Purification and properties of mercuric reductase from Azotobacter chroococcum

Mercury resistance determinants in bacteria are often plasmid-borne or tran sposon-mediated. Mercuric reductase, one of the proteins encoded by the mer cury 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 red ucing power. Mercuric reductase was purified from Azotobacter chroococcum S S2 using Red A dye matrix affinity chromatography. Freshly purified prepara tions of the enzyme showed a single band on polyacrylamide gel electrophore sis under non-denaturing conditions. After SDS-polyacrylamide gel electroph oresis of the freshly prepared enzyme, two protein bands, a major and a min or 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 Hg2+-reductase for HgCl2 was 11. 11 mu mol l(-1) Titration with 5,5'-dithiobis (2-nitrobenzoate) demonstrate d that two enzyme-SH groups become kinetically accessible on reduction with NADPH.