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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
3
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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