Purification and properties of mercuric reductase from Azotobacter chroococcum

Mercury resistance determinants in bacteria are often plasmid‐borne or transposon‐mediated. Mercuric reductase, one of the proteins encoded by the mercury resistance operon, catalyses a unique reaction in which mercuric ions, Hg (II), are reduced to mercury metal Hg(O) using NADPH as a source of reducing power. Mercuric reductase was purified from Azotobacter chroococcum SS 2 using Red A dye matrix affinity chromatography. Freshly purified preparations of the enzyme showed a single band on polyacrylamide gel electrophoresis under non‐denaturing conditions. After SDS‐polyacrylamide gel electrophoresis of the freshly prepared enzyme, two protein bands, a major and a minor one, were observed with molecular weight 69 000 and 54 000, respectively. The molecular weight of the native enzyme as determined by gel filtration in Sephacryl S‐300 was 142 000. The Km of Hg 2+ ‐reductase for HgCl 2 was 11·11 μmol l −1 . Titration with 5,5′‐dithiobis (2‐nitrobenzoate) demonstrated that two enzyme–SH groups become kinetically accessible on reduction with NADPH.