RAPID REDUCTION OF HG(II) BY MERCURIC ION REDUCTASE DOES NOT REQUIRE THE CONSERVED C-TERMINAL CYSTEINE PAIR USING HGBR2 AS THE SUBSTRATE

Conditions are described under which the nonphysiological substrate me rcuric bromide (HgBr2) is rapidly turned over, both by the wild type ( CCCC) and by an active site double mutant (CCAA) of mercuric reductase in which the C-terminal cysteines 557' and 558' are replaced by alani ne and only the redox-active pair Cys135 and Cys140 are available for catalysis. A maximum rate of turnover k(cat)(app) Of approximate to 18 s(-1) (at 3 degrees C) for both enzymes is observed, and at high [HgB r2]/[enzyme] ratios, inhibition is found. The UV-vis spectral changes during turnover are closely similar in both enzymes, indicating that c atalysis follows the same enzymatic mechanism. Single-turnover analysi s of the mutant enzyme shows that after binding of HgBr2, two further rapid events ensue, followed by reduction of the metal ion (k(obs) app roximate to 23.5 s(-1)). It is shown that under multiple-turnover cond itions, completion of the catalytic cycle must occur via an ordered me chanism where rapid binding of a new molecule of HgBr2 to EH2. NADP(+) precedes exchange of the pyridine nucleotide. Binding of HgBr2 to the active site triple mutant C135A/C557A/C558A (ACAA) is ca. 100-fold sl ower compared to that of the CCAA mutant and results in no detectible turnover. It is concluded that in the reducible enzyme . Hg(II) comple x, the metal ion is coordinated to Cys135 and Cys140 and that for effi cient catalysis both residues are required. Furthermore, the data impl y that binding to EH2. NADPH occurs via initial rate-limiting attack o f Cys135, followed by reaction with Cys140.