Rapid reduction of Hg(II) by mercuric ion reductase does not require the conserved C-terminal cysteine pair using HgBr sub(2) as the substrate

• Published in: Biochemistry (Easton) Vol. 37; no. 33; pp. 11496 - 11507
• Main Authors: Engst, S, Miller, S M
• Format: Journal Article
• Language: English
• Published: 01.08.1998
• ISSN: 0006-2960

Conditions are described under which the nonphysiological substrate mercuric bromide (HgBr sub(2)) 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 alanine and only the redox-active pair Cys135 and Cys140 are available for catalysis. A maximum rate of turnover k sub(cat) super(app) of approximately 18 s super(-1) (at 3 degree C) for both enzymes is observed, and at high [HgBr sub(2)]/[enzyme] ratios, inhibition is found. The UV-vis spectral changes during turnover are closely similar in both enzymes, indicating that catalysis follows the same enzymatic mechanism. Single-turnover analysis of the mutant enzyme shows that after binding of HgBr sub(2), two further rapid events ensue, followed by reduction of the metal ion (k sub(obs) approximately 23.5 s super(-1)). It is shown that under multiple-turnover conditions, completion of the catalytic cycle must occur via an ordered mechanism where rapid binding of a new molecule of HgBr sub(2) to EH sub(2) times NADP super(+) precedes exchange of the pyridine nucleotide. Binding of HgBr sub(2) to the active site triple mutant C135A/C557A/C558A (ACAA) is ca. 100-fold slower compared to that of the CCAA mutant and results in no detectable turnover. It is concluded that in the reducible enzyme times Hg(II) complex, the metal ion is coordinated to Cys135 and Cys140 and that for efficient catalysis both residues are required. Furthermore, the data imply that binding to EH sub(2) times NADPH occurs via initial rate-limiting attack of Cys135, followed by reaction with Cys140.