Purification and characterization of cytosolic mercuric reductase from Klebsiella pneumoniae

Mercuric reductase, a cytosolic flavoprotein which catalyzes the NADPHdependent reduction of mercuric ion to metallic mercury, has been purified about 82-fold from Klebsiella pneumoniae cells and kinetic properties were investigated. The purification method consisted of rapid (two-step) and straightforward procedure involving dye matrix affinity chromatography. The freshly native protein exhibited a molecular weight of approximately 190 kDa. The enzyme is an homotrimeric protein composed of identical subunits of 62 kDa. The optimum pH of purified enzyme was 7.5. Studies on the effect of temperature on enzyme activity revealed an optimal value of about 40 °C. The apparent Km values for HgCl2 was estimated to be 75 μM and the value obtained for Vmax was 9 U/mg. The isoelectric point of the enzyme was found to be 5. The presence of oxidationreduction active cysteine residues at the active site was confirmed by appearance of two additional thiols upon reduction with NADPH.