Purification and characterization of (per)chlorate reductase from the chlorate-respiring strain GR-1

Abstract

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.

FIG. 1

SDS-PAGE of the (per)chlorate reductase from strain GR-1. Lanes 1 and 8, set of marker proteins with their molecular masses indicated; lane 2, soluble fraction (115 μg of protein); lane 3, S-Sepharose pool (10.5 μg of protein); lanes 4 and 5, hydroxyapatite pool (9.6 μg of protein); lanes 6 and 7, Superdex 200 pool (3.6 μg of protein). Proteins were stained with Coomassie brilliant blue R250.

FIG. 2

S = 1/2 EPR spectra of [3Fe-4S] and [4Fe-4S] clusters in chlorate reductase. The enzyme concentration was 5.3 mg/ml (39 μM αβ dimer) in 50 mM potassium phosphate buffer (pH 7.0)–10% (vol/vol) glycerol. The upper trace was obtained after anaerobic incubation with 0.4 mM potassium ferricyanide for 4 min at ambient temperature. Similarly, the lower trace was obtained after 5 min of incubation with 5 mM sodium dithionite. EPR conditions: microwave frequency, 9,414 MHz; microwave power, 5 mW; modulation amplitude, 0.5 mT; temperature, 14 K. OX, oxidized; RED, reduced; X", the imaginary part of the magnetic susceptibility; B, magnetic field.

FIG. 3

Low-field S = 3/2 signals from [4Fe-4S] in dithionite-reduced chlorate reductase. The sample was the same as that for Fig. 2, trace RED. The EPR conditions were also the same, except for a higher electronic gain and a microwave power of 126 mW. X" and B are as defined in the legend to Fig. 2.

FIG. 4

Putative Mo(V) EPR signals from chlorate reductase as isolated (6 mg/ml). The spectrum was simulated as a sum of two signals. Parameters for one signal: g = 2.091, 2.016, and 2.016; isotropic line width, 1.5 mT; parameters for the other signal: g = 1.999, 1.976, and 1.950; molybdenum hyperfine splitting, 0.65, 0.5, and 0.83 mT; line width, 0.25, 0.2, and 0.3 mT. EPR conditions: microwave frequency, 9,396 MHz; microwave power, 0.5 mW; modulation amplitude, 0.32 mT; temperature, 40 K. EXP, experimental; SIM, simulated. X" and B are as defined in the legend to Fig. 2.