A bacterial flavin reductase system reduces chromate to a soluble chromium(III)–NAD+ complex

doi:10.1016/S0006-291X(02)00438-2

Biochemical and Biophysical Research Communications

Volume 294, Issue 1, 31 May 2002, Pages 76-81

https://doi.org/10.1016/S0006-291X(02)00438-2 Get rights and content

Abstract

Biological reduction of carcinogenic chromate has been extensively studied in eukaryotic cells partly because the reduction produces stable chromium(III)-DNA adducts, which are mutagenic. Microbial reduction of chromate has been studied for bioremediation purposes, but little is known about the reduction mechanism. In eukaryotic cells chromate is mainly reduced non-enzymatically by ascorbate, which is usually absent in bacterial cells. We have characterized the reduction of chromate by a flavin reductase (Fre) from Escherichia coli with flavins. The Fre-flavin system rapidly reduced chromate, whereas chemical reduction by NADH and glutathione was very slow. Thus, enzymatic chromate reduction is likely the dominant mechanism in bacterial cells. Furthermore, the end-product was a soluble and stable Cr(III)–NAD⁺ complex, instead of Cr(III) precipitate. Since intracellularly generated Cr(III) forms adducts with DNA, protein, glutathione, and ascorbate in eukaryotic cells, we suggest that the produced Cr(III) is primarily complexed to NAD⁺, DNA, and other cellular components inside bacteria.

Section snippets

Materials and methods

Chromate reduction by the Fre system. The production and purification of Fre were done as previously reported [26]. One unit of Fre was defined as the amount of Fre required to catalyze the consumption of 1 nmol of NADH per min with $10 μM$ FMN. The specific activity of the purified Fre was 49,600 $U mg^{−1}$ of protein. Chromate reduction was assayed under both aerobic and anaerobic conditions. Reaction mixtures contained 38 U Fre, $400 μM$ NADH, and $250 μM$ chromate in 1 ml of 40 mM KPi buffer (pH 7.0) with $10 μM$

Reduction of chromate by a Fre–flavin system

The ability of Fre, an E. coli general flavin reductase [24], to reduce chromate was tested. The recombinant Fre overproduced in E. coli was purified as previously reported [26]. Fre, together with riboflavin, FMN, or FAD rapidly reduced chromate under both aerobic and anaerobic conditions (Fig. 1). Chromate reduction was faster under anaerobic conditions than under aerobic conditions. Chromate reduction by NADH without the enzyme or without flavins was not apparent (Fig. 1). When NADH was

Discussion

Chromate reduction in eukaryotic cells is primarily through non-enzymatic reduction by ascorbate [13]. Since bacterial cells normally do not contain ascorbate, glutathione becomes the major reductant [12]. However, chromate reduction by glutathione is much slower than by the Fre-flavin system (Fig. 1) [21], suggesting that chromate reduction is primarily enzymatic inside cells. Fre, a general flavin reductase in E. coli, does not contain bound flavins [24]. As shown here, the Fre system is

Acknowledgements

This research was funded by the Natural and Accelerated Bioremediation Research (NABIR) program, Biological and Environmental Research (BER), US Department of Energy (Grant #DEFG0398ER62693). GJP was partly supported by the National Science Foundation (NSF) Integrative Graduate Education and Research Training (IGERT) program at Washington State University (Grant DGE-9972817). D.M.K. and A.G.R. were supported by a Herman Frasch Foundation Award. We thank Tai-Man Louie, Judah Friese, and Dr. S.B.

References (35)

C. Vasant et al.
Biochem. Biophys. Res. Commun.
(2001)
A. Flores et al.
Toxicol. Appl. Pharmacol.
(1999)
S.N. Mattagajasingh et al.
J. Biol. Chem.
(1996)
B.M. Tebo et al.
FEMS Microbiol. Lett.
(1998)
B.R. Bochner et al.
J. Biol. Chem.
(1982)
S. Langard
Am. J. Ind. Med.
(1990)
World Health Organization, in: Guidelines for Drinking-water Quality, Geneva, 1996, pp....
J. Dragun
K.D. Sugden et al.
J. Enviorn. Pathol. Toxicol. Oncol.
(2000)
D.M. Stearns et al.
Biochemistry
(1995)

V. Voitkun et al.

Nucleic Acids Res.

(1998)

J. Xu et al.

Carcinogenesis

(1996)

T. Norseth

Br. J. Ind. Med.

(1986)

P.H. Connett et al.

Struct. Bonding

(1983)

A.M. Standeven et al.

Carcinogenesis

(1992)

B.R. James et al.

J. Soil Contam.

(1997)

H. Shen et al.

Appl. Environ. Microbiol.

(1993)

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A bacterial flavin reductase system reduces chromate to a soluble chromium(III)–NAD+ complex

Abstract

Section snippets

Materials and methods

Reduction of chromate by a Fre–flavin system

Discussion

Acknowledgements

Biochem. Biophys. Res. Commun.

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

FEMS Microbiol. Lett.

J. Biol. Chem.

Am. J. Ind. Med.

J. Enviorn. Pathol. Toxicol. Oncol.

Biochemistry

Nucleic Acids Res.

Carcinogenesis

Br. J. Ind. Med.

Struct. Bonding

Carcinogenesis

J. Soil Contam.

Appl. Environ. Microbiol.

A bacterial flavin reductase system reduces chromate to a soluble chromium(III)–NAD⁺ complex