Direct Measurement of Mercury(II) Removal from Organomercurial Lyase (MerB) by Tryptophan Fluorescence: NmerA Domain of Coevolved gamma-Proteobacterial Mercuric Ion Reductase (MerA) Is More Efficient Than MerA Catalytic Core or Glutathione

作者：Hong Baoyu; Nauss Rachel; Harwood Ian M; Miller Susan M^*

来源：Biochemistry, 2010, 49(37): 8187-8196.

DOI：10.1021/bi100802k

Aerobic and facultative bacteria and archaea harboring mer loci exhibit resistance to the toxic effects of Hg(II) and organomercurials [RHg(I)]. In broad spectrum resistance, RHg(I) is converted to less toxic Ha(0) in the cytosol by the sequential action of organomercurial lyase (MerB: RHg(I) --> RH + Hg(II)) and mercuric ion reductase (MerA: Hg(II) --> Hg(0)) enzymes, requiring transfer of Hg(II) from MerB to MerA. Although previous studies with gamma-proteobacterial versions of MerA and a nonphysiological Hg(II)-DTT-MerB complex qualitatively support a pathway for direct transfer between proteins, assessment of the relative efficiencies of Hg(II) transfer to the two different dicysteine motifs in gamma-proteobacterial MerA and to competing cellular thiol is lacking. Here we show the intrinsic tryptophan fluorescence of gamma-proteobacterial MerB is sensitive to Hg(II) binding and use this to probe the kinetics of Hg(II) removal from MerB by the N-terminal domain (NmerA) and catalytic core C-terminal cysteine pairs of its coevolved MerA and by glutathione (GSH), the major competing cellular thiol in gamma-proteobacteria. At physiologically relevant concentrations, reaction with a 10-fold excess of NmerA over HgMerB removes 92% Hg(II), while similar extents of reaction require more than 1000-fold excess of GSH. Kinetically, the apparent second-order rate constant for Hg(II) transfer from MerB to NmerA, at (2.3 +/- 0.1) x 10(4) M(-1) s(-1), is 100-fold greater than that for GSH ((1.2 +/- 0.2) x 10(2) M(-1) s(-1)) or the MerA catalytic core (1.2 x 10(2) M(-1) s(-1)), establishing transfer to the metallochaperone-like NmerA domain as the kinetically favored pathway in this coevolved system.

• 出版日期2010-9-21