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Enzymatic reduction of hexavalent chromium in bacteria

Abhipsa Sarangi and Chandraraj Krishnan*
Biomass Conversion and Bioremediation Lab
Department of Biotechnology
Bhupat and Jyothi Mehta School of Biosciences Building
Indian Institute of Technology
Chennai-600 036.
*e-mail: kcraj@iitm.ac.in


Hexavalent chromium is mobile, highly toxic, carcinogenic and mutagenic. It is released into the environment from various industrial processes. In contrast trivalent chromium is less toxic and immobile. Hence, Cr(VI) can be detoxified by converting it to Cr(III). Reduction of Cr(VI) can be achieved by chemical or biological means. The microbial bioconversion of Cr(VI) is eco-friendly and is preferred over chemical method. Several bacteria are tolerant to chromate and reduce Cr(VI) using enzymes. Here we describe the bacterial reduction of Cr(VI) and their potential in bioremediation.

Keywords: Hexavalent   chromium,   chromate reductase, bioremediation, heavy metal.

Hexavalent chromium (Cr(VI)) is released to environment from various industrial processes like electroplating, paint, refractories, wood preservation, nuclear weapon manufacturing, etc. Cr(VI) is a toxic, carcinogenic and mutagenic agent and causes irritation to skin, eye and respiratory tract. Acute exposure of Cr(VI) causes contact dermatitis, nasal perforations, rhinitis, kidney damage and abnormalities in liver and reproductive system (Codd et al, 2001). Cr(VI) is highly soluble in aqueous medium at physiological pH and is transported across the cell membrane into the cells by SO2-4 transporters. Since Cr(VI) is highly stable and soluble, it is easily mobilized from the origin of emission to other locations. The solubility of Cr(VI) also leads to its penetration into reservoir of ground water, a source of drinking water, which can cause serious health problems.

In contrast to Cr(VI), trivalent chromium is insoluble which results in localization of Cr(III) and is less toxic. In addition, 0(111) cannot be transported into cells. Therefore, transformation of Cr(VI) to Cr(III) would be a strategy for detoxification of Cr(VI). Conversion of Cr(VI) to Cr(Ifl) can be carried out using chemicals or biocatalysts. Chemical means of Cr(VI) reduction to Cr(IH) at large scale is uneconomical and not eco-friendly since it generates chemical sludge which again causes disposal problem. On the other hand, biocatalysts are environmental friendly. Biotransformation of Cr(VT) has been reported by plants, fungi, algae and bacteria (Ramirez-Diaz et al., 2007). Of those, bacteria are predominantly known and have been studied extensively. However, effective biocatalysts for Cr(VT) detoxification in industry have not been developed so far. There are several issues that need to be addressed for application of biocatalysts in environmental clean up. This can be alleviated by modification of existing biocatalysts by genetic engineering or protein engineering methods, which would lead to improvement of bioremediation potential. Bacteria are potential candidates for development of chromate remediation because of their amenability to modifications. In this short review the mechanism of Cr(VT) reduction in bacteria and their potential in bioremediation is described.
Bacterial reduction of Cr(VI)
Reduction of Cr(VI) has been reported from bacteria belonging to diverse genera such as Enterobacter, Bacillus, Ochrobacterium, Escherichia, Pseudomonas, Arthrobacter, Streptomyces, Providencia, Exiguobaterium and Leucobacter (Cheung and Gu, 2007; Sarangi and Krishnan, 2008). Cr(VT) reduction in bacteria can be chemical or enzymatic. Chemical reduction of Cr(VT) involves reduced compounds like cysteine, glutathione, sulfite and thiosulfates (Donati et al., 2003). The enzymatic reduction of Cr(VI) involves soluble and membrane bound reductases.

Enzymatic Cr(VI) reduction
Several bacteria reduce Cr(VI) through membrane bound reductases such as flavin reductase, cytochromes and hydrogenases. These enzymes can be part of electron transport system and use chromate as the terminal electron acceptor. In Shewanella, Enterobacter   and   sulfate reducing bacteria the terminal electron acceptors like nitrate and sulphate are replaced by chromate (Myers et al., 2000; Chardin et al, 2003). Some of the membrane bound chromate reductases are associated with cellular energy generation (Francis et al., 2000).
Bacterial strains of various genera like Bacillus, Pseudomonas, Streptomyces, Artho-bacter, Ochrobacterium, Burkholderia, Providencia, Exiguobacterium, Leucobacter etc. possess soluble chromate reductase activity in cytosol. The chromate reductases use NAD(P)H as electron donors to reduce Cr(VI). Compared to membrane bound chromate reductases, soluble reductases are suitable for development of biocatalyst for bioremediation since those are more amenable to protein engineering to suit environmental conditions of contaminated sites.

Substrate specificity

Though soluble chromate reductases have been reported from numerous bacteria, only a few of them have been purified and characterized. As seen in Table 1, these reductases are not specific to chromate and possess multiple activities. The substrates range from organic compounds to inorganic metal ions.

Therefore these reductases are also of fundamental interest to understand structure-function relationship. Sequence homology studies indicated that chromate reductase activity is probably not the primary function of these enzymes. The chromate reductase of Pseudomonas putida belongs to putative flavin binding quinone reductases. Similarly, YieF of Escherichia coli reduces Cr(VI) and quinone has a broad substrate specificity, whereas, the chromate reductase of Pseudomonas ambigua reduces nitrate which is absent in chromate reductase of P. putida. Accordingly, the amino acid sequence of chromate reductase of P. putida differ from that of P. ambigua. The chromate reductase of P. ambigua is similar to nitroreductase of E. coli which can reduce Cr(VI) (Ackerley et al., 2004a). Recently a chromate reductase isolated from Thermus scotoductus showed similarity to old yellow enzyme that reduces xenobiotics (Opperman et al., 2008). In addition, sugar catabolic enzymes like dihydrolipoyl dehydrogenase (DLD) a component of pyruvate dehydrogenase complex reduces chromate. Therefore, it appears that chromium reductase activity is actually secondary function with different primary catalytic functions.

Fungi are the important siderophore producing microorganisms next to bacteria. Some important siderophore producing fungi includes Aspergillus nidulans, A. versicolor, Penicillium chrysogenum, P. citrinum, Mucor, Rhizopus, Trametes versicolor. Ustilago sphaerogina, Saccharomyces cerivisiae, Rhodotorula minuta and Debaromyces species.

Table 1. Properties of purified bacterial chromate reducing enzymes


electron donor

Mol. Wt (KDa)and subunits

P. ambigua Gl Chr Chromate, Nitrocompounds FMN NAD(P) 65  Dimer Suzukis al, 1992
P. Putida Chr Quinones, chromate,2,6 Dichloroindophenol, Potassium ferricyanide FMN NAD(P)H 50   Dimer Parked al, 2000
E. Coli Chr Chromate NAD(P)H 84   Dimer Baeetal., 2005
E. Coli YieF Chromate, quinone, 2,6 -Dichloroindo Phenol, Potassium Ferricyanide, V (V) , Mo(VI) FMN NAD(P)H 50   Dimer Ackerley et al., 2004a
E. Coli NfsA Chromate,Nitrocompounds FMN NADH 50   Dimer Ackerley et al., 2004b
T. Scotoductus Chr Chromate FMN NAD(P)H 72   Dimer Opperman et al., 2008
R. sphaeroides Chr Chromate NADH 42   Monomer Nepple et al., 2000
V. harveyi NfsA Nitrofurazone, Trinitrotoluene Chromate FMN NADH 50  Monomer
Kwak et al., 2003
Oxidative stress

Enzymatic reduction of Cr(VI) to Cr(III) involves transfer of electrons from NAD(P)H. During Cr(VI) reduction the bacterial cells are subjected to oxidative stress due to simultaneous generation of reactive oxygen species (ROS) (Cheung et al., 2007). The oxidative stress affects viability of cells and the efficiency of Cr(VI) reduction. The magnitude of oxidative stress depends on the mode of Cr(VI) reduction by the reductases. Two reaction mechanisms have been proposed. Type I "tight" mechanism involves one step transfer of three electrons from dimeric flavoenzymes to Cr(VT) with one electron transfer to oxygen resulting in ROS. This was demonstrated by absence of Cr(V) formation during reaction as well as minimal ROS generation during chromium reduction (Ackerley et al., 2004b). The tight mechanism is exhibited by YieF from E. coli. In Type II "semi tight" mechanism Cr(VI) is reduced in two steps; first step involves one electron transfer forming Cr(V) with another electron donated to oxygen forming ROS and second step involves two electrons transfer to Cr(V) to form Cr(III). The Cr(V) species thus formed is oxidized back to Cr(VT) by transfer of an electron to oxygen resulting in ROS. Thus, Cr(V) enters oxidative cycle and generates ROS continuously. The chromate reductases with two step mechanism lead to higher oxidative stress. The semi tight mechanism is observed in chromate reductase of P. putida and nitroreductase of £. coli. Similar mechanism has been proposed for chromate reductase of P. ambigua (Suzuki et al., 1992). Among the chromate reductases those forming less ROS are suggested for bioremediation since the viability of detoxifying cells are not affected. The quinone reductase activity of chromate reductase improves tolerance to peroxide. When the chromate reductase was overexpressed in remediating cells, it not only increased the rate of Cr(VI) reduction but improved cell viability by minimizing oxidative stress. The results indicate the possibility of employing chromate reductases of this kind for development of bacteria for bioremediation. The presence of other co-pollutant metal ions in contaminated sites would affect the bioremediation potential of chromate reductases. Engineering chromate redactase to improve tolerance to other metal ions is inevitable. Barak et al. (2006) have shown that mutation of chromate reductase by directed evolution markedly improved the capacity of reduction of two toxic metal ions Cr(VT) and U(VI).

Bacteria reducing Cr(VI) through soluble reductase enzymes are potential candidates for bioremediation. Cellular Cr(VI) reduction by enzymes is found to be associated with formation of Cr(V) intermediate which redox cycles to generate oxidative stress (Codd et al., 2001). Chromate reductase activity of enzymes appears to be secondary function with different primary function. The multiple function of chromate reducing enzymes would be advantageous to cells for effective bioremediation. Chromate reductase with quinone reductase activity has been demonstrated to detoxify chromate with improvement of cellular tolerance to oxidative stress (Gonzalez et al., 2005). Protein engineering resulted in remediation of co-pollutants such as uranyl. Therefore the soluble chromate reducing enzymes would be promising for development of biocatalysts for chromate bioremediation.

We acknowledge the financial support and facility granted by Indian Institute of Technology (IIT), Madras.


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Ackerley, D. F, Gonzalez, C. F, Keyhan, M., Blake, R. and Matin A. (2004b) Mechanism of chromate reduction by the Escherichia coli protein, NfsA, and the role of different chromate reductases in minimizing oxidative stress during chromate reduction. Environ. Microbiol. 6,851-860.

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Kwak, Y. H., Lee, D. S. and Kim, H. B. (2003) Vibrio harveyi nitroreductase is also a chromate reductase. Appl. Environ. Microbiol. 69, 4390-395.

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