Purification and characterization of (per)chlorate reductase from the chlorate-respiring strain GR-1.
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(3)beta(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 K(m) values for perchlorate and chlorate were 27 and <5 microM, 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.
Kengen
S W
SW
Laboratory of Microbiology, Department of Biomolecular Sciences, Wageningen Agricultural University, NL-6703 CT Wageningen, The Netherlands. serve.kengen@algemeen.micr.wau.nl
Rikken
G B
GB
Hagen
W R
WR
van Ginkel
C G
CG
Stams
A J
AJ
D000595:Amino Acid Sequence; D002384:Catalysis; D002704:Chlorates / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000254:growth & development; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010472:Perchlorates / Q000378:metabolism*; D017670:Sodium Compounds / Q000378:metabolism*; D013057:Spectrum Analysis
D002704:Chlorates; D010472:Perchlorates; D017670:Sodium Compounds; C031068:sodium perchlorate; D010088:Oxidoreductases; C021777:chlorate reductase