Chromate reduction by Rhodobactor sphaeroides

Rhodobacter sphaeroides grew in the presence of up to 43 muM chromate and reduced hexavalent chromium to the trivalent form under both aerobic and anaerobic conditions. Reduced chromium remained in the external medium. Reductase activity was present in cells of Rb. sphaeroides independent of whether chromate was present or not in the growth medium. The reducing activity was found in the cytoplasmic cell fraction and was dependent on NADH. The chromate-reducing enzyme was purified by anion exchange, hydroxyapatite and hydrophobic interaction chromatography, and gel filtration. The molecular weight of the enzyme was 42 kDa as determined by gel filtration. The optimum of the reaction is at pH 7.0 and 30 degreesC. The enzyme activity showed a hyperbolic dependence on the concentrations of both substrates, NADH and chromate, with a maximum velocity at 0.15 mM NADH. A K-m of 15+/-1.3 muM CrO42- and a V-max of 420+/-50 mu mol min(-1) mg protein(-1) was determined for the enzyme isolated from anaerobically grown cells and 29+/-6.4 muM CrO42- and 100+/-9.6 mu mol CrO42- min(-1) mg protein(-1) for the one from aerobically grown ones.