Direct measurement of mercury(II) removal from organomercurial lyase (MerB) by tryptophan fluorescence: NmerA domain of coevolved γ-proteobacterial mercuric ion reductase (MerA) is more efficient than MerA catalytic core or glutathione .
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 γ-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 γ-proteobacterial MerA and to competing cellular thiol is lacking. Here we show the intrinsic tryptophan fluorescence of γ-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 γ-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.
Hong
Baoyu
B
Department of Pharmaceutical Chemistry, University of California San Francisco,San Francisco, California 94158-2517, USA.
Nauss
Rachel
R
Harwood
Ian M
IM
Miller
Susan M
SM
D001419:Bacteria / Q000187:drug effects / Q000378:metabolism; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D005453:Fluorescence; D020563:Gammaproteobacteria / Q000378:metabolism; D005978:Glutathione / Q000494:pharmacology; D007477:Ions / Q000494:pharmacology; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D008628:Mercury / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D056511:Metallochaperones; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism*; D014364:Tryptophan / Q000494:pharmacology
D007477:Ions; D056511:Metallochaperones; D014364:Tryptophan; D010088:Oxidoreductases; C021551:mercuric reductase; D008190:Lyases; C050953:alkylmercury lyase; D008628:Mercury; D005978:Glutathione; D003545:Cysteine