Influence of the sulfur species reactivity on biofilm conformation during pyrite colonization by Acidithiobacillus thiooxidans

Massive pyrite (FeS₂) electrodes were potentiostatically modified by means of variable oxidation pulse to induce formation of diverse surface sulfur species (S _n ^2?, S⁰). The evolution of reactivity of the resulting surfaces considers transition from passive (e.g., Fe_1?x S₂) to active sulfur species (e.g., Fe_1?x S_2?y , S⁰). Selected modified pyrite surfaces were incubated with cells of sulfur-oxidizing Acidithiobacillus thiooxidans for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the attached cells density and their exopolysaccharides were analyzed by confocal laser scanning microscopy (CLMS) and atomic force microscopy (AFM) on bio-oxidized surfaces; additionally, S _n ^2?/S⁰ speciation was carried out on bio-oxidized and abiotic pyrite surfaces using Raman spectroscopy. Our results indicate an important correlation between the evolution of S _n ^2?/S⁰ surface species ratio and biofilm formation. Hence, pyrite surfaces with mainly passive-sulfur species were less colonized by A. thiooxidans as compared to surfaces with active sulfur species. These results provide knowledge that may contribute to establishing interfacial conditions that enhance or delay metal sulfide (MS) dissolution, as a function of the biofilm formed by sulfur-oxidizing bacteria.     

Pyrite accumulation in salt marshes in the Eastern Scheldt,southwest Netherlands

Pore water composition, pyrite distribution and pyrite crystal morphology of sediments from salt marshes in the Eastern Scheldt, southwestern Netherlands, were examined from July 1984 to October 1986.Hydrology and marsh vegetation were the chief determinants of pyrite accumulation. In the bare sediments of pans in the low marsh, highly reducing conditions prevailed just below the surface. At these sites, practically all the incoming detrital pyrite (0.5–1% FeS₂) was preserved. The in-situ formation of pyrites was negligible in these anoxic sediments.All incoming detrital pyrite was oxidized in the surface layers (0–10 cm) of the medium-high marsh overgrown withSpartina anglica. Pyrite was formed at a rate of 2.6–3.8 mol S-FeS₂m^–2yr^–1 in a narrow range of depths (15–20cm), at the interface of the oxidizing and underlying reducing sediment. At this interface the concentration profiles of Fe²⁺ and dissolved S intersected. The role of the rhizosphere is discussed in connection with pyrite formation. No further pyrite formation occurred deeper in the sediment. This resulted in the build up of high concentrations of dissolved S and acid volatile sulfides (AVS). The decrease with depth in oxalate-extractable Fe indicated that most of the iron oxyhydroxides (70–80%) had been transformed to pyrite. Another 10–20% of oxalate-extractable Fe was present as AVS. The abundance of framboidal pyrite particles and the high concentrations of AVS and dissolved S indicated that the formation of pyrite occurred via iron monosulfide intermediatesThere was a linear relationship between the organic carbon and the S-FeS₂ content in theSpartina overgrown reducing sediment. The mean C/S ratio was 4.2.     

A two [4Fe-4S]-cluster-containing ferredoxin as an alternative electron donor for 2-hydroxyglutaryl-CoA dehydratase from<Emphasis Type="Italic"> Acidaminococcus fermentans</Emphasis>

The key step in the fermentation of glutamate by Acidaminococcus fermentans is a reversible syn-elimination of water from (R)-2-hydroxyglutaryl-CoA to (E)-glutaconyl-CoA catalyzed by 2-hydroxyglutaryl-CoA dehydratase, a two-component enzyme system. The actual dehydration is mediated by component D, which contains 1.0 [4Fe-4S]²⁺ cluster, 1.0 reduced riboflavin-5′-phosphate and about 0.1 molybdenum (VI) per heterodimer. The enzyme has to be activated by the extremely oxygen-sensitive [4Fe-4S]^1+/2+-cluster-containing homodimeric component A, which generates Mo(V) by an ATP/Mg²⁺-induced one-electron transfer. Previous experiments established that the hydroquinone state of a flavodoxin (m=14.6 kDa) isolated from A. fermentans served as one-electron donor of component A, whereby the blue semiquinone is formed. Here we describe the isolation and characterization of an alternative electron donor from the same organism, a two [4Fe-4S]^1+/2+-cluster-containing ferredoxin (m=5.6 kDa) closely related to that from Clostridium acidiurici. The protein was purified to homogeneity and almost completely sequenced; the magnetically interacting [4Fe-4S] clusters were characterized by EPR and Mössbauer spectroscopy. The redox potentials of the ferredoxin were determined as ?405 mV and ?340 mV. Growth experiments with A. fermentans in the presence of different iron concentrations in the medium (7–45 μM) showed that flavodoxin is the dominant electron donor protein under iron-limiting conditions. Its concentration continuously decreased from 3.5 μmol/g protein at 7 μM Fe to 0.02 μmol/g at 45 μM Fe. In contrast, the concentration of ferredoxin increased stepwise from about 0.2 μmol/g at 7–13 μM Fe to 1.1±0.1 μmol/g at 17–45 μM Fe.     

Measurement of bacterial sulfate reduction in sediments: Evaluation of a single-step chromium reduction method

A procedure which includes the Total Reduced Inorganic Sulfur (TRIS) in a single distillation step is described for the radiotracer measurement of sulfate reduction in sediments. The TRIS includes both Acid Volatile Sulfide (AVS: H₂S + FeS) and the remaining Chromium Reducible Sulfur (CRS: S⁰, FeS₂). The single-step distillation was simpler and faster than the consecutive distillations of AVS and CRS. It also resulted in higher (4–50%) sulfate reduction rates than those obtained from the sum of³⁵S in AVS and CRS. The difference was largest when the sediment had been dried after AVS but before CRS distillation. Relative to the³⁵S-AVS distillation alone, the³⁵S-TRIS single-step distallation yielded 8–87% higher reduction rates. The separation and recovery of FeS, S⁰ and FeS₂ was studied under three distillation conditions: 1) cold acid, 2) cold acid with Cr²⁺, and 3) hot acid with Cr²⁺. The FeS was recovered by cold acid alone while pyrite was recovered by cold acid with Cr²⁺. A smaller S⁰ fraction, presumably of the finer crystal sizes, was recovered also in the cold acid with Cr²⁺ while most of the S⁰ required hot acid with Cr²⁺ for reduction to H₂S.     

酸性硫酸盐土中硫形态转化过程的水分制约作用

分别设土壤田间持水量的30%恒定(FH1)、土壤田间持水量的70%恒定(FH2),一直淹水(INU)、风干后放置(DRY,作为对照)、自然风干(NAD)5个处理进行酸性硫酸盐土室内模拟实验。实验结果显示,水分条件对酸性硫酸盐土中水溶性硫、交换性硫和黄铁矿硫的形态转化有显着的制约作用。淹水环境和过分干燥环境都不利于黄铁矿的氧化及水溶性硫和交换性硫的形成,潮湿但含水量不饱和环境有利于黄铁矿硫向水溶性硫和交换性硫的转换。模拟试验期内,水溶性硫含量的增加速度排列为:FH2>FH1>INU,交换性硫含量的增加速度排列为:FH1>FH2>INU,黄铁矿硫含量的下降速度排列为:FH2>FH1>INU,原状土自然风干(NAD)过程中,水溶性硫、交换性硫和黄铁矿硫之间发生了明显的转化。对不同处理中黄钾铁矾硫、有机硫和元素硫的动态变化也进行了分析。     

The McClure and Weiss models of Fe–O2 bonding for oxyhemes, and the HbO2 + NO reaction

For the Fe–O₂(S = 0) linkages of oxyhemes, valence bond (VB) structures are re-presented for the McClure [Fe^II(S = 1) + O₂(S = 1)], Pauling–Coryell [Fe^II(S = 0) + O₂*(S = 0)], and Weiss [Fe^III(S = ½) + O₂ ^?(S = ½)] models of bonding. The VB structures for the McClure and Weiss models are of the increased-valence type, with more electrons participating in bonding than occur in their component Lewis structures. The Fe–O bond number and O–O bond order for the McClure structure are correlated with measured Fe–O and O–O bond lengths for oxymyoglobin. Back-bonding from O ₂ ^? to Fe^III of the Weiss structure gives a restricted form of the McClure structure. The McClure and Weiss increased-valence structures are used to provide VB formulations of mechanisms for the oxyhemoglobin + NO reaction. The products of these two formulations are Hb⁺ and NO₃ ^? (where Hb is hemoglobin) and Hb⁺ and OONO^?, respectively. Because Hb⁺ and NO₃ ^? are the observed products, they provide an experimental procedure for distinguishing the McClure and Weiss models. It is also shown that the same type of agreement between McClure-type theory and experiment occurs for oxycoboglobin + NO, cytochrome P450 monooxygenases, and related hydrogen atom transfer reactions. In the appendices, the results of density functional theory and multireference molecular orbital calculations for oxyhemes are related to one formulation of the increased-valence wavefunction for the McClure model, and theory is presented for the calculation of approximate weights for the Lewis structures that are components of the McClure increased-valence structure.     

Spectroscopic evidence for an all-ferrous [4Fe–4S]<Superscript>0</Superscript> cluster in the superreduced activator of 2-hydroxyglutaryl-CoA dehydratase from <Emphasis Type="Italic">Acidaminococcus </Emphasis><Emphasis Type="Italic">fermentans</Emphasis>

The key enzyme of the fermentation of glutamate by Acidaminococcus fermentans, 2-hydroxyglutarylcoenzyme A dehydratase, catalyzes the reversible syn-elimination of water from (R)-2-hydroxyglutaryl-coenzyme A, resulting in (E)-glutaconylcoenzyme A. The dehydratase system consists of two oxygen-sensitive protein components, the activator (HgdC) and the actual dehydratase (HgdAB). Previous biochemical and spectroscopic studies revealed that the reduced [4Fe–4S]⁺ cluster containing activator transfers one electron to the dehydratase driven by ATP hydrolysis, which activates the enzyme. With a tenfold excess of titanium(III) citrate at pH 8.0 the activator can be further reduced, yielding about 50% of a superreduced [4Fe–4S]⁰ cluster in the all-ferrous state. This is inferred from the appearance of a new Mössbauer spectrum with parameters δ = 0.65 mm/s and ΔE _Q = 1.51–2.19 mm/s at 140 K, which are typical of Fe(II)S₄ sites. Parallel-mode electron paramagnetic resonance (EPR) spectroscopy performed at temperatures between 3 and 20 K showed two sharp signals at g = 16 and 12, indicating an integer-spin system. The X-band EPR spectra and magnetic Mössbauer spectra could be consistently simulated by adopting a total spin S _t = 4 for the all-ferrous cluster with weak zero-field splitting parameters D = ?0.66 cm^?1 and E/D = 0.17. The superreduced cluster has apparent spectroscopic similarities with the corresponding [4Fe–4S]⁰ cluster described for the nitrogenase Fe-protein, but in detail their properties differ. While the all-ferrous Fe-protein is capable of transferring electrons to the MoFe-protein for dinitrogen reduction, a similar physiological role is elusive for the superreduced activator. This finding supports our model that only one-electron transfer steps are involved in dehydratase catalysis. Nevertheless we discuss a common basic mechanism of the two diverse systems, which are so far the only described examples of the all-ferrous [4Fe–4S]⁰ cluster found in biology.     

Reactive oxygen species generated in the presence of fine pyrite particles and its implication in thermophilic mineral bioleaching

In the tank bioleaching process, maximising solid loading and mineral availability, the latter through decreasing particle size, are key to maximising metal extraction. In this study, the effect of particle size distribution on bioleaching performance and microbial growth was studied through applying knowledge based on medical geology research to understand the adverse effects of suspended fine pyrite particles. Small-scale leaching studies, using pyrite concentrate fractions (106–75, 75–25, ?25 μm fines), were used to confirm decreasing performance with decreasing particle size (D ₅₀ <40 μm). Under equivalent experimental conditions, the generation of the reactive oxygen species (ROS), hydrogen peroxide and hydroxyl radicals from pyrite was illustrated. ROS generation measured from the different pyrite fractions was found to increase with increasing pyrite surface area loading (1.79–74.01 m² L^?1) and Fe²⁺ concentration (0.1–2.8 g?L^?1) in solution. The highest concentration of ROS was measured from the finest fraction of pyrite (0.85 mM) and from the largest concentration of Fe²⁺ (0.78 mM). No ROS was detected from solutions containing only Fe³⁺ under the same conditions tested. The potential of ROS to inhibit microbial performance under bioleaching conditions was demonstrated. Pyrite-free Sulfolobus metallicus cultures challenged with hydrogen peroxide (0.5–2.5 mM) showed significant decrease in both cell growth and Fe²⁺ oxidation rates within the concentration range 1.5–2.5 mM. In combination, the results from this study suggest that conditions of large pyrite surface area loading, coupled with high concentrations of dissolved Fe²⁺, can lead to the generation of ROS, resulting in oxidative stress of the microorganisms.     

不同土壤水分条件下酸性硫酸盐土硫形态转化特征

以4种土壤湿度、3种干湿交替周期和原状土自然风干8个处理进行酸性硫酸盐土室内模拟实验,对模拟过程内土壤6种硫形态和pH等指标的动态变化过程的跟踪测定和分析结果表明,水分条件对酸性硫酸盐土中水溶性硫、交换性硫和黄铁矿硫的形态转化制约作用显著．淹水环境和过分干燥环境都不利于黄铁矿的氧化及水溶性硫和交换性硫的形成．潮湿但含水量不饱和环境有利于黄铁矿硫向水溶性硫和交换性硫的转换．在3种周期的干湿交替处理中,单一排干期水溶性硫和交换性硫土壤含量平均上升幅度分别为0．90～1．63g·kg^-1和0．58～1．47g·kg^-1,而黄铁矿硫含量下降幅度为1．29～3．20g·kg^-1．淹水期黄铁矿硫含量相对稳定而水溶性硫和交换性硫含量明显下降,并在排水过程中造成总硫的部分流失．硫形态转化量和硫的淋失量受到干湿交替周期的显著影响．同时分析了黄钾铁钒硫、有机硫和元素硫的动态．     

Seasonal Variation of Acid Volatile Sulfide and Simultaneously Extracted Metals in Sediment Cores from the Pearl River Estuary

The simultaneously extracted metals/acid volatile sulfide (SEM/AVS) method is widely used to estimate the toxicity of metals in sediment. In this study, SEM and AVS concentrations were obtained by the cold-acid purge-and-trap technique during spring (April) and winter (December) at six sites in the Pearl River estuary. Total organic carbon, grain size distribution, and total metals were also measured in winter samples. AVS concentrations in spring sediments were slightly higher than those in winter sediments, except at site 2. AVS concentrations showed more distinct locational and temporal variation compared with SEM concentrations. Generally, AVS contents increased downcore, whereas SEM concentrations decreased slightly with depth. Higher SEM concentrations were observed in the west shoal (sites 2, 6, and 7) and site 4 (1.24 ～ 4.28 μmol?g^?1) compared with those at sites 1 and 3 (0.73 ～ 2.14 μmol?g^?1) in the middle shoal. Most SEM metals have a significant linear positive correlation with the total metals, especially for Cd, Zn, and Pb, which were easily extracted by 1 M HCl compared with Cr and Ni. According to the toxicity threshold value of 1.7 μmol?g^?1 for the difference of SEM-AVS, a toxic effect is expected at sites 1, 2, and 4 in spring and at sites 4, 6, and 7 in winter, which also indicates a relatively obvious seasonal variation in heavy metal bioavailability. However, comparisons between the total heavy metal concentrations in winter sediments with the sediment quality guidelines of ERLs/ERMs and TELs/PELs showed that adverse biological effects may occasionally occur at sites 2, 4, 6, and 7. Therefore, SEM/AVS prediction in conjunction with sediment quality guidelines can give a more reliable evaluation of the bioavailability of heavy metal in sediments.     

Changes in biofilm structure during the colonization of chalcopyrite by Acidithiobacillus thiooxidans

Biofilms of Acidithiobacillus thiooxidans were grown on the surface of massive chalcopyrite electrodes (MCE) where different secondary sulfur phases were previously formed by potentiostatic oxidation of MCE at 0.780?≤?E _an?≤?0.965 V (electrooxidized MCE, eMCE). The formation of mainly S⁰ and minor amounts of CuS and S _n ^2? were detected on eMCEs. The eMCEs were incubated with A. thiooxidans cells for 1, 12, 24, 48, and 120 h in order to temporally monitor changes in eMCE's secondary phases, biofilm structure, and extracellular polymeric substance (EPS) composition (lipids, proteins, and polysaccharides) using microscopic, spectroscopic, electrochemical, and biochemical techniques. The results show significant cell attachments with stratified biofilm structure since the first hour of incubation and EPS composition changes, the most important being production after 48–120 h when the highest amount of lipids and proteins were registered. During 120 h, periodic oxidation/formation of S⁰/S _n ^2? was recorded on biooxidized eMCEs, until a stable CuS composition was formed. In contrast, no evidence of CuS formation was observed on the eMCEs of the abiotic control, confirming that CuS formation results from microbial activity. The surface transformation of eMCE induces a structural transformation of the biofilm, evolving directly to a multilayered biofilm with more hydrophobic EPS and proteins after 120 h. Our results suggest that A. thiooxidans responded to the spatial and temporal distribution and chemical reactivity of the S _n ^2?/S⁰/CuS phases throughout 120 h. These results suggested a strong correlation between surface speciation, hydrophobic domains in EPS, and biofilm organization during chalcopyrite biooxidation by A. thiooxidans.     

Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability

Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD⁺ and their low operational stability in the presence of reactive organic compounds such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD⁺ binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k _cat/K _m) with NADP⁺. The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00?±?0.13 U?mg^?1 and a K _{m, NADP} ⁺ of 0.147?±?0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP⁺-accepting FDH (v _max, 10.25?±?1.63 U?mg^?1). However, the half-saturation constant for NADP⁺ (K _{m, NADP} ⁺ , 0.92?±?0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.     

Decolorization of azo dyes by Geobacter metallireducens

Geobacter metallireducens was found to be capable of decolorizing several azo dyes with different structures to various extents. Pyruvate, ethanol, acetate, propionate, and benzoate could support 66.3?±?2.6?93.7?±?2.1 % decolorization of 0.1 mM acid red 27 (AR27) in 40 h. The dependence of the specific decolorization rate on AR27 concentration (25 to 800 μM) followed Michaelis–Menten kinetics (K _m?=?186.9?±?1.4 μΜ, V _max?=?0.65?±?0.02 μmol?mg protein^?1 h^?1). Enhanced AR27 decolorization was observed with the increase of cell concentrations ranging from 7.5 to 45 mgL^?1. AR27 decolorization by G. metallireducens was retarded by the presence of goethite, which competed electrons with AR27 and was reduced to Fe(II). The addition of low concentrations of humic acid (1?100 mgL^?1) or 2-hydroxy–1,4-naphthoquinone (0.5?50 μM) could improve the decolorization performance of G. metallireducens. High-performance liquid chromatography analysis suggested reductive pathway to be responsible for decolorization. This was the first study on azo dye decolorization by Geobacter strain and might improve our understanding of natural attenuation and bioremediation of environments polluted by azo dyes.     

The influence of redox conditions in soils on extraplasmatic Fe-loading of plant roots

Loading of extraplasmatic Fe, as a potential storage pool for Fe nutrition, was studied in roots of maize grown under hypoxic conditions in soil culture. Extraplasmatic Fe loading was investigated depending on (i) duration of flooding (0, 1, 2 or 4 days) and (ii) microbial activity as affected by graduated addition of carbon sources (0, 2 or 10 g each starch and cellulose kg^?1 soil). Maize plants were grown in a soil culture system with root systems enclosed in membrane bags to avoid Fe contamination of the root surface by soil particles. Due to the high redox buffer capacity of the Haplic Luvisol employed for the experiments, flooding treatments induced only moderately reducing conditions (～?300 mV) and a slight increase of extraplasmatic Fe loading (41\to165 mg kg^?1 d.m.). Strongly reducing conditions (?100 mV) associated with a high Fe²⁺ concentration in the soil solution and a significant increase of extraplasmatic Fe (1190 mg kg^?1 d.m.) were obtained only after application of high amounts of organic carbon (10 g starch and 10 g cellulose kg^?1 soil), which accompanied by unrealistic reducing conditions due to intense stimulation of microbial growth. The expression of effects only under extremely high application level of organic carbon (～?33 t C ha^?1) suggest that similar to aerobic conditions, extraplasmatic Fe-loading under transient hypoxia is probably of limited ecological significance for the iron nutrition of higher plants, at least in soils with a high redox buffer capacity as employed in the present study. Abbreviations: DHA – dehydrogenase activity; d.m. – dry matter; DOC – dissolved organic carbon; Eh – redox potential; PIXE – proton-induced X-ray emission; STIM – scanning transmission ion microscopy.     

Fe(III) oxides protect fermenter–methanogen syntrophy against interruption by elemental sulfur via stiffening of Fe(II) sulfides produced by sulfur respiration

Thermosipho globiformans (rod-shaped thermophilic fermenter) and Methanocaldococcus jannaschii (coccal hyperthermophilic hydrogenotrophic methanogen) established H₂-mediated syntrophy at 68 °C, forming exopolysaccharide-based aggregates. Electron microscopy showed that the syntrophic partners connected to each other directly or via intercellular bridges made from flagella, which facilitated transfer of H₂. Elemental sulfur (S⁰) interrupted syntrophy; polysulfides abiotically formed from S⁰ intercepted electrons that were otherwise transferred to H⁺ to produce H₂, resulting in the generation of sulfide (sulfur respiration). However, Fe(III) oxides significantly reduced the interruption by S⁰, accompanied by stiffening of Fe(II) sulfides produced by the reduction of Fe(III) oxides with the sulfur respiration-generated sulfide. Sea sand replacing Fe(III) oxides failed to generate stiffening or protect the syntrophy. Several experimental results indicated that the stiffening of Fe(II) sulfides shielded the liquid from S⁰, resulting in methane production in the liquid. Field-emission scanning electron microscopy showed that the stiffened Fe(II) sulfides formed a network of spiny structures in which the microorganisms were buried. The individual fermenter rods likely produced Fe(II) sulfides on their surface and became local centers of a core of spiny structures, and the connection of these cores formed the network, which was macroscopically recognized as stiffening.     

Characterization of a newly synthesized carbonyl reductase and construction of a biocatalytic process for the synthesis of ethyl (S)-4-chloro-3-hydroxybutanoate with high space-time yield

A carbonyl reductase (SCR2) gene was synthesized and expressed in Escherichia coli after codon optimization to investigate its biochemical properties and application in biosynthesis of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), which is an important chiral synthon for the side chain of cholesterol-lowering drug. The recombinant SCR2 was purified and characterized using ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. The specific activity of purified enzyme was 11.9 U mg^?1. The optimum temperature and pH for enzyme activity were 45 °C and pH 6.0, respectively. The half-lives of recombinant SCR2 were 16.5, 7.7, 2.2, 0.41, and 0.05 h at 30 °C, 35 °C, 40 °C, 45 °C, and 50 °C, respectively, and it was highly stable in acidic environment. This SCR2 displayed a relatively narrow substrate specificity. The apparent K _m and V _max values of purified enzyme for COBE are 6.4 mM and 63.3 μmol min^?1 mg^?1, respectively. The biocatalytic process for the synthesis of (S)-CHBE was constructed by this SCR2 in an aqueous–organic solvent system with a substrate fed-batch strategy. At the final COBE concentration of 1 M, (S)-CHBE with yield of 95.3 % and e.e. of 99 % was obtained after 6-h reaction. In this process, the space-time yield per gram of biomass (dry cell weight, DCW) and turnover number of NADP⁺ to (S)-CHBE were 26.5 mmol L^?1 h^?1 g^?1 DCW and 40,000 mol/mol, respectively, which were the highest values as compared with other works.     

HdrC2 from Acidithiobacillus ferrooxidans Owns Two Iron–Sulfur Binding Motifs But Binds Only One Variable Cluster Between [4Fe–4S] and [3Fe–4S]

The heterodisulfide reductase complex HdrABC from Acidithiobacillus ferrooxidans was suggested to own novel features that act in reverse to convert the sulfane sulfur of GS _n H species (n > 1) into sulfite in sulfur oxidation. The HdrC subunit is potentially encoded by two different highly upregulated genes sharing only 29 % identity in A. ferrooxidans grown in sulfur-containing medium, which were named as HdrC1 and HdrC2, respectively and had been confirmed to contain iron–sulfur cluster by expression and characterization, especially the HdrC1 which had been showed to bind only one [4Fe–4S] cluster by mutations. However, the mutations of the HdrC2 remain to be done and the detailed binding information of it is still unclear. Here, we report the expression, mutations, and molecular modeling of the HdrC2 from A. ferrooxidans. This HdrC2 had two identical motifs (Cx₂Cx₂Cx₃C) containing total of eight cysteine residues potentially for iron–sulfur cluster binding. This purified HdrC2 was exhibited to contain one variable cluster converted between [4Fe–4S] and [3Fe–4S] according to different conditions by the UV-scanning and EPR spectra. The site-directed mutagenesis results of these eight residues further confirmed that the HdrC2 in reduction with Fe²⁺ condition loaded only one [4Fe–4S]⁺ with spin S = 1/2 ligated by the residues of Cys73, Cys109, Cys112, and Cys115; the HdrC2 in natural aeration condition lost the Fe atom ligated by the residue of Cys73 and loaded only one [3Fe–4S]⁰ with spin S = 0; the HdrC2 in oxidation condition loaded only one [3Fe–4S]⁺ with spin S = 1/2. Molecular modeling results were also in line with the experiment results.     

The "prismane" protein resolved: X-ray structure at 1.7 Å and multiple spectroscopy of two novel 4Fe clusters

The three-dimensional structure of the native "putative prismane" protein from Desulfovibrio vulgaris (Hildenborough) has been solved by X-ray crystallography to a resolution of 1.72?Å. The molecule does not contain a [6Fe-6S] prismane cluster, but rather two 4Fe clusters some 12?Å apart and situated close to the interfaces formed by the three domains of the protein. Cluster 1 is a conventional [4Fe-4S] cubane bound, however, near the N-terminus by an unusual, sequential arrangement of four cysteine residues (Cys 3, 6, 15, 21). Cluster 2 is a novel 4Fe structure with two μ₂-sulfido bridges, two μ₂-oxo bridges, and a partially occupied, unidentified μ₂ bridge X. The protein ligands of cluster 2 are widely scattered through the second half of the sequence and include three cysteine residues (Cys 312, 434, 459), one persulfido-cysteine (Cys 406), two glutamates (Glu 268, 494), and one histidine (His 244). With this unusual mixture of bridging and external type of ligands, cluster 2 is named the "hybrid" cluster, and its asymmetric, open structure suggests that it could be the site of a catalytic activity. X-ray absorption spectroscopy at the Fe K-edge is readily interpretable in terms of the crystallographic model when allowance is made for volume contraction at 10?K; no Fe··Fe distances beyond 3.1?Å could be identified. EPR, Mössbauer and MCD spectroscopy have been used to define the oxidation states and the magnetism of the clusters in relation to the crystallographic structure. Reduced cluster 1 is a [4Fe-4S]¹⁺ cubane with S?=?3/2; it is the first biological example of a "spin-admixed" iron-sulfur cluster. The hybrid cluster 2 has four oxidation states from (formally) all Fe^III to three Fe^II plus one Fe^III. The four iron ions are exchange coupled resulting in the system spins S?=?0, 9/2, 0 (and 4), 1/2, respectively, for the four redox states. Resonance Raman spectroscopy suggests that the bridging ligand X which could not be identified unambiguously in the crystal structure is a solvent-exchangeable oxygen.     

Acute Toxicity of Arsenic under Different Temperatures and Salinity Conditions on the White Shrimp Litopenaeus vannamei

The aim of this study was to determine acute toxicity in the post larvae of the white shrimp Litopenaeus vannamei after 96 h of exposure to dissolved arsenic under three different temperatures and salinity conditions. Recent reports have shown an increase in the presence of this metalloid in coastal waters, estuaries, and lagoons along the Mexican coast. The white shrimp stands out for its adaptability to temperature and salinity changes and for being the main product for many commercial fisheries; it has the highest volume of oceanic capture and production in Mexican shrimp farms. Lethal concentrations (LC₅₀–96 h) were obtained at nine different combinations (3?×?3 combinations in total) of temperature (20, 25, and 30 °C) and salinity (17, 25, and 33) showing mean LC₅₀–96 h values (±standard error) of 9.13?±?0.76, 9.17?±?0.56, and 6.23?±?0.57 mgAs?L^?1(at 20 °C and 17, 25, and 33 salinity); 12.29?±?2.09, 8.70?±?0.82, and 8.03?±?0.59 mgAs?L^?1 (at 25 °C and 17, 25, and 33 salinity); and 7.84?±?1.30, 8.49?±?1.40, and 7.54?±?0.51 mgAs?L^?1 (at 30 °C and 17, 25, and 33 salinity), respectively. No significant differences were observed for the optimal temperature and isosmotic point of maintenance (25 °C–S 25) for the species, with respect to the other experimental conditions tested, except for at 20 °C–S 33, which was the most toxic. Toxicity under 20 °C–S 33 conditions was also higher than 25 °C–S 17 and 20 °C (S 17 or 25). The least toxic condition was 25 °C–S 17. All this suggests that the toxic effect of arsenic is not affected by temperature changes; it depends on the osmoregulatory pattern developed by the shrimp, either hyperosmotic at low salinity or hiposmotic at high salinity, as observed at least on the extreme salinity conditions here tested (17 and 33). However, further studies testing salinities near the isosmotic point (between 20 and 30 salinities) are needed to clarify these mechanisms.     

Influence of the surface speciation on biofilm attachment to chalcopyrite by Acidithiobacillus thiooxidans

Surfaces of massive chalcopyrite (CuFeS₂) electrodes were modified by applying variable oxidation potential pulses under growth media in order to induce the formation of different secondary phases (e.g., copper-rich polysulfides, S _n ^2?; elemental sulfur, S⁰; and covellite, CuS). The evolution of reactivity (oxidation capacity) of the resulting chalcopyrite surfaces considers a transition from passive or inactive (containing CuS and S _n ^2?) to active (containing increasing amounts of S⁰) phases. Modified surfaces were incubated with cells of sulfur-oxidizing bacteria (Acidithiobacillus thiooxidans) for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the density of cells attached to chalcopyrite surfaces, the structure of the formed biofilm, and their exopolysaccharides and nucleic acids were analyzed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy coupled to dispersive X-ray analysis (SEM-EDS). Additionally, CuS and S _n ^2?/S⁰ speciation, as well as secondary phase evolution, was carried out on biooxidized and abiotic chalcopyrite surfaces using Raman spectroscopy and SEM-EDS. Our results indicate that oxidized chalcopyrite surfaces initially containing inactive S _n ^2? and S _n ^2?/CuS phases were less colonized by A. thiooxidans as compared with surfaces containing active phases (mainly S⁰). Furthermore, it was observed that cells were partially covered by CuS and S⁰ phases during biooxidation, especially at highly oxidized chalcopyrite surfaces, suggesting the innocuous effect of CuS phases during A. thiooxidans performance. These results may contribute to understanding the effect of the concomitant formation of refractory secondary phases (as CuS and inactive S _n ^2?) during the biooxidation of chalcopyrite by sulfur-oxidizing microorganisms in bioleaching systems.