DNA damage by sulfite autoxidation catalyzed by cobalt complexes

DNA damage was investigated in the presence of sulfite, dissolved oxygen and cobalt(II) complexes with glycylglycylhistidine, glycylhistidyllysine, glycylglycyltyrosylarginine and tetraglycine. These studies indicated that only Co(II) complexed with glycylglycylhistidine (GGH) induced DNA strand breaks at low sulfite concentrations (1-80 mu M) via strong oxidants formed in the reaction. In the presence of the other complexes, some damage occurred only in the presence of high sulfite concentrations (0.1-2.0 mM) after incubation for 4 h. In the presence of GGH, Co(II) and dissolved O(2), DNA damage must involve a reactive high-valent cobalt complex. The damaging effect was increased by adding S(IV), due to the oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by the complex. SO(3)(center dot)-S-, HO(center dot) and H(center dot) radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline N-oxide). The results indicate that Co(II) binds O2 in the presence of GGH, and leads to the formation of a DMPO-HO(center dot) adduct without first forming free superoxide or hydroxyl radical, supporting the participation of a reactive high-valent cobalt complex.

Fermentation kinetics for xylitol production by a Pichia stipitis D-Xylulokinase mutant previously grown in spent sulfite liquor

Spent sulfite pulping liquor (SSL) contains lignin, which is present as lignosulfonate, and hemicelluloses that are present as hydrolyzed carbohydrates. To reduce the biological oxygen demand of SSL associated with dissolved sugars, we studied the capacity of Pichia stipitis FPL-YS30 (xyl3 Delta) to convert these sugars into useful products. FPL-YS30 produces a negligible amount of ethanol while converting xylose into xylitol. This work describes the xylose fermentation kinetics of yeast strain P.stipitis FPL-YS30. Yeast was grown in rich medium supplemented with different carbon sources: glucose, xylose, or ammonia-base SSL. The SSL and glucose-acclimatized cells showed similar maximum specific growth rates (0.146 h(-1)). The highest xylose consumption at the beginning of the fermentation process occurred using cells precultivated in xylose, which showed relatively high specific activity of glucose-6-phosphate dehydrogenase (EC 1.1.1.49). However, the maximum specific rates of xylose consumption (0.19 g(xylose)/g(cel) h) and xylitol production (0.059 g(xylitol)/g(cel) h) were obtained with cells acclimatized in glucose, in which the ratio between xylose reductase (EC 1.1.1.21) and xylitol dehydrogenase (EC 1.1.1.9) was kept at higher level (0.82). In this case, xylitol production (31.6 g/l) was 19 and 8% higher than in SSL and xylose-acclimatized cells, respectively. Maximum glycerol (6.26 g/l) and arabitol (0.206 g/l) production were obtained using SSL and xylose-acclimatized cells, respectively. The medium composition used for the yeast precultivation directly reflected their xylose fermentation performance. The SSL could be used as a carbon source for cell production. However, the inoculum condition to obtain a high cell concentration in SSL needs to be optimized.

Alkaline-sulfite chemithermomechanical pulping of Eucalyptus grandis biotreated by Ceriporiopsis subvermispora under varied culture conditions

The effect of different culture conditions have been evaluated concerning the extracellular enzyme activities of the white-rot fungus Ceriporiopsis subvermispora growing on Eucalyptus grandis wood. The consequence of the varied fungal pretreatment on a subsequent chemithermomechanical pulping (CTMP) was addressed. In all cultures, manganese peroxidase (MnP) and xylanase were the predominant extracellular enzymes. The biopulping efficiency was evaluated based on the amount of fiber bundles obtained after the first fiberizing step and the fibrillation levels of refined pulps. It was found that the MnP levels in the cultures correlated positively with the biopulping benefits. On the other hand, xylanase and total oxalate levels did not vary significantly. Accordingly, it was not possible to determine whether MnP accomplishes the effect alone or depends on synergic action of other extracellular agents. Pulp strength and fiber size distribution were also evaluated. The average fiber length of CTMP pulps prepared from untreated wood chips was 623 mu m. Analogous values were observed for most of the biopulps; however, significant amounts of shorter fibers were found in the biopulp prepared from wood chips biotreated in cultures supplemented with glucose plus corn-steep liquor. Despite evidence of reduced average fiber length, biopulps prepared from these wood chips presented the highest improvement in tensile indexes (+28% at 23 degrees Schopper-Riegler).

Sulfite : Cytochrome c oxidoreductase from Thiobacillus novellus - Purification, characterization, and molecular biology of a heterodimeric member of the sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotrophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidoreductase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellus is a periplasmically located alpha beta heterodimer, consisting of a 40.6-kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytochrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The organic component of the molybdenum cofactor was identified as molybdopterin contained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagnetic resonance spectroscopy revealed the presence of a sulfite-inducible Mo(V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-dependent changes in the electron paramagnetic resonance signal were not detected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanism involving two reactive sites. K-m values for sulfite and cytochrome c(550) were determined to be 27 and 4 mu M, respectively; the enzyme was found to be reversibly inhibited by sulfate and various buffer ions. The sorAB genes, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits significant similarities to known sequences of eukaryotic and bacterial sulfite:acceptor oxidoreductases, SorB does not appear to be closely related to any known c-type cytochromes.

Enzymology and molecular biology of prokaryotic sulfite oxidation

Despite its toxicity, sulfite plays a key role in oxidative sulfur metabolism and there are even some microorganisms which can use it as sole electron source. Sulfite is the main intermediate in the oxidation of sulfur compounds to sulfate, the major product of most dissimilatory sulfur-oxidizing prokaryotes. Two pathways of sulfite oxidation are known: (1) direct oxidation to sulfate catalyzed by a sulfite: acceptor oxidoreductase, which is thought to be a molybdenum-containing enzyme; (2) indirect oxidation under the involvement of the enzymes adenylylsulfate (APS) reductase and ATP sulfurylase and/or adenylylsulfate phosphate adenylyltransferase with APS as an intermediate. The latter pathway allows substrate phosphorylation and occurs in the bacterial cytoplasm. Direct oxidation appears to have a wider distribution; however, a redundancy of pathways has been described for diverse photo- or chemotrophic, sulfite-oxidizing prokaryotes. In many pro- and also eukaryotes sulfite is formed as a degradative product from molecules containing sulfur as a heteroatom. In these organisms detoxification of sulfite is generally achieved by direct oxidation to sulfate. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Functional deficiencies of sulfite oxidase: Differential diagnoses in neonates presenting with intractable seizures and cystic encephalomalacia

Sulfite oxidase is a mitochondrial enzyme encoded by the SUOX gene and essential for the detoxification of sulfite which results mainly from the catabolism of sulfur-containing amino acids. Decreased activity of this enzyme can either be due to mutations in the SUOX gene or secondary to defects in the synthesis of its cofactor, the molybdenum cofactor. Defects in the synthesis of the molybdenum cofactor are caused by mutations in one of the genes MOCS1, MOCS2, MOCS3 and GEPH and result in combined deficiencies of the enzymes sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Although present in many ethnic groups, isolated sulfite oxidase deficiency and molybdenum cofactor deficiency are rare inborn errors of metabolism, which makes awareness of key clinical and laboratory features of affected individuals crucial for early diagnosis. We report clinical, radiologic, biochemical and genetic data on a Brazilian and on a Turkish child with sulfite oxidase deficiency due to the isolated defect and impaired synthesis of the molybdenum cofactor, respectively. Both patients presented with early onset seizures and neurological deterioration. They showed no sulfite oxidase activity in fibroblasts and were homozygous for the mutations c.1136A>G in the SUOX gene and c.667insCGA in the MOCS1 gene, respectively. Widely available routine laboratory tests such as assessment of total homocysteine and uric acid are indicated in children with a clinical presentation resembling that of hypoxic ischemic encephalopathy and may help in obtaining a tentative diagnosis locally, which requires confirmation by specialized laboratories. (C) 2009 Published by Elsevier B.V.

Direct electrochemistry of a bacterial sulfite dehydrogenase

Sulfite dehydrogenase from Starkeya novella is an alphabeta heterodimer comprising a 40.6 kDa subunit (containing the Mo cofactor) and a smaller 8.8 kDa heme c subunit. The enzyme catalyses the oxidation of sulfite to sulfate with the natural electron acceptor being cytochrome c(550). Its catalytic mechanism is thought to resemble that found in eukaryotic sulfite oxiclases. Using protein film voltammetry and redox potentiometry, we have identified both Mo- and heme-centered redox responses from the enzyme immobilized on a pyrolytic graphite working electrode: E-m,E-8 (Fe-III/II) +177 mV; E-m,E-8 (Mo-VI/V) +211 mV and E(m,)8 (Mo-V/IV) -118 mV vs NHE; Upon addition of sulfite to the electrochemical cell a steady-state voltammogram is observed and an apparent Michaelis constant (K-m) of 26(l) muM was determined for the enzyme immobilized on the working electrode surface, which is comparable with the value obtained from solution assays.

Intramolecular electron transfer in a bacterial sulfite dehydrogenase

Sulfite dehydrogenase (SDH) from Starkeya novella, a sulfite-oxidizing molybdenum-containing enzyme, has a novel tightly bound αβ-heterodimeric structure in which the Mo cofactor and the c-type heme are located on different subunits. Flash photolysis studies of intramolecular electron transfer (IET) in SDH show that the process is first-order, independent of solution viscosity, and not inhibited by sulfate, which strongly indicates that IET in SDH proceeds directly through the protein medium and does not involve substantial movement of the two subunits relative to each other. The IET results for SDH contrast with those for chicken and human sulfite oxidase (SO) in which the molybdenum domain is linked to a b-type heme domain through a flexible loop, and IET shows a remarkable dependence on sulfate concentration and viscosity that has been ascribed to interdomain docking. The results for SDH provide additional support for the interdomain docking hypothesis in animal SO and clearly demonstrate that dependence of IET on viscosity and sulfate is not an inherent property of all sulfite-oxidizing molybdenum enzymes.

Crystallographic and biochemical studies on dissimilatory sulfite reductases

Thesis dissertation presented to obtain a PhD degree in Biochemistry at Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa

Sulfite Oxidase Deficiency – An Unusual Late and Mild Presentation

Introduction: Sulfite oxidase deficiency (SOD) is an autosomal recessive inherited disease usually presenting in the neonatal period with severe neurological symptoms including seizures, often refractory to anticonvulsant therapy, and a rapidly progressive encephalopathy resembling neonatal hypoxic ischemia, with premature death. Most patients develop dislocated ocular lenses. Later or milder presentations of SOD are being reported with increasing frequency. These presentations include neurological regression with loss of previously acquired milestones or movement disorders. Case report: We report a four years old girl presenting with intermittent ataxia and uncoordinated limb movements. A similar episode of ataxia had occurred previously, one year before, with complete neurologic recovery and normal developmental milestones. Bilateral lens dislocation had been recently diagnosed. Cranial MRI demonstrated bilateral globus pallidus enhancement. Low homocysteine was found in plasma and SulfitestR was positive. Further investigations led to confirmation of isolated sulfite oxidase deficiency with no enzyme activity detected on skin fibroblasts culture. Discussion: This case illustrates the clinical variability of SOD and it is not only atypical but also seems to be the mildest form described so far. The association of ectopia lentis with a movement disorder, even without psychomotor regression, should prompt us to look for this diagnosis.

Keratin degradation by dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump.

Millions of people suffer from superficial infections caused by dermatophytes. Intriguingly, these filamentous fungi exclusively infect keratin-rich host structures such as hair, nails, and skin. Keratin is a hard, compact protein, and its utilization by dermatophytes for growth has long been discussed as a major virulence attribute. Here, we provide strong support for the hypothesis that keratin degradation is facilitated by the secretion of the reducing agent sulfite, which can cleave keratin-stabilizing cystine bonds. We discovered that sulfite is produced by dermatophytes from environmental cysteine, which at elevated concentrations is toxic for microbes and humans. We found that sulfite formation from cysteine relies on the key enzyme cysteine dioxygenase Cdo1. Sulfite secretion is supported by the sulfite efflux pump Ssu1. Targeted mutagenesis proved that dermatophyte mutants in either Cdo1 or Ssu1 were highly growth-sensitive to cysteine, and mutants in Ssu1 were specifically sensitive to sulfite. Most notably, dermatophyte mutants in Cdo1 and Ssu1 were specifically growth-defective on hair and nails. As keratin is rich in cysteine, our identified mechanism of cysteine conversion and sulfite efflux supports both cysteine and sulfite tolerance per se and progression of keratin degradation. These in vitro findings have implications for dermatophyte infection pathogenesis.

Factors Supporting Cysteine Tolerance and Sulfite Production in Candida albicans.

The amino acid cysteine has long been known to be toxic at elevated levels for bacteria, fungi, and humans. However, mechanisms of cysteine tolerance in microbes remain largely obscure. Here we show that the human pathogenic yeast Candida albicans excretes sulfite when confronted with increasing cysteine concentrations. Mutant construction and phenotypic analysis revealed that sulfite formation from cysteine in C. albicans relies on cysteine dioxygenase Cdg1, an enzyme with similar functions in humans. Environmental cysteine induced not only the expression of the CDG1 gene in C. albicans, but also the expression of SSU1, encoding a putative sulfite efflux pump. Accordingly, the deletion of SSU1 resulted in enhanced sensitivity of the fungal cells to both cysteine and sulfite. To study the regulation of sulfite/cysteine tolerance in more detail, we screened a C. albicans library of transcription factor mutants in the presence of sulfite. This approach and subsequent independent mutant analysis identified the zinc cluster transcription factor Zcf2 to govern sulfite/cysteine tolerance, as well as cysteine-inducible SSU1 and CDG1 gene expression. cdg1Δ and ssu1Δ mutants displayed reduced hypha formation in the presence of cysteine, indicating a possible role of the newly proposed mechanisms of cysteine tolerance and sulfite secretion in the pathogenicity of C. albicans. Moreover, cdg1Δ mutants induced delayed mortality in a mouse model of disseminated infection. Since sulfite is toxic and a potent reducing agent, its production by C. albicans suggests diverse roles during host adaptation and pathogenicity.

Electrodeposition of gold from formaldehyde-sulfite baths: bath stability and deposits characterization

It was investigated Au(I)-sulfite baths containing formaldehyde. As a result, high stability was achieved for baths containing formaldehyde concentration close to 10 mL L-1 with a lifetime superior to 600 days. On the other hand, cyclic voltammograms indicated that the increase of formaldehyde concentration in the bath promotes decreasing of the maximum cathodic current, so that, if the formaldehyde concentration is high, the surface areal concentration of gold will be low. Also, the lowest surface roughness was obtained for 10 mL L-1 of formaldehyde.

Effect of some antioxidants on oxidative DNA damage induced by autoxidation of microquantities of sulfite in the presence of Ni(II)/Gly-Gly-L-His

Ni(II)GGH (GGH, glycylglycyl-L-histidine) reacts rapidly with S(IV), in air-saturated solution, to produce Ni(III)GGH. A mechanism is proposed where Ni(III) oxidizes SO(3)(2-) to SO(3)(center dot-), which reacts with dissolved oxygen to produce SO(5)(center dot-), initiating radical chain reactions. DNA strand breaks and 8-oxo-7,8-dihydro-20-deoxyguanosine (8-oxodGuo) formation were observed in air-saturated solutions containing micromolar concentrations of nickel(II) and S(IV). The efficacies of melatonin, (-)-epigallocatechin-gallate (from green tea), resveratrol, tannic, and ascorbic acids in terms of their inhibitory activities of DNA strand breaks and 8-oxodGuo formation were evaluated.