Functional and evolutionary characterization of Ohr proteins in eukaryotes reveals many active homologs among pathogenic fungi.

Ohr and OsmC proteins comprise two subfamilies within a large group of proteins that display Cys-based, thiol dependent peroxidase activity. These proteins were previously thought to be restricted to prokaryotes, but we show here, using iterated sequence searches, that Ohr/OsmC homologs are also present in 217 species of eukaryotes with a massive presence in Fungi (186 species). Many of these eukaryotic Ohr proteins possess an N-terminal extension that is predicted to target them to mitochondria. We obtained recombinant proteins for four eukaryotic members of the Ohr/OsmC family and three of them displayed lipoyl peroxidase activity. Further functional and biochemical characterization of the Ohr homologs from the ascomycete fungus Mycosphaerella fijiensis Mf_1 (MfOhr), the causative agent of Black Sigatoka disease in banana plants, was pursued. Similarly to what has been observed for the bacterial proteins, we found that: (i) the peroxidase activity of MfOhr was supported by DTT or dihydrolipoamide (dithiols), but not by ß-mercaptoethanol or GSH (monothiols), even in large excess; (ii) MfOhr displayed preference for organic hydroperoxides (CuOOH and tBOOH) over hydrogen peroxide; (iii) MfOhr presented extraordinary reactivity towards linoleic acid hydroperoxides (k=3.18 (±2.13)×108M-1s-1). Both Cys87 and Cys154 were essential to the peroxidase activity, since single mutants for each Cys residue presented no activity and no formation of intramolecular disulfide bond upon treatment with hydroperoxides. The pKa value of the Cysp residue was determined as 5.7±0.1 by a monobromobimane alkylation method. Therefore, eukaryotic Ohr peroxidases share several biochemical features with prokaryotic orthologues and are preferentially located in mitochondria.

Expression of a thioredoxin peroxidase in insulin-producing cells.

The presence of thioredoxin peroxidase (TPx), also known as thiol specific antioxidant (TSA), was investigated in neonatal and adult rat islets, and in the beta-cell line HIT-T15. Western blotting of extracts from neonatal and adult pancreatic islets and from the tumoral cell line HIT-T15 revealed the presence of a 25 kDa protein that comigrated with purified yeast TPx. Endocrine pancreatic TPx accounted for approximately 0.01% of the total protein content. Treatment with H2O2 for 3 h increased the expression of TPx in HIT-T15 cells. The distribution of TPx throughout the islet cells was confirmed by immunocytochemistry. Since pancreatic beta-cells possess a weak antioxidant enzyme defense system, especially with regard to hydrogen peroxidase-decomposing enzymes, the presence of a TPx analog in islets suggests that this enzyme may play a role in protecting pancreatic cells against reactive oxygen species.

Cytosolic thioredoxin peroxidase I is essential for the antioxidant defense of yeast with dysfunctional mitochondria.

The specific role of cytosolic thioredoxin peroxidase I (cTPx I), encoded by TSA1 (thiol-specific antioxidant), was investigated in the oxidative stress response of Saccharomyces cerevisiae. In most cases, deletion of TSA1 has showed only a slight effect on hydrogen peroxide sensitivity. However, when the functional state of the mitochondria was compromised, the necessity of TSA1 in cell protection against this oxidant was much more evident. All the procedures used to disrupt the mitochondrial respiratory chain promoted increases in the generation of H(2)O(2) in cells, which could be related to their elevated sensitivity to oxidative stress. In fact, TSA1 is highly expressed when cells with respiratory deficiency are exposed to H(2)O(2). In conclusion, our results indicate that cTPx I is a key component of the antioxidant defense in respiratory-deficient cells.

Selectively labeling the heterologous protein in Escherichia coli for NMR studies: a strategy to speed up NMR spectroscopy.

Nuclear magnetic resonance is an important tool for high-resolution structural studies of proteins. It demands high protein concentration and high purity; however, the expression of proteins at high levels often leads to protein aggregation and the protein purification step can correspond to a high percentage of the overall time in the structural determination process. In the present article we show that the step of sample optimization can be simplified by selective labeling the heterologous protein expressed in Escherichia coli by the use of rifampicin. Yeast thioredoxin and a coix transcription factor Opaque 2 leucine zipper (LZ) were used to show the effectiveness of the protocol. The (1)H/(15)N heteronuclear correlation two-dimensional NMR spectrum (HMQC) of the selective (15)N-labeled thioredoxin without any purification is remarkably similar to the spectrum of the purified protein. The method has high yields and a good (1)H/(15)N HMQC spectrum can be obtained with 50 ml of M9 growth medium. Opaque 2 LZ, a difficult protein due to the lower expression level and high hydrophobicity, was also probed. The (15)N-edited spectrum of Opaque 2 LZ showed only the resonances of the protein of heterologous expression (Opaque 2 LZ) while the (1)H spectrum shows several other resonances from other proteins of the cell lysate. The demand for a fast methodology for structural determination is increasing with the advent of genome/proteome projects. Selective labeling the heterologous protein can speed up NMR structural studies as well as NMR-based drug screening. This methodology is especially effective for difficult proteins such as hydrophobic transcription factors, membrane proteins, and others.

Catalases and thioredoxin peroxidase protect Saccharomyces cerevisiae against Ca(2+)-induced mitochondrial membrane permeabilization and cell death.

The involvement of reactive oxygen species in Ca(2+)-induced mitochondrial membrane permeabilization and cell viability was studied using yeast cells in which the thioredoxin peroxidase (TPx) gene was disrupted and/or catalase was inhibited by 3-amino-1,2, 4-triazole (ATZ) treatment. Wild-type Saccharomyces cerevisiae cells were very resistant to Ca(2+) and inorganic phosphate or t-butyl hydroperoxide-induced mitochondrial membrane permeabilization, but suffered an immediate decrease in mitochondrial membrane potential when treated with Ca(2+) and the dithiol binding reagent phenylarsine oxide. In contrast, S. cerevisiae spheroblasts lacking the TPx gene and/or treated with ATZ suffered a decrease in mitochondrial membrane potential, generated higher amounts of hydrogen peroxide and had decreased viability under these conditions. In all cases, the decrease in mitochondrial membrane potential could be inhibited by ethylene glycol-bis(beta-aminoethyl ether) N,N, N',N'-tetraacetic acid, dithiothreitol or ADP, but not by cyclosporin A. We conclude that TPx and catalase act together, maintaining cell viability and protecting S. cerevisiae mitochondria against Ca(2+)-promoted membrane permeabilization, which presents similar characteristics to mammalian permeability transition.

The thiol-specific antioxidant enzyme prevents mitochondrial permeability transition. Evidence for the participation of reactive oxygen species in this mechanism.

Mitochondrial swelling and membrane protein thiol oxidation associated with mitochondrial permeability transition induced by Ca2+ and inorganic phosphate are inhibited in a dose-dependent manner either by catalase, the thiol-specific antioxidant enzyme (TSA), a protein recently demonstrated to present thiol peroxidase activity, or ebselen, a selenium-containing heterocycle which also possesses thiol peroxidase activity. This inhibition of mitochondrial permeability transition is due to the removal of mitochondrial-generated H2O2 which can easily diffuse to the extramitochondrial space. Whereas ebselen required the presence of reduced glutathione as a reductant to grant its protective effect, TSA was fully reduced by mitochondrial components. Decrease in the oxygen concentration of the reaction medium also inhibits mitochondrial permeabilization and membrane protein thiol oxidation, in a concentration-dependent manner. The results presented in this report confirm that mitochondrial permeability transition induced by Ca2+ and inorganic phosphate is reactive oxygen species-dependent. The possible importance of TSA as an intracellular antioxidant, avoiding the onset of mitochondrial permeability transition, is discussed in the text.

The iron-catalyzed oxidation of dithiothreitol is a biphasic process: hydrogen peroxide is involved in the initiation of a free radical chain of reactions.

Dithiothreitol (DTT) is the most common agent used to reduce disulfide bonds in proteins. In the presence of transition metals and O2, however, DTT can induce oxidative damage in biomolecules. By means of polarographic measurements, it was established that the DTT oxidation catalyzed by Fe3+ is a biphasic process, characterized by a lag phase of several minutes during which O2 is consumed at a slow rate, by a mechanism involving the DTT-dependent reduction of Fe3+ to Fe2+ and the conversion of O2 to H2O2. Some lines of evidence indicate that the reduction of Fe3+ is the rate-limiting step: (i) The replacement of Fe3+ with Fe2+ leads to a decrease in the length of the lag phase. (ii) The rate of Fe2+ formation by DTT is the same as the initial rate of O2 uptake, (iii) The rate of sulfhydryl oxidation under anaerobic conditions is very slow. EDTA stimulates the iron-catalyzed oxidation of DTT probably by accelerating Fe3+ reduction. The lag phase is followed by a rapid uptake of O2 involving both O2.(-)-dependent and O2.(-)-independent free radical reactions, which can proceed, albeit more slowly, in the absence of H2O2 (i.e., in the presence of catalase). Glutamine synthetase is inactivated faster when added during the phase of rapid O2 uptake than when added before the beginning of the reaction. In both cases, catalase protects the enzyme, suggesting that only reactive species generated by H2O2 decomposition are able to induce the inactivation.

Identification of C8-methylguanine in the hydrolysates of DNA from rats administered 1,2-dimethylhydrazine. Evidence for in vivo DNA alkylation by methyl radicals.

C8-Methylguanine was identified in the neutral hydrolysates of DNA isolated from the liver or colon tissue of rats administered 1,2-dimethylhydrazine. In all the samples examined, the biologically isolated adducts were characterized by co-elution with synthetic C8-methylguanine under different high pressure liquid chromatography conditions. The sample isolated from liver DNA was also identified by UV spectroscopy at different pH values and by mass spectrometry. The estimated yields of C8-methylguanine obtained in hydrolysates of DNA from the liver or colon tissue were comparable to those of O6-methylguanine. C8-Methylguanine was not detected when the spin trap alpha-(4-pyridyl-1-oxide)-N-tert- butylnitrone was administered together with 1,2-dimethylhydrazine. The spin trap also inhibited N7-methylguanine and O6-methylguanine yields, although to a lesser extent. These results constitute the first evidence that DNA alkylation by carbon-centered radicals can occur in vivo.

DNA alkylation by carbon-centered radicals.

1. Over the last two decades, the prevalent view in chemical carcinogenesis has been that most free radicals do not bind to DNA. Recent studies, however, are demonstrating formation of adducts between DNA and free radicals such as hydroxyl radicals and aromatic cation radicals. 2. Within this context, we discuss the recent work from our group demonstrating DNA alkylation by carbon-centered radicals formed during biotransformation of genotoxic hydrazine derivatives both in vitro and in vivo. 3. The mutagenic potential of the identified methyl radical adduct, C8-methylguanine, is discussed, and other possible biological sources of carbon-centered radicals are presented.

DNA alkylation by carbon-centered radicals

1. Over the last two decades, the prevalent view in chemical carcinogenesis has been that most free radicals do not bind to DNA. Recent studies, however, are demonstrating formation of adducts between DNA and free radicals such as hydroxyl radicals and aromatic cation radicals. 2. Within this context, we discuss the recent work from our group demonstrating DNA alkylation by carbon-centered radicals formed during biotransformation of genotoxic hydrazine derivatives both in vitro and in vivo. 3. The mutagenic potential of the identified methyl radical adduct, C8-methylguanine, is discussed, and other possible biological sources of carbon-centered radicals are presented

Iron(III) binding in DNA solutions: complex formation and catalytic activity in the oxidation of hydrazine derivatives.

A strong interaction between iron(III) and calf thymus DNA at pH 7.4 was demonstrated in the present study by separation of the complex by column chromatography and by the slow kinetics of iron(III) removal from DNA by disodium-1,2-dihydroxybenzene-3,5-disulfonate (Tiron). An equilibrium constant of 2.1 x 10(14) was calculated by measurements of bound iron(III) by flame atomic absorption spectroscopy and assuming a one iron to two nucleotide stoichiometry. Graphic analysis of the interaction however, indicated that DNA has two binding sites for iron(III) characterized by a stoichiometry of one iron to 12 nucleotides and one iron to 2 nucleotides, and association constants of 4.8 x 10(12) and 2.3 x 10(11), respectively. The DNA-iron(III) complex isolated by column chromatography was shown to catalyze the oxidation of both 2-phenylethylhydrazine and methylhydrazine by spin-trapping experiments with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN). By contrast, oxidation of 1,2-dimethylhydrazine was not catalyzed. Catalysis of 2-phenylethylhydrazine oxidation was confirmed by oxygen consumption studies. The results suggest that iron chelated to DNA may be significant in DNA damage induced by oxidizable chemicals.

Hemoglobin-mediated oxidation of the carcinogen 1,2-dimethylhydrazine to methyl radicals.

Oxidation of 1,2-dimethylhydrazine (SDMH) catalyzed by hemoglobin is investigated by oxygen consumption studies, ESR spin-trapping experiments, and gas chromatography. Kinetic analysis and the study of the effects of superoxide dismutase, catalase, and azide on reaction rates indicate that SDMH oxidation is primarily dependent on ferric hemoglobin and autoxidatively formed H2O2. SDMH oxidation generates both methyl and hydroxyl radicals as ascertained by spin-trapping experiments with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone, 5,5-dimethyl-1-pyrroline-N-oxide, and tert-nitrosobutane. Quantitative estimates indicate that the yield of the methyl radical trapped by alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone is about 8% of the consumed oxygen. Analysis of the gaseous products by gas chromatography shows methane formation at a yield 10 times lower than that obtained for the spin-trap methyl adduct. These results are discussed within the context of the spin-trapping technique. The relative efficiencies of oxyhemoglobin in catalyzing SDMH, 2-phenylethylhydrazine, and phenylhydrazine oxidation, defined as Vmax/KM, are estimated as 1, 13, and 386, respectively. The higher efficiency obtained for the monosubstituted derivatives leads us to suggest that hemoglobin-catalyzed oxidation could be a detoxification route for these compounds. By contrast, SDMH oxidation requires a peroxidase-like activity, a fact that may be related to the efficacy and specificity of this carcinogen.

Enzymatic activation of the carcinogens 2-phenylethylhydrazine and 1,2-dimethylhydrazine to carbon-centered radicals.

Spin-trapping experiments demonstrate that oxidation of 1,2-dimethylhydrazine and 2-phenylethylhydrazine generates a comparable yield of carbon-centered radicals when catalyzed by horseradish peroxidase - H2O2. Using oxyhemoglobin as the catalyst, 2-phenylethylhydrazine oxidation generates ten times more carbon-centered radicals than 1,2-dimethylhydrazine oxidation. This result is in agreement with oxygen consumption studies from which the apparent KM values of 8.0 mM and 72 mM were calculated for the oxyhemoglobin-catalyzed oxidation of 2-phenylethylhydrazine and 1,2-dimethylhydrazine, respectively. These differences in metabolic activation of mono- and disubstituted hydrazines may be of importance regarding the carcinogenic properties of these derivatives.

Enzymatic activation of the carcinogens 2 phenylethylhydrazine and 1,2 dimethylhydrazine to carbon centered radicals

Spin-trapping experiments demonstrate that oxidation of 1,2-dimethylhydrazine and 2-phenylethylgydrazine generates a comparable yield of carbon-centered radicals when catalyzed by horseradish peroxidase-H2O2. Using oxyhemoglobin as the catalyst, 2-phenylethylgydrazine oxidation generates ten times carbon-centered radicals than 1,2-dimethylhidrazine oxidation. This results is in agreement with oxygen consumption studies from which the apparent KM values of 8.0 mM and 72 mM were calculated for the oxyhemoglobin-catalyzed oxidation of 2-phenylethylhydrazine and 1,2-dimethylhydrazine, respectively. These differences in metabolic activation of mono- and disubstituted hydrazines may be of importance regarding the carcinogenic properties of these derivatives