Chromate Reductase Activity Research Articles

Proteogenomic and functional analysis of chromate reduction in Acidiphilium cryptum JF-5, an Fe(III)-respiring acidophile

Acidiphilium cryptum JF-5, an acidophilic iron-respiring Alphaproteobacterium, has the ability to reduce chromate under aerobic and anaerobic conditions, making it an intriguing and useful model organism for the study of extremophilic bacteria in bioremediation applications. Genome sequence annotation suggested two potential mechanisms of Cr(VI) reduction, namely, a number of c-type cytochromes, and a predicted NADPH-dependent Cr(VI) reductase. In laboratory studies using pure cultures of JF-5, an NADPH-dependent chromate reductase activity was detected primarily in soluble protein fractions, and a periplasmic c-type cytochrome (ApcA) was also present, representing two potential means of Cr(VI) reduction. Upon further examination, it was determined that the NADPH-dependent activity was not specific for Cr(VI), and the predicted proteins were not detected in Cr(VI)-grown cultures. Proteomic data did show measureable amounts of ApcA in cells grown with Cr(VI). Purified ApcA is reducible by menadiol, and in turn can reduce Cr(VI), suggesting a means to obtain electrons from the respiratory chain and divert them to Cr(VI). Electrochemical measurements confirm that Cr reduction by ApcA is pH dependent, with low pH being favored. Homology modeling of ApcA and comparison to a known Cr(VI)-reducing c-type cytochrome structure revealed basic amino acids which could interact with chromate ion. From these studies, it can be concluded that A. cryptum has the physiologic and genomic capability to reduce Cr(VI) to the less toxic Cr(III). However, the expected chromate reductase mechanism may not be the primary means of Cr(VI) reduction in this organism.

Chromate reductase activity in Streptomyces sp. MC1

Biological transformation of Cr(VI) to Cr(III) by enzymatic reduction may provide a less costly and more environmentally friendly approach to remediation. In a previous report a Cr(VI) resistant actinomycete strain, Streptomyces sp. MC1, was able to reduce Cr(VI) present in a synthetic medium, soil extract and soil samples. This is the first time optimal conditions such as pH, temperature, growth phase and electron donor have been elucidated in vitro for Cr(VI) reduction by a streptomycete. Chromate reductase of Streptomyces sp. MC1 is a constitutive enzyme which was mainly associated with biomass and required NAD(P)H as an electron donor. It was active over a broad temperature (19-39 degrees C) and pH (5-8) range, and optimum conditions were 30 degrees C and pH 7. The enzyme was present in supernatant, pellet and cell free extract. Bioremediation with the enzyme was observed in non-compatible cell reproduction systems, conditions frequently found in contaminated environments.

A putative azoreductase gene is involved in the Shewanella oneidensis response to heavy metal stress

The Shewanella oneidensis MR-1 gene SO3585, which is annotated as a putative flavin mononucleotide-dependent azoreductase, shares 28% sequence identity with Bacillus subtilis azoreductase and Pseudomonas putida ChrR, a soluble flavoprotein exhibiting chromate reductase activity. Reverse transcription polymerase chain reaction demonstrated that the SO3585 gene is co-transcribed with two downstream open reading frames: SO3586 (a glyoxalase family protein) and SO3587 (a predicted membrane-associated hypothetical protein). The transcriptional start site of the so3585 transcript was localized using 5' rapid amplification of complementary DNA ends analysis. To investigate the cellular function of SO3585, an in-frame deletion of the so3585 locus was generated in MR-1, and the phenotype of the resulting mutant was characterized. The so3585 deletion mutant was comparable to the parental strain in its ability to decolorize two sulfonated azo dyes (Orange II, Direct Blue 15) under aerobic conditions. By contrast, growth of the so3585 deletion mutant was sensitive to different exogenous transition heavy metals [Cr(VI), Cd(II), Cu(II), and Zn(II)], while the most severe growth deficiencies were observed in the presence of Cd(II) and Cu(II). In addition, the rate of extracellular chromate disappearance by the deletion strain was initially impaired, although both the so3585 mutant and MR-1 wild type reduced Cr(VI) within the same time period.

Biosorption of hexavalent chromium by Termitomyces clypeatus biomass: Kinetics and transmission electron microscopic study

Biosorption of Cr +6 by Termitomyces clypeatus has been investigated involving kinetics, transmission electron microscopy (TEM) and Fourier transform infrared spectroscopic (FTIR) studies. Kinetics experiments reveal that the uptake of chromium by live cell involves initial rapid surface binding followed by relatively slow intracellular accumulation. Of the different chromate analogues tested, only sulfate ion reduces the uptake of chromium to the extent of ∼30% indicating chromate ions accumulation into the cytoplasm using sulfate transport system. Metabolic inhibitors, e.g. N,N′-dicyclohexylcarbodiimide, 2,4-ditrophenol and sodium azide inhibit chromate accumulation by ∼30% in live cell. This indicates that accumulation of chromium into the cytoplasm occurs through the active transport system. TEM-EDXA analysis reveals that the chromium localizes in the cell wall and also in the cytoplasm. Reduction of chromate ions takes place by chromate reductase activity of cell-free extracts of T. clypeatus. FTIR study indicates that chromate ions accumulate into the cytoplasm and then reduced to less toxic Cr +3 compounds.

Chromium-induced in vivo DNA changes in marine algae Oscillatoria willei BDU 130511 (Cyanophyta)

This work has been devised to resolve and examine the fate of chromosomal DNA of marine cyanobacterium (Cyanophyta) Oscillatoria willei BDU 130511 upon chromium exposure. The organism exhibited ability to grow at 1 mM hexavalent chromium. Decreased intensity at base stacking and 3 nm red-shift in the helicity band of circular dichroism (CD) spectrum coupled with decreased DNA viscosity; which demonstrated the change in structure of DNA through possible electrostatic interaction of Cr (III) ions as an adaptive mechanism. Increased chromate reductase activity provided evidence for the conversion of toxic Cr (VI) to less toxic Cr (III).

Hexavalent chromate reductase activity in cytosolic fractions of Pseudomonas sp. G1DM21 isolated from Cr(VI) contaminated industrial landfill

Hexavalent chromate reductase activity was localized and characterized in vitro in cytosolic fraction of a newly isolated Pseudomonas sp. G1DM21. The suspended culture of the bacterium reduced 99.7% of 500 μM Cr(VI) and 93.06% of 1000 μM Cr(VI) in 48 h. The suspended culture repeatedly reduced 100 μM Cr(VI) within 6 h up to four consecutive inputs. The permeabilized cells of the bacterium reduced 92% of 100 μM Cr(VI) within 6 h. The cell-free extracts (CFE) reduced 90% of 100 μM Cr(VI) in 120 min. The K m and V max determined for chromate reductase activity in the CFE were 175 μM Cr(VI) and 1.6 μmoles/min/mg of protein, respectively, the K m and V max determined in the presence of 0.5 mM NADH were 150 μM Cr(VI) and 2.0 μmoles/min/mg of protein, respectively. Hexavalent chromate reductase activity was maximum at 30 °C and pH 7.0. Relative molecular mass ( M r) of the native Cr(VI) reductase in the cytosolic fraction was estimated as 61.7 kDa. The Cr(VI) reductase activity increased in the presence of metal ions like Cu 2+, Mg 2+, Na + and electron donors like citrate, succinate, acetate and was significantly inhibited in the presence of metal ions like Hg 2+, Ag +, Cd 2+, disulfide reducers like 2-mercaptoethanol, while the respiratory inhibitors had a minute effect on the activity. Scanning probe atomic force microscopy (AFM) analysis indicates that exposure of Pseudomonas sp. G1DM21 to 1 mM Cr(VI) for 24 h, leads to an increase in cell length and height.

Evaluation of In vitro Cr(VI) reduction potential in cytosolic extracts of three indigenous Bacillus sp. isolated from Cr(VI) polluted industrial landfill

Three efficient Cr(VI) reducing bacterial strains were isolated from Cr(VI) polluted landfill and characterized for in vitro Cr(VI) reduction. Phylogenetic analysis using 16S rRNA gene sequencing revealed that the newly isolated strains G1DM20, G1DM22 and G1DM64 were closely related to Bacillus cereus, Bacillus fusiformis and Bacillus sphaericus, respectively. The suspended cultures of all Bacillus sp. exhibited more than 85% reduction of 1000 μM Cr(VI) within 30 h. The suspended culture of Bacillus sp. G1DM22 exhibited an ability for continuous reduction of 100 μM Cr(VI) up to seven consecutive inputs. Assays with the permeabilized cells and cell-free extracts from each of Bacillus sp. demonstrated that the hexavalent chromate reductase activity was mainly associated with the soluble fraction of cells and expressed constitutively. The Cr(VI) reduction by the cell-free extracts of Bacillus sp. G1DM20 and G1DM22 was maximum at 30 °C and pH 7 whereas, Bacillus sp. G1DM64 exhibited maximum Cr(VI) reduction at pH 6. Addition of 1 mM NADH enhanced the Cr(VI) reductase activity in the cell-free extracts of all three isolates. Amongst all three isolates tested, crude cell-free extracts of Bacillus sp. G1DM22 exhibited the fastest Cr(VI) reduction rate with complete reduction of 100 μM Cr(VI) within 100 min. The apparent K m and V max of the chromate reductase activity in Bacillus sp. G1DM22 were determined to be 200 μM Cr(VI) and 5.5 μmol/min/mg protein, respectively. The Cr(VI) reductase activity in cell-free extracts of all the isolates was stable in presence of different metal ions tested except Hg 2+ and Ag +.

Prooxidant Cytotoxicity of Chromate in Mammalian Cells: The Opposite Roles of DT-Diaphorase and Glutathione Reductase

The geno- and cytotoxicity of chromate, an important environmental pollutant, is partly attributed to the flavoenzyme-catalyzed reduction with the concomitant formation of reactive oxygen species. The aim of this work was to characterize the role of NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2) and glutathione reductase (GR, EC 1.6.4.2) in the mammalian cell cytotoxicity of chromate, which was evidenced controversially so far. The chromate reductase activity of NQO1 was higher than that of GR, but lower than that of lipoamide dehydrogenase (EC 1.6.4.3), ferredoxin:NADP+ reductase (EC 1.18.1.2), and NADPH: cytochrome P-450 reductase (EC 1.6.2.4). The reduction of chromate by NQO1 was accompanied by the formation of reactive oxygen species. The concentration of chromate for 50% survival of bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) during a 24-h incubation was (22 +/- 4) microM. The cytotoxicity was partly prevented by desferrioxamine, the antioxidant N,N'-diphenyl-p-phenylene diamine and by an inhibitor of NQO1, dicumarol, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), which inactivates GR. The NADPH-dependent chromate reduction by digitonin-permeabilized FLK cells was partly inhibited by dicumarol and not affected by BCNU. Taken together, these data indicate that the oxidative stress-type cytotoxicity of chromate in FLK cells may be partly attributed to its reduction by NQO1, but not by GR. The effect of BCNU on the chromate cytotoxicity may indicate that the general antioxidant action of reduced glutathione is more important than its prooxidant activities arising from the reactions with chromate.

Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry.

A Cr(VI)-resistant yeast was isolated from tanning liquors from a leather factory in Leon, Guanajuato, Mexico. Based on morphological and physiological analyses and the D1/D2 domain sequence of the 26S rDNA, the yeast was identified as Candida maltosa. Resistance of the strain to high Cr(VI) concentrations and its ability to chemically reduce chromium was studied. When compared to the three laboratory yeasts Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica, the C. maltosa strain was found to tolerate chromate concentrations as high as 100 micro g/ml. In addition to this phenotypic trait, the C. maltosa strain showed ability to reduce Cr(VI). Chromate reduction occurred both in intact cells (grown in culture medium or in soil containing chromate) as well as in cell-free extracts. NADH-dependent chromate reductase activity was found associated with soluble protein and, to a lesser extent, with the membrane fraction.

Amperometric cytochrome c 3-based biosensor for chromate determination

The chromate reductase activity of cytochrome c 3 (Cyt c 3, M r 13 000), isolated from the sulfate-reducing bacterium Desulfomicrobium norvegicum, was used to develop an amperometric biosensor to measure chromate (CrO 4 2−) bioavailability. The performance of various biosensor configurations for qualitative and quantitative determination of Cr(VI) was studied. Biosensor properties depend on the technique used to immobilize the enzyme on the electrode (glassy carbon electrode). Immobilization of Cyt c 3 by entrapment in poly 3,4-ethylenedioxythiophene films denatured the enzyme, while application of an adsorption technique did not affect enzyme activity but the detection range was limited. The best results were obtained with dialysis membranes, which allowed the determination of Cr(VI) from 0.20 to 6.84 mg l −1 (3.85–132 μM) with a sensitivity of 35 nA mg −1 l (1.82 nA μM −1). No interference was observed with As(V), As(III) and Fe(III). Only a small amount of Cyt c 3 (372 ng of protein) was needed for this biosensor.

Chromate reductase activity ofEnterobacter aerogenesis induced by nitrite

A chromate resistant mutant of Enterobacter aerogenes manifested its chromate resistance only under aerobic conditions. Both parent and mutant showed substantial levels of anaerobic chromate reductase activity when grown on glycerol plus fumarate. The chromate reductase was further induced by growth in the presence of nitrite but was repressed by nitrate. The chromate reductase activity paralleled that of the formate-linked nitrite reductase. There was no significant difference in chromate reductase levels between the parent and its chromate resistant mutant, indicating that this enzyme activity is not, in fact responsible for chromate resistance as was suggested previously by others.

Chromate reductase activity of Enterobacter aerogenes is induced by nitrite

Chromate reductase activity of Enterobacter aerogenes is induced by nitrite

Chromium reduction in Pseudomonas putida

Reduction of hexavalent chromium (chromate) to less-toxic trivalent chromium was studied by using cell suspensions and cell-free supernatant fluids from Pseudomonas putida PRS2000. Chromate reductase activity was associated with soluble protein and not with the membrane fraction. The crude enzyme activity was heat labile and showed a Km of 40 microM CrO4(2-). Neither sulfate nor nitrate affected chromate reduction either in vitro or with intact cells.

Chromate reductase activity: characterization of Cr(VI) to Cr(III) conversion

In order to understand chromate reductase activity (CRA), related to cellular carcinogenecity and metal toxicity in ecological systems, we began studying the reduction of chromium(VI) salts using cysteine, ascorbic acid and several monosaccharides mainly through EPR and absorption spectroscopic methods. The present study indicates Cr(V) as a definite intermediate and also shows the relative trends of reducing capabilities among various reducing agents employed. Considerable progress has been achieved in the isolation of intermediates and final products as well.

Membrane-associated chromate reductase activity from Enterobacter cloacae

Washed cells of Enterobacter cloacae HO1 reduced hexavalent chromium (chromate: CrO4(2-) anaerobically. Chromate reductase activity was preferentially associated with the membrane fraction of the cells. Right-side-out membrane vesicles prepared from E. cloacae cells showed high chromate reductase activities when ascorbate-reduced phenazine methosulfate was added as an electron donor.

Chromium(V) is produced upon reduction of chromate by mitochondrial electron transport chain complexes

Incubation of chromate with isolated rat liver submitochondrial particles under anaerobic conditions in vitro results in reduction of chromium(VI) and formation of chromium(V). In the presence of NADH, submitochondrial particles (SMPs) were active in reducing chromate as shown by UV-vis spectroscopic studies, and forming a chromium(V) species which was detectable by electron paramagnetic resonance spectroscopy. In the presence of succinate, SMPs were less effective in reducing chromate and forming chromium(V) relative to their NADH-dependent activity. However, SMPs showed a higher rate of oxygen depletion with NADH as compared to succinate as substrate, suggesting that differences in the NADH-dependent versus succinate-dependent chromate-reductase activity of SMPs is probably due to differences in efficiency of electron donation by succinate and NADH. The use of specific electron transport chain inhibitors allowed the sites of chromium(VI) reduction and chromium(V) formation in SMPs to be determined. Rotenone, antimycin and cyanide all produced approximately 40% inhibition of the NADH-dependent chromate-reductase activity. Thus, complex I (NADH:ubiquinone oxidoreductase) appears to be responsible for the inhibitor-insensitive, and complex IV (ferrocytochrome c:oxygen oxidoreductase) for the inhibitor-sensitive NADH-dependent chromium(VI) reduction and chromium(V) formation. Cyanide and antimycin produced approximately 50% inhibition of the succinate-dependent chromate-reductase activity of SMPs, while no detectable inhibition was observed with rotenone. These results confirm the chromate-reductase activity of complex IV, and suggest that complex II (succinate:ubiquinone oxidoreductase) is responsible for the inhibitor-insensitive succinate-dependent chromate-reductase activity of SMPs. Since chromium(VI) is effectively metabolized by electron transport chain complexes of the mitochondrial inner membrane in vitro, and chromium(V) is formed as an intermediate in the process, mitochondria may play a role in chromium(VI) carcinogenesis.

Chromate resistance and reduction in Pseudomonas fluorescens strain LB300

Pseudomonas fluorescens LB300 is a chromateresistant strain isolated from chromium-contaminated river sediment. Chromate resistance is conferred by the plasmid pLHB1. Strain LB300 grew in minimal salts medium with as much as 1000 μg of K2CrO4 ml−1, and actively reduced chromate to Cr(III) while growing aerobically on a variety of substrates. Chromate was also reduced during anaerobic growth on acetate, the chromate serving as terminal electron acceptor. P. fluorescens LB303, a plasmidless, chromatesensitive variant of P. fluorescens LB300, did not grow in minimal salts medium with more than 10 μg of K2CrO4 ml−1. However, resting cells of strain LB303 grown without chromate reduced chromate as well as strain LB300 cells grown under the same conditions. Furthermore, resting cells of chromate-sensitive Pseudomonas putida strain AC10, also catalyzed chromate reduction. Evidently chromate resistance and chromate reduction in these organisms are unrelated. Comparison of the rates of chromate reduction by chromate grown cells and cells grown without chromate indicated that the chromate reductase activity is constitutive. Studies with cell-free extracts show that the reductase is membrane-associated and can mediate the transfer of electrons from NADH to chromate.

Mitochondrial reduction of the carcinogen chromate: formation of chromium(V).

Incubation of chromate with isolated rat liver mitochondria in vitro resulted in the uptake and reduction of chromium(VI), as well as the formation of chromium(V) species. Chromate was rapidly taken up and reduced by intact mitochondria. The rate of reduction of chromate by intact mitochondria was increased upon addition of succinate or malate plus glutamate, substrates for the electron-transport chain, but was decreased upon addition of cyanide, an inhibitor of the electron-transport chain. Incubation of chromate with mitochondria in the presence or absence of malate, glutamate, and succinate resulted in a steady increase in the level of chromium(V) over time. The extent of chromium(V) formation was increased upon addition of malate, glutamate, and succinate but was inhibited upon addition of the electron-transport chain inhibitors, antimycin, cyanide, or rotenone, to whole mitochondria. High levels of glutamate plus malate inhibited chromium(V) formation; however, high concentrations of succinate or sulfate had no effect. These studies suggest that the chromate-reductase activity in mitochondria is due to the electron-transport chain as well as other mitochondrial reducing systems which are insensitive to inhibitors of the electron-transport chain. Since chromium(VI) is effectively metabolized by mitochondria in vitro and chromium(V) "reactive intermediates" are formed in the process, mitochondria may play a role in chromium(VI) carcinogenesis.