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 a cceptor. The organism performs a complete reduction of chlorate or perchlor ate to chloride and oxygen, with the intermediate formation of chlorite, Th is 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 ac tivity. 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 a lpha(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, I n accordance, quantitative electron paramagnetic resonance spectroscopy sho wed the presence of one [3Fe-4S] cluster and two [4Pe-4S] clusters. Further more, two different signals were ascribed to Mo(V). The K-m values for perc hlorate and chlorate were 27 and <5 mu M, respectively, Besides perchlorate and chlorate, nitrate, iodate, and bromate were also reduced at considerab le rates. The resemblance of the enzyme to nitrate reductases, formate dehy drogenases, and selenate reductase is discussed.