Mercuric reductase from R-plasmid NR1: characterization and mechanistic study

Mercuric reductase, a flavoprotein which catalyzes the NADPH-dependent reduction of mercuric ion to metallic mercury, has been purified from Escherichia coli containing the cloned mercury resistance genes from plasmid NR1. The purified enzyme has a molecular weight of 110,000 and is composed of two identical subunits of 56,000 each. Anaerobic titration of the enzyme with NADPH indicated that the enzyme-bound FAD could not be fully reduced to FADH sub(2) unless arsenite was included in the reaction mixture. In the absence of arsenite, NADPH formed a charge-transfer complex with partially reduced enzyme with an absorbance maximum around 540 nm. The similarity of these spectra with glutathione reductase suggested the presence of oxidation-reduction active cysteine residues at the active site. In the presence of thiol reagents there was marked biphasic kinetics characterized by an initial rapid reaction velocity which slowly reaches a much slower steady-state rate. This was shown to be a hysteretic phenomenon induced by sulfhydryl compounds.