Evaluating the stability of disulfide bridges in proteins: a torsional potential energy surface for diethyl disulfide

Disulfide bonds formed by the oxidation of cysteine residues in proteins are the major form of intra- and inter-molecular covalent linkages in the polypeptide chain. To better understand the conformational energetics of this linkage, we have used the MP2(full)/6-31G(d) method to generate a full potential energy surface (PES) for the torsion of the model compound diethyl disulfide (DEDS) around its three critical dihedral angles (χ₂, χ₃, χ₂′). The use of ten degree increments for each of the parameters resulted in a continuous, fine-grained surface. This allowed us to accurately predict the relative stabilities of disulfide bonds in high resolution structures from the Protein Data Bank. The MP2(full) surface showed significant qualitative differences from the PES calculated using the Amber force field. In particular, a different ordering was seen for the relative energies of the local minima. Thus, Amber energies are not reliable for comparison of the relative stabilities of disulfide bonds. Surprisingly, the surface did not show a minimum associated with χ₂～ ? 60°, χ₃～90, χ₂′～ ? 60°. This is due to steric interference between Hα atoms. Despite this, significant populations of disulfides were found to adopt this conformation. In most cases this conformation is associated with an unusual secondary structure motif, the cross-strand disulfide. The relative instability of cross-strand disulfides is of great interest, as they have the potential to act as functional switches in redox processes.     

Characterization of Microbial Arsenate Reduction in the Anoxic Bottom Waters of Mono Lake,California

Dissimilatory reduction of arsenate (DAsR) occurs in the arsenic-rich, anoxic water column of Mono Lake, California, yet the microorganisms responsible for this observed in situ activity have not been identified. To gain insight as to which microorganisms mediate this phenomenon, as well as to some of the biogeochemical constraints on this activity, we conducted incubations of arsenate-enriched bottom water coupled with inhibition/amendment studies and Denaturing Gradient Gel Electrophoresis (DGGE) characterization techniques. DAsR was totally inhibited by filter-sterilization and by nitrate, partially inhibited (~50%) by selenate, but only slightly (~25%) inhibited by oxyanions that block sulfate-reduction (molybdate and tungstate). The apparent inhibition by nitrate, however, was not due to action as a preferred electron acceptor to arsenate. Rather, nitrate addition caused a rapid, microbial re-oxidation of arsenite to arsenate, which gave the overall appearance of no arsenate loss. A similar microbial oxidation of As(III) was also found with Fe(III), a fact that has implications for the recycling of As(V) in Mono Lake's anoxic bottom waters. DAsR could be slightly (10%) stimulated by substrate amendments of lactate, succinate, malate, or glucose, but not by acetate, suggesting that the DAsR microflora is not electron donor limited. DGGE analysis of amplified 16S rDNA gene fragments from incubated arsenate-enriched bottom waters revealed the presence of two bands that were not present in controls without added arsenate. The resolved sequences of these excised bands indicated the presence of members of the epsilon ( Sulfurospirillum ) and delta ( Desulfovibrio ) subgroups of the Proteobacteria , both of which have representative species that are capable of anaerobic growth using arsenate as their electron acceptor.     

Microbial arsenic: from geocycles to genes and enzymes

Arsenic compounds have been abundant at near toxic levels in the environment since the origin of life. In response, microbes have evolved mechanisms for arsenic resistance and enzymes that oxidize As(III) to As(V) or reduce As(V) to As(III). Formation and degradation of organoarsenicals, for example methylarsenic compounds, occur. There is a global arsenic geocycle, where microbial metabolism and mobilization (or immobilization) are important processes. Recent progress in studies of the ars operon (conferring resistance to As(III) and As(V)) in many bacterial types (and related systems in Archaea and yeast) and new understanding of arsenite oxidation and arsenate reduction by respiratory-chain-linked enzyme complexes has been substantial. The DNA sequencing and protein crystal structures have established the convergent evolution of three classes of arsenate reductases (that is classes of arsenate reductases are not of common evolutionary origin). Proposed reaction mechanisms in each case involve three cysteine thiols and S-As bond intermediates, so convergent evolution to similar mechanisms has taken place.     

Simultaneous Bio-reduction of Nitrate,Perchlorate, Selenate,Chromate, Arsenate,and Dibromochloropropane Using a Hydrogen-based Membrane Biofilm Reactor

We tested the hypothesis that the H₂-based membrane biofilm reactor (MBfR) is capable of reducing multiple oxidized contaminants, a common situation for groundwater contamination. We conducted bench-scale experiments with three groundwater samples collected from California’s San Joaquin Valley and on two synthetic groundwaters containing selenate and chromate. The actual groundwater sources had nitrate levels exceeding 10 mg-N l⁻¹ and different combinations of anthropogenic perchlorate + chlorate, arsenate, and dibromochloropropane (DBCP). For all actual groundwaters, the MBfR reduced nitrate to less than 0.01 mg-N l⁻¹. Present in two groundwaters, perchlorate + chlorate was reduced to below the California Notification Level, 6 μg-ClO₄ l⁻¹. As(V) was substantially reduced to As(III) for two groundwaters samples, which had influent As(V) concentrations from 3 to 8.8 μg-As l⁻¹. DBCP, present in one groundwater at 1.4 μg l⁻¹, was reduced to below its detection limit of 0.01 μg l⁻¹, which is well below California’s 0.2 μg l⁻¹ MCL for DBCP. For the synthetic groundwaters, two MBfRs initially reduced Se(VI) or Cr(VI) stably to Se° or Cr(III). When we switched the influent oxidized contaminants, the new oxidized contaminant was reduced immediately, and its reduction soon was approximately the same or greater than it had been reduced in its original MBfR. These results support that the H₂-based MBfR can reduce multiple oxidized contaminants simultaneously.     

Pelovirga terrestris gen. nov., sp. nov., anaerobic,alkaliphilic, fumarate-, arsenate-, Fe(III)- and sulfur-reducing bacterium isolated from a terrestrial mud volcano

A novel anaerobic, mesophilic, alkaliphilic, chemoorganotrophic bacterium (strain M08fum^T) was isolated from a salsa lake of a terrestrial mud volcano (Taman Peninsula, Russia). Cell of strain M08fum^T were Gram-stain-negative, rod-shaped, non-spore forming motile rods. The temperature range for growth was 10–45 °C (optimum at 30 °C). The pH range for growth was 7.0–11.0, with an optimum at pH 8.5–9.0. The isolate was capable of organic acids fermentation and anaerobic respiration with elemental sulfur, Fe(III) and arsenate. The end products of fumarate fermentation were succinate, acetate and CO₂. The closest phylogenetic relatives of strain M08fum^T were members of the family Geopsychrobacteraceae, class Desulfuromonadia. The genome of strain M08fum^T had a size of 3.10 Mb with a DNA G + C content of 53.1% (WGS). Genome analysis revealed the presence of genes involved in fumarate fermentation, arsenate reduction and resistance, sulfur respiration and Fe (III) reduction. Based on the phenotypic, genotypic and phylogenetic characteristics we propose to assign strain M08fum^T to a new species of a novel genus Pelovirga terrestris gen. nov., sp. nov. within the family Geopsychrobacteraceae. The type strain of Pelovirga terrestris is M08fum^T (=KCTC 15919^T = VKM B-3407^T). This is the first representative of the class Desulfuromonadia, isolated in pure culture from a mud volcano and the first alkaliphile in the family Geopsychrobacteraceae.     

Arsenate reductase activity in roots from the arsenic hyperaccumulator Pteris vittata utilizes both glutathione and dithiothreitol as reductants

Abstract

The role of glutathione and dithiothreitol as reductants supporting arsenate reductase activity in root extract from the arsenic hyperaccumulator Pteris vittata was examined. The two reductants in combination enhanced arsenate reduction in vitro more than glutathione alone. The implications of these results for in vivo arsenate reduction are discussed.     

Bacterial respiration of arsenic and selenium

Oxyanions of arsenic and selenium can be used in microbial anaerobic respiration as terminal electron acceptors. The detection of arsenate and selenate respiring bacteria in numerous pristine and contaminated environments and their rapid appearance in enrichment culture suggest that they are widespread and metabolically active in nature. Although the bacterial species that have been isolated and characterized are still few in number, they are scattered throughout the bacterial domain and include Gram-positive bacteria, beta, gamma and epsilon Proteobacteria and the sole member of a deeply branching lineage of the bacteria, Chrysiogenes arsenatus. The oxidation of a number of organic substrates (i.e. acetate, lactate, pyruvate, glycerol, ethanol) or hydrogen can be coupled to the reduction of arsenate and selenate, but the actual donor used varies from species to species. Both periplasmic and membrane-associated arsenate and selenate reductases have been characterized. Although the number of subunits and molecular masses differs, they all contain molybdenum. The extent of the environmental impact on the transformation and mobilization of arsenic and selenium by microbial dissimilatory processes is only now being fully appreciated.     

Effects of arsenate on the growth and microcystin production of Microcystis aeruginosa isolated from Taiwan as influenced by extracellular phosphate

Arsenic pollution and eutrophication are both prominent issues in the aquaculture ponds of Taiwan. It is important to study the effects of arsenic on algal growth and toxin production in order to assess the ecological risk of arsenic pollution, or at least to understand naturally occurring ponds. The sensitivity of algae to arsenate has often been linked to the structural similarities between arsenate and phosphate. Thus, in this study we examined the effects of arsenate (10⁻⁸ to 10⁻⁴ M) on Microcystis aeruginosa TY-1 isolated from Taiwan, under two phosphate regimes. The present study showed that M. aeruginosa TY-1 was arsenate tolerant up to 10⁻⁴ M, and that this tolerance was not affected by extracellular phosphate. However, it seems that extracellular phosphate contributed to microcystin production and leakage by M. aeruginosa in response to arsenate. Under normal phosphate conditions, total toxin yields after arsenate treatment followed a typical inverted U-shape hormesis, with a peak value of 2.25 ± 0.06 mg L⁻¹ in the presence of 10⁻⁷ M arsenate, whereas 10⁻⁸ to 10⁻⁶ M arsenate increased leakage of ∼75% microcystin. Under phosphate starvation, total toxin yields were not affected by arsenate, while 10⁻⁶ and 10⁻⁵ M arsenate stimulated microcystin leakage. It is suggested that arsenate may play a role in the process of microcystin biosynthesis and excretion. Given the arsenic concentrations in aquaculture ponds in Taiwan, arsenate favors survival of toxic M. aeruginosa in such ponds, and arsenate-stimulated microcystin production and leakage may have an impact on the food chain.     

A comparison of arsenic mobility in Phaseolus vulgaris, Mentha aquatica, and Pteris cretica rhizosphere

The ability of Phaseolus vulgaris, Mentha aquatica, and Pteris cretica to release arsenic (As) species from contaminated soil was tested in rhizobox experiments in three soils differing in their physicochemical parameters and total and mobile As concentration. Relatively low uptake of arsenic by P. vulgaris and M. aquatica resulted in very low and ambiguous changes in rhizosphere soil compared to bulk soil. However, there were observed differences in the distribution of the mobile As portion in soil to individual As species as affected by plant species and/or plantation conditions of these plants. Higher percentage of mobile arsenite in mint rhizosphere seems to be related to more reducing conditions during cultivation of these wetland plants. P. cretica planted in the soils containing between 36 and 1436 mg As kg⁻¹ was able to accumulate between 80 and 500 mg As kg⁻¹ in aboveground biomass. The extractable concentrations of As compounds in rhizosphere soil of P. cretica showed a clear depletion of arsenate (representing more than 90% of extractable arsenic) with the distance from plant roots. However, the As uptake mechanisms, as well as As transformation within hyperaccumulating fern plants, differ substantially from those in higher plants. Therefore the finding of suitable higher plant tolerant to the As soil contamination with good ability to accumulate As in aboveground biomass remains for the further research.