Distinct iron binding property of two putative iron donors for the iron-sulfur cluster assembly: IscA and the bacterial frataxin ortholog CyaY under physiological and oxidative stress conditions

Frataxin, a small mitochondrial protein linked to the neurodegenerative disease Friedreich ataxia, has recently been proposed as an iron donor for the iron-sulfur cluster assembly. An analogous function has also been attributed to IscA, a key member of the iron-sulfur cluster assembly machinery found in bacteria, yeast, and humans. Here we have compared the iron binding property of IscA and the frataxin ortholog CyaY from Escherichia coli under physiological and oxidative stress conditions. In the presence of the thioredoxin reductase system, which emulates the intracellular redox potential, CyaY fails to bind any iron even at a 10-fold excess of iron in the incubation solution. Under the same physiologically relevant conditions, IscA efficiently recruits iron and transfers the iron for the iron-sulfur cluster assembly in a proposed scaffold IscU. In the presence of hydrogen peroxide, however, IscA completely loses its iron binding activity, whereas CyaY becomes a competent iron-binding protein and attenuates the iron-mediated production of hydroxyl free radicals. Hydrogen peroxide appears to oxidize the iron binding thiol groups in IscA, thus blocking the iron binding in the protein. Once the oxidized thiol groups in IscA are re-reduced with the thioredoxin reductase system, the iron binding activity of IscA is fully restored. On the other hand, hydrogen peroxide has no effect on the iron binding carboxyl groups in CyaY, allowing the protein to bind iron under oxidative stress conditions. The results suggest that IscA is capable of recruiting intracellular iron for the iron-sulfur cluster assembly under normal physiological conditions, whereas CyaY may serve as an iron chaperon to sequester redox active free iron and alleviate cellular oxidative damage under oxidative stress conditions.     

Manganese import is a key element of the OxyR response to hydrogen peroxide in Escherichia coli

Very little manganese is imported into Escherichia coli under routine growth conditions: the import system is weakly expressed, the manganese content is low, and a manganese-dependent enzyme is not correctly metallated. Mutants that lack MntH, the importer, grow at wild-type rates, indicating that manganese plays no critical role. However, MntH supports the growth of iron-deficient cells, suggesting that manganese can substitute for iron in activating at least some metalloenzymes. MntH is also strongly induced when cells are stressed by hydrogen peroxide. This adaptation is essential, as E. coli cannot tolerate peroxide stress if mntH is deleted. Other workers have observed that manganese improves the ability of a variety of microbes to tolerate oxidative stress, and the prevailing hypothesis is that manganese does so by chemically scavenging hydrogen peroxide and/or superoxide. We found that manganese does not protect peroxide-stressed cells by scavenging peroxide. Instead, the beneficial effects of manganese correlate with its ability to metallate mononuclear enzymes. Because iron-loaded enzymes are vulnerable to the Fenton reaction, the substitution of manganese may prevent protein damage. Accordingly, during H₂O₂ stress, mutants that cannot import manganese and/or are unable to sequester iron suffer high rates of protein oxidation.     

Iron and oxidative stress in bacteria

The appearance of oxygen on earth led to two major problems: the production of potentially deleterious reactive oxygen species and a drastic decrease in iron availability. In addition, iron, in its reduced form, potentiates oxygen toxicity by converting, via the Fenton reaction, the less reactive hydrogen peroxide to the more reactive oxygen species, hydroxyl radical and ferryl iron. Conversely superoxide, by releasing iron from iron-containing molecules, favors the Fenton reaction. It has been assumed that the strict regulation of iron assimilation prevents an excess of free intracellular iron that could lead to oxidative stress. Studies in bacteria supporting that view are reviewed. While genetic studies correlate oxidative stress with increase of intracellular free iron, there are only few and sometimes contradictory studies on direct measurements of free intracellular metal. Despite this weakness, the strict regulation of iron metabolism, and its coupling with regulation of defenses against oxidative stress, as well as the role played by iron in regulatory protein in sensing redox change, appear as essential factors for life in the presence of oxygen.     

Isc1p plays a key role in hydrogen peroxide resistance and chronological lifespan through modulation of iron levels and apoptosis

The inositolphosphosphingolipid phospholipase C (Isc1p) of Saccharomyces cerevisiae belongs to the family of neutral sphingomyelinases that generates the bioactive sphingolipid ceramide. In this work the role of Isc1p in oxidative stress resistance and chronological lifespan was investigated. Loss of Isc1p resulted in a higher sensitivity to hydrogen peroxide that was associated with an increase in oxidative stress markers, namely intracellular oxidation, protein carbonylation, and lipid peroxidation. Microarray analysis showed that Isc1p deficiency up-regulated the iron regulon leading to increased levels of iron, which is known to catalyze the production of the highly reactive hydroxyl radicals via the Fenton reaction. In agreement, iron chelation suppressed hydrogen peroxide sensitivity of isc1Δ mutants. Cells lacking Isc1p also displayed a shortened chronological lifespan associated with oxidative stress markers and aging of parental cells was correlated with a decrease in Isc1p activity. The analysis of DNA fragmentation and caspase-like activity showed that Isc1p deficiency increased apoptotic cell death associated with oxidative stress and aging. Furthermore, deletion of Yca1p metacaspase suppressed the oxidative stress sensitivity and premature aging phenotypes of isc1Δ mutants. These results indicate that Isc1p plays an important role in the regulation of cellular redox homeostasis, through modulation of iron levels, and of apoptosis.     

Oxygen toxicity and microbial evolution

It is postulated that the role of oxygen toxicity in the evolution of life strongly depends on the origin of molecular oxygen, due to the strong redox buffering capacity of Precambrian waters containing large amounts of ferrous and manganese cations. The critical selective pressure could be observed only after aerobic photosynthesis had been developed, due to the high local concentration of oxygen in close vicinity of photosynthesizing cells. It is also postulated that early oxygen-evolving organisms excreted a substantial part of this element in the form of hydrogen peroxide. As a consequence of the high reactivity of this compound with ferrous and manganese cations, an important percentage of iron deposits were produced with H2O2 as a major oxidant after the development of aerobic photosynthesis. It is postulated that negatively charged extracellular polymers of simple pro- and eukaryotic organisms function as sacrificial targets of hydroxyl radicals and at the same time as extracellular equivalents of superoxide dismutases, in these two ways protecting cellular membranes against oxidative damage. The role of oxygen toxicity in developing aerobic mechanisms of iron uptake is also discussed.     

Disruption of sitA compromises Sinorhizobium meliloti for manganese uptake required for protection against oxidative stress

During the initial stages of symbiosis with the host plant Medicago sativa, Sinorhizobium meliloti must overcome an oxidative burst produced by the plant in order for proper symbiotic development to continue. While identifying mutants defective in symbiosis and oxidative stress defense, we isolated a mutant with a transposon insertion mutation of sitA, which encodes the periplasmic binding protein of the putative iron/manganese ABC transporter SitABCD. Disruption of sitA causes elevated sensitivity to the reactive oxygen species hydrogen peroxide and superoxide. Disruption of sitA leads to elevated catalase activity and a severe decrease in superoxide dismutase B (SodB) activity and protein level. The decrease in SodB level strongly correlates with the superoxide sensitivity of the sitA mutant. We demonstrate that all free-living phenotypes of the sitA mutant can be rescued by the addition of exogenous manganese but not iron, a result that strongly implies that SitABCD plays an important role in manganese uptake in S. meliloti.     

Iron and its complexation by phenolic cellular metabolites: From oxidative stress to chemical weapons

Iron is a transition metal that forms chelates and complexes with various organic compounds, also with phenolic plant secondary metabolites. The ligands of iron affect the redox potential of iron. Electrons may be transferred either to hydroxyl radicals, hydrogen peroxide or molecular oxygen. In the first case, oxidative stress is decreased, in the latter two cases, oxidative stress is increased. This milieu-dependent mode of action may explain the non-linear mode of action of juglone and other secondary metabolites. Attention to this phenomenon may help to explain idiosyncratic and often nonlinear effects that result in biological assays. Current chemical assays are discussed that help to explore these aspects of redox chemistry.Key words: Fenton reaction, catechin, juglone, siderosphores, invasive plants, redox homeostasis     

Intracellular Hydrogen Peroxide and Superoxide Poison 3-Deoxy-d-Arabinoheptulosonate 7-Phosphate Synthase,the First Committed Enzyme in the Aromatic Biosynthetic Pathway of Escherichia coli

In Escherichia coli, aromatic compound biosynthesis is the process that has shown the greatest sensitivity to hydrogen peroxide stress. This pathway has long been recognized to be sensitive to superoxide as well, but the molecular target was unknown. Feeding experiments indicated that the bottleneck lies early in the pathway, and the suppressive effects of fur mutations and manganese supplementation suggested the involvement of a metalloprotein. The 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHP synthase) activity catalyzes the first step in the pathway, and it is provided by three isozymes known to rely upon a divalent metal. This activity progressively declined when cells were stressed with either oxidant. The purified enzyme was activated more strongly by ferrous iron than by other metals, and only this metalloform could be inactivated by hydrogen peroxide or superoxide. We infer that iron is the prosthetic metal in vivo. Both oxidants displace the iron atom from the enzyme. In peroxide-stressed cells, the enzyme accumulated as an apoprotein, potentially with an oxidized cysteine residue. In superoxide-stressed cells, the enzyme acquired a nonactivating zinc ion in its active site, an apparent consequence of the repeated ejection of iron. Manganese supplementation protected the activity in both cases, which matches the ability of manganese to metallate the enzyme and to provide substantial oxidant-resistant activity. DAHP synthase thus belongs to a family of mononuclear iron-containing enzymes that are disabled by oxidative stress. To date, all the intracellular injuries caused by physiological doses of these reactive oxygen species have arisen from the oxidation of reduced iron centers.     

Free Radical Inactivation of Rabbit Muscle Creatine Kinase: Catalysis by Physiological and Hydrolyzed ICRF-187 (ICRF-198) Iron Chelates

Creatine kinase is a sulfhydryl containing enzyme that is particularly susceptible to oxidative inactivation. This enzyme is potentially vulnerable to inactivation under conditions when it would be used as a diagnostic marker of tissue damage such as during cardiac ischemia/reperfusion or other oxidative tissue injury. Oxidative stress in tissues can induce the release of iron from its storage proteins, making it an available catalyst for free radical reactions. Although creatine kinase inactivation in a heart reperfusion model has been documented, the mechanism has not been fully described, particularly with regard to the role of iron. We have investigated the inactivation of rabbit muscle creatine kinase by hydrogen peroxide and by xanthine oxidase generated superoxide or Adriamycin radicals in the presence of iron catalysts. As shown previously, creatine kinase was inactivated by hydrogen peroxide. Ferrous iron enhanced the inactivation. In addition, micromolar levels of iron and iron chelates that were reduced and recycled by superoxide or Adriamycin radicals were effective catalysts of creatine kinase inactivation. Of the physiological iron chelates studied, Fe(ATP) was an especially effective catalyst of inactivation by what appeared to be a site-localized reaction. Fe(ICRF-198), a non-physiological chelate of interest because of its putative role in alleviating Adriamycin-induced cardiotoxicity, also catalyzed the inactivation. Scavenger studies implicated hydroxyl radical as the oxidant involved in iron-dependent creatine kinase inactivation. Loss of protein thiols accompanied loss of creatine kinase activity. Reduced glutathione (GSH) provided marked protection from oxidative inactivation, suggesting that enzyme inactivation under physiological conditions would occur only after GSH depletion.     

Glucose-6-phosphate dehydrogenase deficiency: a new hypothesis for an old disease

Summary

We have previously shown insulinoma (HIT-T15 and RINm5F) cells in culture to be very sensitive, in comparison with a reference cell line (J-774), to the oxidative stress that is created when alloxan reacts extracellularly with reducing agents, forming superoxide and hydrogen peroxide. The toxic effects are prevented by catalase added to the medium, suggesting that alloxan does not need to be taken up in order to affect cells. Rather, alloxan seems to exert its action through extracellular formation of hydrogen peroxide that influences the stability of the cells' lysosomes following diffusion into them. To further analyse the mechanisms in operation, we studied the influence of induced autophagocytosis on the sensitivity to ensuing oxidative stress. Starvation for 60–120 min in PBS at 37°C markedly enhanced autophagocytosis and, in parallel, increased the cytotoxic effect and lysosomal vulnerability of ensuing exposure to hydrogen peroxide, while not significantly changing the antioxidative status or the energy balance. Autophagocytosis increased the size of the intralysosomal pool of reactive, low-molecular-weight, iron, probably by degradation of metallo-proteins, as shown by autometallography and HPLC demonstration of desferrioxamine-reactive intracellular iron. Moreover, exposure to the iron-chelator desferrioxamine before treatment with hydrogen peroxide prevented lysosomal destabilization and cellular death of both starved and control cells, further proving the importance of intralysosomal iron for the response to oxidative stress. We hypothesize that β-cells which, like insulinoma cells, have a weak antioxidative defence system under conditions of enhanced general autophagocytosis, or crinophagy, might become vulnerable to even low, or moderate, oxidative stress.     

Impact of Hydrogen Peroxide on the Activity, Structure, and Conformational Stability of the Oxidized Protein Repair Enzyme Methionine Sulfoxide Reductase A

The oxidized protein repair methionine sulfoxide reductase (Msr) system has been implicated in aging, in longevity, and in the protection against oxidative stress. This system is made of two different enzymes (MsrA and MsrB) that catalyze the reduction of the two diastereoisomers S- and R-methionine sulfoxide back to methionine within proteins, respectively. Due to its role in cellular protection against oxidative stress that is believed to originate from its reactive oxygen species scavenging ability in combination with exposed methionine at the surface of proteins, the susceptibility of MsrA to hydrogen-peroxide-mediated oxidative inactivation has been analyzed. This study is particularly relevant to the oxidized protein repair function of MsrA in both fighting against oxidized protein formation and being exposed to oxidative stress situations. The enzymatic properties of MsrA indeed rely on the activation of the catalytic cysteine to the thiolate anion form that is potentially susceptible to oxidation by hydrogen peroxide. The residual activity and the redox status of the catalytic cysteine were monitored before and after treatment. These experiments showed that the enzyme is only inactivated by high doses of hydrogen peroxide. Although no significant structural modification was detected by near- and far-UV circular dichroism, the conformational stability of oxidized MsrA was decreased as compared to that of native MsrA, making it more prone to degradation by the 20S proteasome. Decreased conformational stability of oxidized MsrA may therefore be considered as a key factor for determining its increased susceptibility to degradation by the proteasome, hence avoiding its intracellular accumulation upon oxidative stress.     

Pro- or anti-oxidant manganese: a suggested mechanism for reconciliation

The neurodegeneration induced by manganese has been attributed to its ability to undergo redox cycling, and catalysis of reactive oxygen species (ROS) formation, as with other transition metals. However, the characterization of manganese as a pro-oxidant is confounded by increasing evidence that the metal may scavenge superoxide anions and protect cells from oxidative damage. The current study was designed to address conflicting reports pertaining to the oxidative capacity of manganese. We found that the metal has distinctive redox dynamics in which the divalent reduced form, unlike iron, possessed no intrinsic oxidative capacity. The apparent ability of Mn(2+) to promote the formation of ROS within a cortical mitochondrial-synaptosomal fraction was quenched by the depletion of contaminating nanomolar concentrations of trivalent metals. The addition of manganic ions at trace concentrations dose-dependently restored the oxidative capacity attributed to divalent manganese, whereas the presence of the ferric ion retarded the rate of ROS generation. This result was paralleled by the spectrophotometric demonstration that the kinetics of iron oxidation is accelerated by trivalent but not divalent manganese. The markedly different capacities of the lower and higher valence states of manganese to promote free-radical formation in cortical fractions and to modulate the process of iron oxidation may account for earlier contradictory reports of anti- and pro-oxidant properties of manganese.     

Enhancement of cytotoxicity of hydrogen peroxide by hyperthermia in chinese hamster ovary cells: role of antioxidant defenses

Regional hyperthermia has potential for human cancer treatment, particularly in combination with systemic chemotherapy or radiotherapy. The mechanisms involved in heat-induced cell killing are currently unknown. Hyperthermia may increase oxidative stress in cells, and thus, oxidative stress could have a role in the mechanism of cell death. We use hydrogen peroxide as a model oxidant to improve understanding of interactions between heat and oxidative stress. Heat increased cytotoxicity of hydrogen peroxide in Chinese hamster ovary cells. Altered levels of cellular antioxidants should create an imbalance between prooxidant and antioxidant systems, thus modifying cytotoxic responses to heat and to oxidants. We determine the involvement of the two cellular antioxidant defenses against peroxides, catalase and the glutathione redox cycle, in cellular sensitivity to heat, to hydrogen peroxide, and to heat combined with the oxidant. Defense systems were either inhibited or increased. For inhibition studies, intracellular glutathione was diminished to less than 15% of its initial level by treatment with L-buthionine sulfoximine (1 mM, 24 h). Inhibition of catalase was achieved with 3-amino-1,2,4-triazole (20 mM, 2 h), which caused a 80% decrease in endogenous enzyme activity. To increase antioxidants, cells were pretreated with the thiol-containing reducing agents, N-acetyl-L-cysteine, 2-oxo-4-thiazolidine carboxylate, and 2-mercaptoethane sulfonate. These compounds increased intracellular glutathione levels by 30%. Catalase activity was increased by addition of exogenous enzyme to cells. We show that levels of glutathione and catalase affect cellular cytotoxic responses to heat and hydrogen peroxide, either used separately or in combination. These findings are relevant to mechanisms of cell killing at elevated temperatures and suggest the involvement of oxidative stress.     

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.     

The early intracellular production of a reactive oxygen intermediate mediates apoptosis in dexamethasone-treated thymocytes

Many conditions that induce an oxidative stress are capable of evoking apoptosis. This has lead to the proposal of oxidative stress as a mediator of apoptosis. We show that, in murine thymocytes, oxidative stress and apoptosis occur in the same cell. We identified four distinct apoptotic subpopulations that appeared sequentially in time. Catalase protected from dex-amethasone-induced death in the initial stages of apoptosis, while iron chelators and Vitamin E did not. Further studies provided evidence supporting the early production of an intracellular oxidative intermediate as an obligatory step for the efficient induction of apoptosis. We propose that at least one of the molecules capable of filling this role is hydrogen peroxide.     

Stress proteins in oligodendrocytes: differential effects of heat shock and oxidative stress

Heat shock proteins (HSP) or stress proteins serve as biomarkers to identify the contribution of stress situations underlying the pathogenesis of degenerative diseases of the CNS. We have analyzed by immunoblot technique the constitutive and inducible occurrence of stress proteins in cultured rat brain oligodendrocytes subjected to heat shock or oxidative stress exerted by hydrogen peroxide, or a combination of both. The data demonstrate that oligodendrocytes constitutively express HSP32, HSP60 and the cognate form of the HSP70 family of proteins, HSC70. After heat shock, HSP25, alpha B-crystallin and HSP70 were up-regulated, while after oxidative stress the specific induction of HSP32 and alpha B-crystallin was observed. HSP32 represents heme oxygenase 1 (HO-1), a small stress protein with enzymatic activity involved in the oxidative degradation of heme which participates in iron metabolism. The presence of the iron chelators phenanthroline or deferoxamine (DFO), which previously has been shown to protect oligodendrocytes from oxidative stress-induced onset of apoptosis, caused a marked stimulation of HSP32 without affecting HSP70. This indicates that DFO possibly exerts its protective role by directly influencing the antioxidant capacity of HO-1. In summary, HSP in oligodendrocytes are differentially stimulated by heat stress and oxidative stress. Heme oxygenase-1 has been linked to inflammatory processes and oxidative stress, its specific up-regulation after oxidative stress in oligodendrocytes suggests that it is an ideal candidate to investigate the involvement of oxidative stress in demyelinating diseases.