AN EXAFS INVESTIGATION OF HG(II) BINDING TO MERCURIC REDUCTASE - COMPARATIVE-ANALYSIS OF THE WILD-TYPE ENZYME AND A MUTANT ENZYME GENERATED BY SITE-DIRECTED MUTAGENESIS

Bacterial mercuric ion reductase is a unique metal-detoxification biocatalyst in its ability to reduce toxic Hg(II) salts to Hg(0). The enzyme contains FAD, a reducible active-site disulfide (Cys135, Cys140), and a C-terminal pair of cysteines (Cys558, Cys559). Previous studies from this laboratory have shown by mutagenesis that all four cysteines are required for efficient Hg(II) reduction. To determine whether Hg(II) bound at the active site of the EH2 form of the enzyme exists in bis-ligation or higher ligation (tris or tetrakis) to the indicated cysteine residues, we have conducted the first mercury EXAFS studies on both wild-type enzyme (Cys135, Cys140, Cys558, Cys559 = CCCC) and a very low activity C-terminal double alanine mutant (Cys135, Cys140, Ala558, Ala559 = CCAA) form of mercuric reductase. Stoichiometric titration of the wild-type or CCAA enzyme EH2 form was achieved with either Hg(CN)2 or HgBr2, as monitored by quenching of the Cys140 thiolate-FAD charge-transfer-complex absorbance band as well as by alterations in FAD fluorescence. Hg EXAFS of these stoichiometric enzyme-Hg complexes revealed distances, ligand identification, and ligand number consistent with bis-thiol coordination in both the CCAA mutant (as anticipated) and also in the CCCC wild-type mercuric reductase. Implications for the catalytic mechanism are addressed. © 1990, American Chemical Society. All rights reserved.