Direct measurement of mercury(II) removal from organomercurial lyase (MerB) by tryptophan fluorescence: NmerA domain of coevolved ?-proteobacterial mercuric ion reductase (MerA) is more efficient than MerA catalytic core or glutathione .

ABSTRACT: 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 Hg(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 ?-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 ?-proteobacterial MerA and to competing cellular thiol is lacking. Here we show the intrinsic tryptophan fluorescence of ?-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 ?-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) × 10(4) M(-1) s(-1), is ?100-fold greater than that for GSH ((1.2 ± 0.2) × 10(2) M(-1) s(-1)) or the MerA catalytic core (1.2 × 10(2) M(-1) s(-1)), establishing transfer to the metallochaperone-like NmerA domain as the kinetically favored pathway in this coevolved system.

SUBMITTER: Hong B 

PROVIDER: S-EPMC3042367 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature