraquat, a superoxide generator, and H₂O₂, respectively, with practically no cross reactivity. For the test screening, 27 natural compounds including phenolics and flavonoids that are putatively considered anti‐oxidant nutritional supplements were collected and assayed for their capability to alleviate oxidative stress in these bacterial systems. Among them, rutin, kaempferol and quercetin had significant anti‐H₂O₂ activity, and betaine, glycyrrhizic acid and baicalin had weak anti‐superoxide activity. While rutin, kaempferol and quercetin significantly reduced the H₂O₂ stress at low concentrations, betaine, glycyrrhizic acid and baicalin required higher concentration for their anti‐superoxide effects. In vitro, only quercetin protected DNA in a metal‐catalysed oxidation system, suggesting that the other compounds might indirectly exert their anti‐oxidant activities through other biological functions. Finally, quercetin, rutin and kaempferol significantly restored the viability of a superoxide dismutase mutant that has limited viability because of defective defence against oxidative stress. Conclusion: These bacterial systems could provide a more efficient method for measuring the activity of compounds affecting cellular oxidative stress and viability. Significance and Impact of the Study: The demand for anti‐oxidant and anti‐ageing activities is increasing in one of the fastest growing segments of the functional food market, but the screening for these activities is currently very laborious, expensive and time consuming. This study suggests a basis for a high throughput screening method for these activities.     

Agrobacterium tumefaciens ferritins play an important role in full virulence through regulating iron homeostasis and oxidative stress survival

Ferritins are a large family of iron storage proteins, which are used by bacteria and other organisms to avoid iron toxicity and as a safe iron source in the cytosol. Agrobacterium tumefaciens, a phytopathogen, has two ferritin-encoding genes: atu2771 and atu2477. Atu2771 is annotated as a Bfr-encoding gene (Bacterioferritin, Bfr) and atu2477 as a Dps-encoding gene (D NA binding p rotein from s tarved cells, Dps). Three deletion mutants (Δbfr, Δdps, and bfr-dps double-deletion mutant ΔbdF) of these two ferritin-encoding genes were constructed to investigate the effects of ferritin deficiency on the iron homeostasis, oxidative stress resistance, and pathogenicity of A. tumefaciens. Deficiency of two ferritins affects the growth of A. tumefaciens under iron starvation and excess. When supplied with moderate iron, the growth of A. tumefaciens is not affected by the deficiency of ferritin. Deficiency of ferritin significantly reduces iron accumulation in the cells of A. tumefaciens, but the effect of Bfr deficiency on iron accumulation is severer than Dps deficiency and the double mutant ΔbdF has the least intracellular iron content. All three ferritin-deficient mutants showed a decreased tolerance to 3 mM H₂O₂ in comparison with the wild type. The tumour induced by each of three ferritin-deficient mutants is less than that of the wild type. Complementation reversed the effects of ferritin deficiency on the growth, iron homeostasis, oxidative stress resistance, and tumorigenicity of A. tumefaciens. Therefore, ferritin plays an important role in the pathogenesis of A. tumefaciens through regulating iron homeostasis and oxidative stress survival.     

How Escherichia coli Tolerates Profuse Hydrogen Peroxide Formation by a Catabolic Pathway

When Escherichia coli grows on conventional substrates, it continuously generates 10 to 15 μM/s intracellular H₂O₂ through the accidental autoxidation of redox enzymes. Dosimetric analyses indicate that scavenging enzymes barely keep this H₂O₂ below toxic levels. Therefore, it seemed potentially problematic that E. coli can synthesize a catabolic phenylethylamine oxidase that stoichiometrically generates H₂O₂. This study was undertaken to understand how E. coli tolerates the oxidative stress that must ensue. Measurements indicated that phenylethylamine-fed cells generate H₂O₂ at 30 times the rate of glucose-fed cells. Two tolerance mechanisms were identified. First, in enclosed laboratory cultures, growth on phenylethylamine triggered induction of the OxyR H₂O₂ stress response. Null mutants (ΔoxyR) that could not induce that response were unable to grow. This is the first demonstration that OxyR plays a role in protecting cells against endogenous H₂O₂. The critical element of the OxyR response was the induction of H₂O₂ scavenging enzymes, since mutants that lacked NADH peroxidase (Ahp) grew poorly, and those that additionally lacked catalase did not grow at all. Other OxyR-controlled genes were expendable. Second, phenylethylamine oxidase is an unusual catabolic enzyme in that it is localized in the periplasm. Calculations showed that when cells grow in an open environment, virtually all of the oxidase-generated H₂O₂ will diffuse across the outer membrane and be lost to the external world, rather than enter the cytoplasm where H₂O₂-sensitive enzymes are located. In this respect, the periplasmic compartmentalization of phenylethylamine oxidase serves the same purpose as the peroxisomal compartmentalization of oxidases in eukaryotic cells.     

鸭疫里氏杆菌B739＿RS00825基因缺失株抗血红素毒性和氧化应激损伤以及定殖能力分析

【目的】血红素可作为细菌重要的铁离子来源,然而转运过多的血红素也会对细菌造成毒性。细菌通过调节、外排、螯合等多种方式减轻血红素毒性作用。鸭疫里氏杆菌(Riemerella anatipestifer, RA)是一种感染鸭及其他禽类的革兰氏阴性病原菌。前期研究表明,该菌编码血红素转运系统,且能够从血红蛋白获取血红素,然而该菌是否编码血红素解毒蛋白未知。本研究以编码一氧化氮合成酶的基因B739＿RS00825为研究对象,分析其在抗血红素毒性和氧化应激损伤以及定殖能力中的功能。【方法】构建B739＿RS00825缺失株,并通过测定生长曲线、细菌存活率、毒力及定殖等试验方法鉴定其在抗血红素毒性、抗氧化应激损伤、宿主致病中的功能。【结果】与RA CH-1相比,RA CH-1ΔB739＿RS00825在添加过量血红素的培养基中生长不受影响;然而与RACH-1Δfur相比,RACH-1ΔfurΔB739＿RS00825在含血红素培养基中的生长明显受到抑制且对H₂O₂的抵抗力降低;B739＿RS00825基因在氧化应激条件下及fur缺失株中明显上调;与RA ...     

Kinetic simulation studies on the transient formation of the oxo‐iron(IV) porphyrin radical cation during the reaction of iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)‐porphyrin with hydrogen peroxide in aqueous solution

High‐valent oxo‐iron(IV) species are commonly proposed as the key intermediates in the catalytic mechanisms of iron enzymes. Water‐soluble iron(III) tetrakis‐5,10,15,20‐(N‐methyl‐4‐pyridyl)porphyrin (Fe(III)TMPyP) has been used as a model of heme‐enzyme to catalyse the hydrogen peroxide (H₂O₂) oxidation of various organic compounds. However, the mechanism of the reaction of Fe(III)TMPyP with H₂O₂ has not been fully established. In this study, we have explored the kinetic simulation of the reaction of Fe(III)TMPyP with H₂O₂ and of the catalytic reactivity of FeTMPyP in the luminescent peroxidation of luminol. According to the mechanism that has been established in this work, Fe(III)TMPyP is oxidized by H₂O₂ to produce (TMPyP)^·+Fe(IV)=O (k1 = 4.5 × 10⁴/mol/L/s) as a precursor of TMPyPFe(IV)=O. The intermediate, (TMPyP)^·+Fe(IV)=O, represented nearly 2% of Fe(III)TMPyP but it does not accumulate in suf?cient concentration to be detected because its decay rate is too fast. Kinetic simulations showed that the proposed scheme is capable of reproducing the observed time courses of FeTMPyP in various oxidation states and the decay pro?les of the luminol chemiluminescence. It also shows that (TMPyP)^·+Fe(IV)=O is 100 times more reactive than TMPyPFe(IV)=O in most of the reactions. These two species are responsible for the initial sharp and the sustained luminol emissions, respectively. Copyright © 2003 John Wiley & Sons, Ltd.     

The heme-binding protein HbpS regulates the activity of the <Emphasis Type="Italic">Streptomyces reticuli</Emphasis> iron-sensing histidine kinase SenS in a redox-dependent manner

The SenS/SenR system of Streptomyces reticuli regulates the expression of the redox regulator FurS, the catalase-peroxidase CpeB and the heme-binding protein HbpS. SenS/SenR is also proposed to participate in sensing redox changes, mediated by HbpS. Here, we show in vitro that heme-free HbpS represses the autokinase activity of SenS; whereas hemin-treated HbpS considerably enhances SenS autophosphorylation under redox conditions using either H₂O₂ or DTT. The presence of iron ions alone or in combination with H₂O₂ or DTT also leads to significantly increased phosphorylation levels of SenS. Further comparative physiological studies using the S. reticuli WT, a S. reticuli hbpS mutant and a S. reticuli senS-senR mutant corroborates the importance of HbpS and the SenS/SenR system for resistance against high concentrations of iron ions and hemin in vivo. Hence SenS/SenR and HbpS act in concert as a novel three-component system which detects redox stress, mediated by iron ions and heme.     

The YaaA protein of the Escherichia coli OxyR regulon lessens hydrogen peroxide toxicity by diminishing the amount of intracellular unincorporated iron

Hydrogen peroxide (H₂O₂) is commonly formed in microbial habitats by either chemical oxidation processes or host defense responses. H₂O₂ can penetrate membranes and damage key intracellular biomolecules, including DNA and iron-dependent enzymes. Bacteria defend themselves against this H₂O₂ by inducing a regulon that engages multiple defensive strategies. A previous microarray study suggested that yaaA, an uncharacterized gene found in many bacteria, was induced by H₂O₂ in Escherichia coli as part of its OxyR regulon. Here we confirm that yaaA is a key element of the stress response to H₂O₂. In a catalase/peroxidase-deficient (Hpx⁻) background, yaaA deletion mutants grew poorly, filamented extensively, and lost substantial viability when they were cultured in aerobic LB medium. The results from a thyA forward mutagenesis assay and the growth defect of the yaaA deletion in a recombination-deficient (recA56) background indicated that yaaA mutants accumulated high levels of DNA damage. The growth defect of yaaA mutants could be suppressed by either the addition of iron chelators or mutations that slowed iron import, indicating that the DNA damage was caused by the Fenton reaction. Spin-trapping experiments confirmed that Hpx⁻ yaaA cells had a higher hydroxyl radical (HO^•) level. Electron paramagnetic resonance spectroscopy analysis showed that the proximate cause was an unusually high level of intracellular unincorporated iron. These results demonstrate that during periods of H₂O₂ stress the induction of YaaA is a critical device to suppress intracellular iron levels; it thereby attenuates the Fenton reaction and the DNA damage that would otherwise result. The molecular mechanism of YaaA action remains unknown.     

Molecular Insights into Frataxin-Mediated Iron Supply for Heme Biosynthesis in Bacillus subtilis

Iron is required as an element to sustain life in all eukaryotes and most bacteria. Although several bacterial iron acquisition strategies have been well explored, little is known about the intracellular trafficking pathways of iron and its entry into the systems for co-factor biogenesis. In this study, we investigated the iron-dependent process of heme maturation in Bacillus subtilis and present, for the first time, structural evidence for the physical interaction of a frataxin homologue (Fra), which is suggested to act as a regulatory component as well as an iron chaperone in different cellular pathways, and a ferrochelatase (HemH), which catalyses the final step of heme b biogenesis. Specific interaction between Fra and HemH was observed upon co-purification from crude cell lysates and, further, by using the recombinant proteins for analytical size-exclusion chromatography. Hydrogen–deuterium exchange experiments identified the landscape of the Fra/HemH interaction interface and revealed Fra as a specific ferrous iron donor for the ferrochelatase HemH. The functional utilisation of the in vitro-generated heme b co-factor upon Fra-mediated iron transfer was confirmed by using the B. subtilis nitric oxide synthase bsNos as a metabolic target enzyme. Complementary mutational analyses confirmed that Fra acts as an essential component for maturation and subsequent targeting of the heme b co-factor, hence representing a key player in the iron-dependent physiology of B. subtilis.