Hexavalent chromate reductase activity was localized and characterized in vitro in cytosolic fraction of a newly isolated Pseudomonas sp. G1DM21. The suspended culture of the bacterium reduced 99.7% of 500 mu M Cr(VI) and 93.06% of 1000 mu M Cr(VI) in 48 h. The suspended culture repeatedly reduced 100 mu M Cr(VI) within 6 h up to four consecutive inputs. The permeabilized cells of the bacterium reduced 92% of 100 mu M Cr(VI) within 6 h. The cell-free extracts (CFE) reduced 90% of 100 mu M Cr(VI) in 120 min. The K(m) and V(max) determined for chromate reductase activity in the CFE were 175 mu M Cr(VI) and 1.6 mu moles/min/ mg of protein, respectively, the K(m) and V(max) determined in the presence of 0.5 mM NADH were 150 mu M Cr(VI) and 2.0 mu moles/min/mg of protein, respectively. Hexavalent chromate reductase activity was maximum at 30 degrees C and pH 7.0. Relative molecular mass (M(r)) of the native Cr(VI) reductase in the cytosolic fraction was estimated as 61.7 kDa. The Cr(VI) reductase activity increased in the presence of metal ions like Cu(2+), Mg(2+), Na(+) and electron donors like citrate, succinate, acetate and was significantly inhibited in the presence of metal ions like Hg(2+), Ag(+), Cd(2+), disulfide reducers like 2-mercaptoethanol, while the respiratory inhibitors had a minute effect on the activity. Scanning probe atomic force microscopy (AFM) analysis indicates that exposure of Pseudomonas sp. G1DM21 to 1 mM Cr(VI) for 24 h, leads to an increase in cell length and height. (C) 2008 Elsevier Ltd. All rights reserved.