Active Site of Mercuric Reductase Resides at the Subunit Interface and Requires Cys135 and Cys140 from One Subunit and Cys558 and Cys559 from the Adjacent Subunit: Evidence from in Vivo and in Vitro Heterodimer Formation

Mark D. Distefano, Melissa J. Moore, Christopher T. Walsh

Research output: Contribution to journalArticle

59 Scopus citations

Abstract

Mercuric reductase catalyzes the two-electron reduction of Hg(II) to Hg(0) using NADPH as the reductant; this reaction constitutes the molecular basis for detoxification of Hg(II) by bacteria. The enzyme is an α2 homodimer and possesses two pairs of cysteine residues, Cys135 and Cys140 (redox-active pair) and Cys558 and Cys559 (C-terminal pair), which are known to be essential for catalysis. In the present study, we have obtained evidence for an intersubunit active site, consisting of a redox-active cysteine pair from one subunit and a C-terminal pair from the adjacent subunit, by reconstituting catalytic activity both in vivo and in vitro starting with two inactive, mutant enzymes, Ala135Ala140Cys558Cys559 (AACC) and Cys135Cys140Ala558Ala559 (CCAA). Genetic complementation studies were used to show that coexpression of AACC and CCAA in the same cell yielded an HgR phenotype, some 104-fold more resistant than cells expressing only one mutant. Purification and catalytic characterization of a similarly coexpressed protein mixture showed the mixture to have activity levels ca. 25% those of wild type; this is the same as that statistically anticipated for a CCAA-AACC heterodimeric/homodimeric mixture with only one functional active site per heterodimer. Actual physical evidence for the formation of active mutant heterodimers was obtained by chaotrope-induced subunit interchange of inactive pure CCAA and AACC homodimers in vitro followed by electrophoretic separation of heterodimers from homodimers. Taken together, these data provide compelling evidence that the active site in mercuric reductase resides at the subunit interface and contains cysteine residues originating from separate polypeptide chains.

Original languageEnglish (US)
Pages (from-to)2703-2713
Number of pages11
JournalBiochemistry
Volume29
Issue number11
DOIs
StatePublished - Mar 1 1990

Fingerprint Dive into the research topics of 'Active Site of Mercuric Reductase Resides at the Subunit Interface and Requires Cys<sub>135</sub> and Cys<sub>140</sub> from One Subunit and Cys<sub>558</sub> and Cys<sub>559</sub> from the Adjacent Subunit: Evidence from in Vivo and in Vitro Heterodimer Formation'. Together they form a unique fingerprint.

  • Cite this