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Journal of Bacteriology, November 1999, p. 6706-6711, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Purification and Characterization of (Per)Chlorate Reductase from the Chlorate-Respiring Strain GR-1

Servé W. M. Kengen,1,* Geoffrey B. Rikken,1 Wilfred R. Hagen,2 Cees G. van Ginkel,3 and Alfons J. M. Stams1

Laboratory of Microbiology, Department of Biomolecular Sciences, Wageningen Agricultural University, NL-6703 CT Wageningen,1 Laboratory of Biochemistry, Department of Biomolecular Sciences, Wageningen Agricultural University, NL-6703 HA Wageningen,2 and Analytical and Environmental Chemistry Department, Akzo-Nobel Central Research, NL-6800 SB Arnhem,3 The Netherlands

Received 6 April 1999/Accepted 18 August 1999

Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an alpha 3beta 3 composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The Km values for perchlorate and chlorate were 27 and <5 µM, respectively. Besides perchlorate and chlorate, nitrate, iodate, and bromate were also reduced at considerable rates. The resemblance of the enzyme to nitrate reductases, formate dehydrogenases, and selenate reductase is discussed.

* Corresponding author. Mailing address: Laboratory of Microbiology, Department of Biomolecular Sciences, Wageningen Agricultural University, Hesselink van Suchtelenweg 4, NL-6703 CT Wageningen, The Netherlands. Phone: 31-317-483748. Fax: 31-317-483829. E-mail: serve.kengen{at}algemeen.micr.wau.nl.

Journal of Bacteriology, November 1999, p. 6706-6711, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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