Oxidative mutagenesis of doxorubicin-Fe(III) complex

Doxorubicin has a high affinity for inorganic iron, Fe(III), and has potential to form doxorubicin-Fe(III) complexes in biological systems. Indirect involvement of iron has been substantiated in the oxidative mutagenicity of doxorubicin. In this study, however, direct involvement of Fe(III) was evaluated in mutagenicity studies with the doxorubicin-Fe(III) complex. The Salmonella mutagenicity assay with strain TA102 was used with a pre-incubation step. The highest mutagenicity of doxorubicin-Fe(III) complex was observed at the dose of 2.5nmol/plate of the complex. The S9-mix decreased this highest mutagenicity but increased the number of revertants at a higher dose of 10nmol/plate of the complex. On the other hand, the mutagenicity of the doxorubicin-Fe(III) complex at the doses of 0.25, 0.5, 1 and 2nmol/plate was enhanced about twice by the addition of glutathione plus H(2)O(2). This enhanced mutagenicity as well as of the complex itself, the complex plus glutathione, and the complex plus H(2)O(2) were reduced by the addition of ADR-529, an Fe(III) chelator, and potassium iodide, a hydroxyl radical scavenger. These results indicate that doxorubicin-Fe(III) complex exert the mutagenicity through oxidative DNA damage and that Fe(III) is a required element in the mutagenesis of doxorubicin.     

Conversion of a heme-based oxygen sensor to a heme oxygenase by hydrogen sulfide: effects of mutations in the heme distal side of a heme-based oxygen sensor phosphodiesterase (Ec DOS)

The heme-based oxygen-sensor phosphodiesterase from Escherichia coli (Ec DOS), is composed of an N-terminal heme-bound oxygen sensing domain and a C-terminal catalytic domain. Oxygen (O₂) binding to the heme Fe(II) complex in Ec DOS substantially enhances catalysis. Addition of hydrogen sulfide (H₂S) to the heme Fe(III) complex in Ec DOS also remarkably stimulates catalysis in part due to the heme Fe(III)–SH and heme Fe(II)–O₂ complexes formed by H₂S. In this study, we examined the roles of the heme distal amino acids, M95 (the axial ligand of the heme Fe(II) complex) and R97 (the O₂ binding site in the heme Fe(II)–O₂ complex) of the isolated heme-binding domain of Ec DOS (Ec DOS-PAS) in the binding of H₂S under aerobic conditions. Interestingly, R97A and R97I mutant proteins formed an oxygen-incorporated modified heme, verdoheme, following addition of H₂S combined with H₂O₂ generated by the reactions. Time-dependent mass spectroscopic data corroborated the findings. In contrast, H₂S did not interact with the heme Fe(III) complex of M95H and R97E mutants. Thus, M95 and/or R97 on the heme distal side in Ec DOS-PAS significantly contribute to the interaction of H₂S with the Fe(III) heme complex and also to the modification of the heme Fe(III) complex with reactive oxygen species. Importantly, mutations of the O₂ binding site of the heme protein converted its function from oxygen sensor to that of a heme oxygenase. This study establishes the novel role of H₂S in modifying the heme iron complex to form verdoheme with the aid of reactive oxygen species.     

Novel iron complexes and chelators based on cis,cis-1,3,5-triaminocyclohexane: iron-mediated ligand oxidation and biochemical properties

 The interaction of Fe(II) and Fe(III) with the novel Fe(II) chelator N,N′N″-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane (referred to as tachpyr) gives rise to six-coordinate, low-spin, cationic complexes of Fe(II). Tachpyr also displays a cytotoxicity toward cultured bladder cancer cells that is believed to involve coordination of intracellular iron. The anaerobic reaction of tachpyr with Fe(II) salts affords the Fe(II)-tachpyr²⁺ complex, but in presence of oxygen, oxidative dehydrogenation of one or two of the aminomethylene group(s) of the ligand occurs, with formal loss of H₂: R—N(H)—C(H)₂—(2-py) → R—N=C(H)—(2-py)+H₂. The resulting mono- and diimino Fe(II) complexes (denoted as [Fe(tachpyr-H₂)]²⁺ and [Fe(tachpyr-2H₂)]²⁺) are an inseparable mixture, but they may be fully oxidized by H₂O₂ to the known tris(imino) complex Fe(II)[cis,cis-1,3,5-tris(pyridine-2-carboxaldimino)cyclohexane]²⁺ (or [Fe(tachpyr-3H₂)]²⁺). Cyclic voltammetry of the imino complex mixture reveals an irreversible anodic wave at +0.78 V vs. NHE. Tachpyr acts as a reducing agent toward Fe(IIII) salts, affording the same two Fe(II) imino complexes as products. Tachpyr also reductively removes Fe(III) from an Fe(III)(ATP)₃ complex (which is a putative form of intracellular iron), producing the two Fe(II) imino complexes. Novel N-alkylated derivatives of tachpyr have been synthesized. N-Alkylation has two effects on tachpyr: lowering metal affinity through increased steric hindrance, and preventing Fe(III) reduction because oxidative dehydrogenation of nitrogen is blocked. The N-methyl tachpyr derivative binds Fe(II) only weakly as a high-spin complex, and no complexation or reduction of Fe(III) is observed. Corresponding to their inability to bind iron, the N-alkylated chelators are nontoxic to cultured bladder cancer cells. A tach-based chelator with three N-propyleneamino arms is also synthesized. Studies of the chemical and biochemical properties of this chelator further support a relationship between intracellular iron chelation, iron reduction, and cytotoxicity. Received: 23 March 1998 / Accepted: 1 June 1998     

First success of catalytic epoxidation of olefins by an electron-rich iron(III) porphyrin complex and H2O2: imidazole effect on the activation of H2O2 by iron porphyrin complexes in aprotic solvent

An electron-rich iron(III) porphyrin complex (meso-tetramesitylporphinato)iron(III) chloride [Fe(TMP)Cl], was found to catalyze the epoxidation of olefins by aqueous 30% H₂O₂ when the reaction was carried out in the presence of 5-chloro-1-methylimidazole (5-Cl-1-MeIm) in aprotic solvent. Epoxides were the predominant products with trace amounts of allylic oxidation products, indicating that Fenton-type oxidation reactions were not involved in the olefin epoxidation reactions. cis-Stilbene was stereospecifically oxidized to cis-stilbene oxide without giving isomerized trans-stilbene oxide product, demonstrating that neither hydroperoxy radical (HOO·) nor oxoiron(IV) porphyrin [(TMP)Fe^IV=O] was responsible for the olefin epoxidations. We also found that the reactivities of other iron(III) porphyrin complexes such as (meso-tetrakis(2,6-dichlorophenyl)porphinato)iron(III) chloride [Fe(TDCPP)Cl], (meso-tetrakis(2,6-difluorophenyl)porphinato)iron(III) chloride [Fe(TDFPP)Cl], and (meso-tetrakis(pentafluorophenyl)porphinato)iron(III) chloride [Fe(TPFPP)Cl] were significantly affected by the presence of the imidazole in the epoxidation of olefins by H₂O₂. These iron porphyrin complexes did not yield cyclohexene oxide in the epoxidation of cyclohexene by H₂O₂ in the absence of 5-Cl-1-MeIm in aprotic solvent; however, addition of 5-Cl-1-MeIm to the reaction solutions gave high yields of cyclohexene oxide with the formation of trace amounts of allylic oxidation products. We proposed, on the basis of the results of mechanistic studies, that the role of the imidazole is to decelerate the O–O bond cleavage of an iron(III) hydroperoxide porphyrin (or H₂O₂–iron(III) porphyrin adduct) and that the intermediate transfers its oxygen to olefins prior to the O–O bond cleavage.     

Nitric oxide precipitates catastrophic chromosome fragmentation by bolstering both hydrogen peroxide and Fe(II) Fenton reactants in E. coli

Immune cells kill invading microbes by producing reactive oxygen and nitrogen species, primarily hydrogen peroxide (H₂O₂) and nitric oxide (NO). We previously found that NO inhibits catalases in Escherichia coli, stabilizing H₂O₂ around treated cells and promoting catastrophic chromosome fragmentation via continuous Fenton reactions generating hydroxyl radicals. Indeed, H₂O₂-alone treatment kills catalase-deficient (katEG) mutants similar to H₂O₂+NO treatment. However, the Fenton reaction, in addition to H₂O₂, requires Fe(II), which H₂O₂ excess instantly converts into Fenton-inert Fe(III). For continuous Fenton when H₂O₂ is stable, a supply of reduced iron becomes necessary. We show here that this supply is ensured by Fe(II) recruitment from ferritins and Fe(III) reduction by flavin reductase. Our observations also concur with NO-mediated respiration inhibition that drives Fe(III) reduction. We modeled this NO-mediated inhibition via inactivation of ndh and nuo respiratory enzymes responsible for the step of NADH oxidation, which results in increased NADH pools driving flavin reduction. We found that, like the katEG mutant, the ndh nuo double mutant is similarly sensitive to H₂O₂-alone and H₂O₂+NO treatments. Moreover, the quadruple katEG ndh nuo mutant lacking both catalases and efficient respiration was rapidly killed by H₂O₂-alone, but this killing was delayed by NO, rather than potentiated by it. Taken together, we conclude that NO boosts the levels of both H₂O₂ and Fe(II) Fenton reactants, making continuous hydroxyl-radical production feasible and resulting in irreparable oxidative damage to the chromosome.     

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.     

Binding of α-synuclein with Fe(III) and with Fe(II) and biological implications of the resultant complexes

Parkinson’s disease (PD) is hallmarked by the abnormal intracellular inclusions (Lewy bodies or LBs) in dopaminergic cells. Amyloidogenic protein α-synuclein (α-syn) and iron (including both Fe(III) and Fe(II)) are both found to be present in LBs. The interaction between iron and α-syn might have important biological relevance to PD etiology. Previously, a moderate binding affinity between α-syn and Fe(II) (5.8 × 10³ M⁻¹) has been measured, but studies on the binding between α-syn and Fe(III) have not been reported. In this work, electrospray mass spectrometry (ES-MS), cyclic voltammetry (CV), and fluorescence spectroscopy were used to study the binding between α-syn and Fe(II) and the redox property of the resultant α-syn-Fe(II) complex. The complex is of a 1:1 stoichiometry and can be readily oxidized electrochemically and chemically (by O₂) to the putative α-syn-Fe(III) complex, with H₂O₂ as a co-product. The reduction potential was estimated to be 0.025 V vs. Ag/AgCl, which represents a shift by −0.550 V vs. the standard reduction potential of the free Fe(III)/Fe(II) couple. Such a shift allows a binding constant between α-syn and Fe(III), 1.2 × 10¹³ M⁻¹, to be deduced. Despite the relatively high binding affinity, α-syn-Fe(III) generated from the oxidation of α-syn-Fe(II) still dissociates due to the stronger tendency of Fe(III) to hydrolyze to Fe(OH)₃ and/or ferrihydrite gel. The roles of α-syn and its interaction with Fe(III) and/or Fe(II) are discussed in the context of oxidative stress, metal-catalyzed α-syn aggregation, and iron transfer processes.     

Multiple electrophoretic variants of Cu,Zn superoxide dismutase as expression of the enzyme aging. Effects of H2O2, ascorbate and metal ions

Multiple electrophoretic bands, with R_F identical to the natural molecular variants, are produced by treatment of purified Cu, Zn Superoxide dismutase with either H₂O₂ or ascorbate plus Fe(III) EDTA. The ascorbate reaction is also due to H₂O₂ since it is inhibited by catalase. However while H₂O₂ inactivates the enzyme, the electromorphs produced by ascorbate-Fe(III) EDTA have only slightly less activity than the native enzyme and this property parallels the natural situation. It is concluded that oxidative aging can be responsible for the multiple molecular variants of the natural enzyme, under conditions where the oxidant attack is preferentially directed to amino acid side chains outside the active site. Such conditions may occur when a metal ion coordinated to the protein surface undergoes a redox cycle with biological reductants, like ascorbate.     

Mono- and dinuclear Fe(III) complexes with the N2O2 donor 5-chlorosalicylideneimine ligands; synthesis, X-ray structural characterization and magnetic properties

Two novel monomeric [C₁₈H₁₇Cl₃N₂O₂Fe] (1) and dimeric [C₃₈H₃₆N₄O₄Cl₆Fe₂] (2) Fe(III) tetradentate Schiff base complexes have been synthesized and their crystal structures have been determined by single crystal X-ray diffraction analysis. In complex (1) the Schiff base ligand coordinates toward one iron atom in a tetradentate mode and each iron atom is five coordinated with the coordination geometry around iron atom which can be described as a distorted square pyramid. The presence of a short (2.89 Å) non-bonding interatomic Fe···O distances between adjacent monomeric Fe(III) complexes results in the formation of a dimer. Structural analysis of compound (2) shows that the structure is a centrosymmetric dimer in which the six coordinated Fe(III) atoms are linked by μ-phenoxo bridges from one of the phenolic oxygen atoms of each Schiff base ligand to the opposite metal center. The variable-temperature (2-300 K) magnetic susceptibility (χ) data of these two compounds have been investigated. The results show that for both complexes Fe(III) centers are in the high spin configuration (S = 5/2) and indicate antiferromagnetic spin-exchange interaction between Fe(III) ions. The obtained results are briefly discussed using magnetostructural correlations developed for other class of iron(III) complexes.     

Electron transport chain dysfunction by H2O2 is linked to increased reactive oxygen species production and iron mobilization by lipoperoxidation: studies using Saccharomyces cerevisiae mitochondria

The mitochondrial electron transport chain (ETC) contains thiol groups (−SH) which are reversibly oxidized to modulate ETC function during H₂O₂ overproduction. Since deleterious effects of H₂O₂ are not limited to –SH oxidation, due to the formation of other H₂O₂-derived species, some processes like lipoperoxidation could enhance the effects of H₂O₂ over ETC enzymes, disrupt their modulation by –SH oxidation and increase superoxide production. To verify this hypothesis, we tested the effects of H₂O₂ on ETC activities, superoxide production and iron mobilization in mitochondria from lipoperoxidation-resistant native yeast and lipoperoxidation-sensitized yeast. Only complex III activity from lipoperoxidation-sensitive mitochondria exhibited a higher susceptibility to H₂O₂ and increased superoxide production. The recovery of ETC activity by the thiol reductanct β-mercaptoethanol (BME) was also altered at complex III, and a role was attributed to lipoperoxidation, the latter being also responsible for iron release. A hypothetical model linking lipoperoxidation, increased complex III damage, superoxide production and iron release is given.     

Oxidative stress alters global histone modification and DNA methylation

The JmjC domain-containing histone demethylases can remove histone lysine methylation and thereby regulate gene expression. The JmjC domain uses iron Fe(II) and α-ketoglutarate (αKG) as cofactors in an oxidative demethylation reaction via hydroxymethyl lysine. We hypothesize that reactive oxygen species will oxidize Fe(II) to Fe(III), thereby attenuating the activity of JmjC domain-containing histone demethylases. To minimize secondary responses from cells, extremely short periods of oxidative stress (3 h) were used to investigate this question. Cells that were exposed to hydrogen peroxide (H₂O₂) for 3 h exhibited increases in several histone methylation marks including H3K4me3 and decreases of histone acetylation marks including H3K9ac and H4K8ac; preincubation with ascorbate attenuated these changes. The oxidative stress level was measured by generation of 2′,7′-dichlorofluorescein, GSH/GSSG ratio, and protein carbonyl content. A cell-free system indicated that H₂O₂ inhibited histone demethylase activity where increased Fe(II) rescued this inhibition. TET protein showed a decreased activity under oxidative stress. Cells exposed to a low-dose and long-term (3 weeks) oxidative stress also showed increased global levels of H3K4me3 and H3K27me3. However, these global methylation changes did not persist after washout. The cells exposed to short-term oxidative stress also appeared to have higher activity of class I/II histone deacetylase (HDAC) but not class III HDAC. In conclusion, we have found that oxidative stress transiently alters the epigenetic program process through modulating the activity of enzymes responsible for demethylation and deacetylation of histones.     

Ability of Ferric Nitrilotriacetate Complex with Three pH-dependent Conformations to Induce Lipid Peroxidation

This study examined the generation of reactive oxygen species (ROS) and the induction of lipid peroxidation by carcinogenic iron(III)-NTA complex (1:1), which has three conformations with two pKa values (pKa₁≈4, pKa₂≈8). These conformations are type (a) in acidic conditions of pH 1-6, type (n) in neutral conditions of pH 3-9, and type (b) in basic conditions of pH 7-10. The iron(III)-NTA complex was reduced to iron(II) complex under cool-white fluorescent light without the presence of any reducer. The reduction rates of three species of iron(III)-NTA were in the order type (a)?type (n) ? type (b). Iron(III)-NTA-dependent lipid peroxidation was induced in the presence and absence of preformed lipid peroxides (L-OOH) through processes associated with and without photoreduction of iron(III). The order of the abilities of the three species of iron(III)-NTA to initiate the three mechanisms of lipid peroxidation was: (1) type (a) ? type (n) ? type (b) in lipid peroxidation that is induced L-OOH- and H₂O₂-dependently and mediated by the photoreduction of iron(III); (2) type (b) ? type (n) ? type (a) in lipid peroxidation that is induced L-OOH- and H₂O₂-dependently but not mediated by the photoreduction of iron(III); (3) type (n) ? type (b) ? type (a) in lipid peroxidation that is induced peroxide-independently and mediated by the photoactivation but not by the photoreduction of iron(III). The rate of lipid peroxidation induced L-OOH-dependently is faster than that induced H₂O₂-dependently in the mechanism (1), but the rate of lipid peroxidation induced H₂O₂-dependently is faster than that induced L-OOH-dependently in the mechanism (2). In the lag process of mechanism (3), L-OOH and/or some free radical species, not ¹O₂, were generated by photoactivation of iron(III)-NTA. These multiple pro-oxidant properties that depend on the species of iron(III)-NTA were postulated to be a principal cause of its carcinogenicity.     

Copper catechol-driven Fenton reactions and their potential role in wood degradation

Metals can potentially play a role in the non-enzymatic processes involved in wood biodegradation. Dihydroxybenzenes reduce Cu(II)–Cu(I), which then react with H₂O₂ driving a Fenton reaction. In this work the degradation of veratryl alcohol (VA), the simplest non-phenolic lignin model compound, via a cuprous Fenton reaction mediated by 1,2-dihydroxybenzene (catechol, CAT) was studied. A factorial experimental design was performed to assess the impact of several experimental variables including, pH, and CAT, CuCl₂ and H₂O₂ concentrations on VA degradation. Optimized conditions were determined using a response surface modeling methodology (RSM). The greatest amount of VA degradation occurred at a CAT:CuCl₂:H₂O₂ ratio of 0.287:0.313:4.062, a pH of 3.6. A time-course measurement for VA degradation was performed under these experimental conditions and after an 8 h reaction period, 31% of the VA was degraded. Under the same experimental conditions, VA degradation by an iron CAT-driven Fenton reaction was more effective than the copper CAT-driven Fenton reaction. In a similar experiment, carboxymethyl cellulose (CMC) depolymerization was also determined. Only the iron CAT-driven Fenton reaction was found to depolymerize CMC. We suggest that the greater redox potential of the Fe(III)CAT complex compared to the Cu(II)CAT complex would dictate that under most environmental conditions, degradation of VA would occur by the iron complex only. This research has important implications for the mechanisms of brown rot fungal degradation in wood because it eliminates a pathway that had previously been proposed as a mechanism explaining free radical generation in the oxidative depolymerization of cellulose in the cell wall.     

Iron oxidation stimulates organic matter decomposition in humid tropical forest soils

Humid tropical forests have the fastest rates of organic matter decomposition globally, which often coincide with fluctuating oxygen (O₂) availability in surface soils. Microbial iron (Fe) reduction generates reduced iron [Fe(II)] under anaerobic conditions, which oxidizes to Fe(III) under subsequent aerobic conditions. We demonstrate that Fe (II) oxidation stimulates organic matter decomposition via two mechanisms: (i) organic matter oxidation, likely driven by reactive oxygen species; and (ii) increased dissolved organic carbon (DOC) availability, likely driven by acidification. Phenol oxidative activity increased linearly with Fe(II) concentrations (P < 0.0001, pseudo R² = 0.79) in soils sampled within and among five tropical forest sites. A similar pattern occurred in the absence of soil, suggesting an abiotic driver of this reaction. No phenol oxidative activity occurred in soils under anaerobic conditions, implying the importance of oxidants such as O₂ or hydrogen peroxide (H₂O₂) in addition to Fe(II). Reactions between Fe(II) and H₂O₂ generate hydroxyl radical, a strong nonselective oxidant of organic compounds. We found increasing consumption of H₂O₂ as soil Fe(II) concentrations increased, suggesting that reactive oxygen species produced by Fe(II) oxidation explained variation in phenol oxidative activity among samples. Amending soils with Fe(II) at field concentrations stimulated short‐term C mineralization by up to 270%, likely via a second mechanism. Oxidation of Fe(II) drove a decrease in pH and a monotonic increase in DOC; a decline of two pH units doubled DOC, likely stimulating microbial respiration. We obtained similar results by manipulating soil acidity independently of Fe(II), implying that Fe(II) oxidation affected C substrate availability via pH fluctuations, in addition to producing reactive oxygen species. Iron oxidation coupled to organic matter decomposition contributes to rapid rates of C cycling across humid tropical forests in spite of periodic O₂ limitation, and may help explain the rapid turnover of complex C molecules in these soils.     

Mechanisms underlying the cytotoxic effects of Tachpyr—a novel metal chelator

Tachpyr (N,N′N″-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane), a novel metal chelator, was previously shown to deplete intracellular iron and exert a cytotoxic effect on cultured bladder cancer cells. Tachpyr binds Fe(II) and readily reduces Fe(III). The iron(II)–Tachpyr chelate undergoes intramolecular oxidative dehydrogenation resulting in mono- and diimino Fe(II) complexes. The present study investigates the redox-activity of the Tachpyr–iron complex to better define the mechanism of Tachpyr's cytotoxicity. Tachpyr's mechanism of cytotoxicity was studied using cell-free solutions, isolated DNA, and cultured mammalian cells by employing UV–VIS spectrophotometry, oximetry, spin-trapping technique, and electron paramagnetic resonance (EPR) spectrometry. The results show that: (1) Tachpyr by itself after 24 h of incubation had a cytotoxic effect on cultured cells; (2) fully oxidized Tachpyr had no cytotoxic effects on cultured cells even after 24 h of incubation; (3) Tachpyr protected isolated DNA against H₂O₂-induced damage, but not against HX/XO-induced damage; and (4) Tachpyr–Fe(II) chelate slows down but does not block oxidation of Fe(II), allows O₂⁻-induced or Tachpyr-induced reduction of Fe(III), and consequently promotes production of OH through the Haber–Weiss reaction cycle. The results indicate that Tachpyr can protect cells against short-term, metal-mediated damage. However, upon prolonged incubation, Tachpyr exerts cytotoxic effects. Therefore, in addition to iron depletion, low-level oxidative stress, which in part occurs because of redox cycling of the coordinated iron ion, may contribute to the cytotoxic effects of Tachpyr.     

Novel trigonal prismatic iron complexes of expanded hexadentate Jäger type ligands: synthesis and X-ray analysis

The mononuclear iron complexes Fe2a and Fe2b of expanded hexadentate Jäger type ligands H₃2a and H₃2b with [N₄O₂]-donorset were synthesized via reaction of the free ligands with iron(II) acetate. The identity of both compounds was proven by mass spectrometry and micro analysis. Furthermore crystals suitable for X-ray structure determination were obtained in case of Fe2a. The X-ray investigation reveals that this compound is a hexacoordinated iron complex with a rather rare trigonal prismatic geometry of the coordination polyeder: in this complex all six coordination sites are occupied by the donor atoms of the ligand exclusively, which was observed for the first time in iron complexes of Jäger type ligands. Electronic properties of Fe2b were characterized using EPR-spectroscopy and temperature dependent susceptibility measurements, indicating a high-spin Fe(III) complex.     

Inhibition of phagocytic activity of ARPE-19 cells by free radical mediated oxidative stress

Oxidative stress is a main factor responsible for key changes leading to the onset of age-related macular degeneration (ARMD) that occur in the retinal pigment epithelium (RPE), which is involved in phagocytosis of photoreceptor outer segments (POS). In this study, hydrogen peroxide (H₂O₂), H₂O₂ and iron ions (Fe) or rose Bengal (RB) in the presence of NADH and Fe were used to model free radical mediated oxidative stress to test if free radicals and singlet oxygen have different efficiency to inhibit phagocytosis of ARPE-19 cells. Free radical mediated oxidative stress was confirmed by HPLC-EC(Hg) measurements of cholesterol hydroperoxides in treated cells. Electron paramagnetic resonance (EPR) spin trapping was employed to detect superoxide anion. Cell survival was analyzed by the MTT assay. Specific phagocytosis of fluorescein-5-isothiocyanate-labeled POS and non-specific phagocytosis of fluorescent beads were measured by flow cytometry. HPLC analysis of cells photosensitized with RB in the presence of NADH and Fe indicated substantial increase in formation of free radical-dependent 7α/7β-hydroperoxides. EPR spin trapping confirmed the photogeneration of superoxide anion in samples enriched with RB, NADH and Fe. For all three protocols sub-lethal oxidative stress induced significant inhibition of the specific phagocytosis of POS. In contrast, non-specific phagocytosis was inhibited only by H₂O₂ or H₂O₂ and Fe treatment. Inhibition of phagocytosis was transient and recoverable by 24?h. These results suggest that free radicals may exert similar to singlet oxygen efficiency in inhibiting phagocytosis of RPE cells, and that the effect depends on the location where initial reactive species are formed.     

Effects of hydrogen sulfide on the heme coordination structure and catalytic activity of the globin-coupled oxygen sensor <Emphasis Type="Italic">Af</Emphasis>GcHK

AfGcHK is a globin-coupled histidine kinase that is one component of a two-component signal transduction system. The catalytic activity of this heme-based oxygen sensor is due to its C-terminal kinase domain and is strongly stimulated by the binding of O₂ or CO to the heme Fe(II) complex in the N-terminal oxygen sensing domain. Hydrogen sulfide (H₂S) is an important gaseous signaling molecule and can serve as a heme axial ligand, but its interactions with heme-based oxygen sensors have not been studied as extensively as those of O₂, CO, and NO. To address this knowledge gap, we investigated the effects of H₂S binding on the heme coordination structure and catalytic activity of wild-type AfGcHK and mutants in which residues at the putative O₂-binding site (Tyr45) or the heme distal side (Leu68) were substituted. Adding Na₂S to the initial OH-bound 6-coordinate Fe(III) low-spin complexes transformed them into SH-bound 6-coordinate Fe(III) low-spin complexes. The Leu68 mutants also formed a small proportion of verdoheme under these conditions. Conversely, when the heme-based oxygen sensor EcDOS was treated with Na₂S, the initially formed Fe(III)–SH heme complex was quickly converted into Fe(II) and Fe(II)–O₂ complexes. Interestingly, the autophosphorylation activity of the heme Fe(III)–SH complex was not significantly different from the maximal enzyme activity of AfGcHK (containing the heme Fe(III)–OH complex), whereas in the case of EcDOS the changes in coordination caused by Na₂S treatment led to remarkable increases in catalytic activity.     

Examination of Antioxidative System’s Responses in the Different Phases of Drought Stress and During Recovery in Desert Plant <Emphasis Type="Italic">Reaumuria soongorica</Emphasis> (Pall.) Maxim

The aim of this study was to test the protective roles of superoxide dismutases (SODs), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) against oxidative damage and their activities in different phases of the dry down process in Reaumuria soongorica (Pall.) Maxim. leaves. Drought stress was imposed during 100 consecutive days and rewatering after 16, 72, and 100 days. The concentration of hydrogen peroxide (H₂O₂), malondialdehyde, and SODs activities were elevated significantly with progressing drought stress. POD and CAT activities increased markedly in the early phase of drought and decreased significantly with further drought stress continuation, and POD activity was unable to recover after rewatering. Ascorbate, reduced glutathione, APX, and GR activities declined in the initial stages of drought process, elevated significantly with further increasing water deficit progression and recovered after rewatering. These results indicate that: (1) iron SODs-removing superoxide anion is very effective during the whole drought stress; (2) CAT scavenges H₂O₂ in the early phase of drought and enzymes of ascorbate–glutathione cycle scavenge H₂O₂ in further increasing drought stress; and (3) POD does not contribute to protect against oxidative damage caused by H₂O₂ under drought stress.     

Atmospheric hydrogen peroxide and Eoarchean iron formations

It is widely accepted that photosynthetic bacteria played a crucial role in Fe(II) oxidation and the precipitation of iron formations (IF) during the Late Archean–Early Paleoproterozoic (2.7–2.4 Ga). It is less clear whether microbes similarly caused the deposition of the oldest IF at ca. 3.8 Ga, which would imply photosynthesis having already evolved by that time. Abiological alternatives, such as the direct oxidation of dissolved Fe(II) by ultraviolet radiation may have occurred, but its importance has been discounted in environments where the injection of high concentrations of dissolved iron directly into the photic zone led to chemical precipitation reactions that overwhelmed photooxidation rates. However, an outstanding possibility remains with respect to photochemical reactions occurring in the atmosphere that might generate hydrogen peroxide (H₂O₂), a recognized strong oxidant for ferrous iron. Here, we modeled the amount of H₂O₂ that could be produced in an Eoarchean atmosphere using updated solar fluxes and plausible CO₂, O₂, and CH₄ mixing ratios. Irrespective of the atmospheric simulations, the upper limit of H₂O₂ rainout was calculated to be <10⁶ molecules cm^?2 s^?1. Using conservative Fe(III) sedimentation rates predicted for submarine hydrothermal settings in the Eoarchean, we demonstrate that the flux of H₂O₂ was insufficient by several orders of magnitude to account for IF deposition (requiring ~10¹¹ H₂O₂ molecules cm^?2 s^?1). This finding further constrains the plausible Fe(II) oxidation mechanisms in Eoarchean seawater, leaving, in our opinion, anoxygenic phototrophic Fe(II)‐oxidizing micro‐organisms the most likely mechanism responsible for Earth's oldest IF.