title: Reactivity of the two essential cysteine residues of the periplasmic mercuric ion-binding protein, MerP.
abstract: Reactivities of the two essential cysteine residues in the heavy metal binding motif, MTC(14)AAC(17), of the periplasmic Hg(2+)-binding protein, MerP, have been examined. While Cys-14 and Cys-17 have previously been shown to be Hg(2+)-binding residues, MerP is readily isolated in an inactive Cys-14-Cys-17 disulfide form. In vivo results demonstrated that these cysteine residues are reduced in the periplasm of Hg(2+)-resistant Escherichia coli. Denaturation and redox equilibrium studies revealed that reduced MerP is thermodynamically favored over the oxidized form. The relative stability of reduced MerP appears to be related to the lowered thiol pK(a) (5.5) of the Cys-17 side chain. Despite its much lower pK(a), the Cys-17 thiol is far less accessible than Cys-14, reacting 45 times more slowly with iodoacetamide at pH 7.5. This is reminiscent of proteins such as thioredoxin and DsbA, which contain a similar C-X-X-C motif, except in those cases the more exposed thiol has the lowered pK(a). In terms of MerP function, electrostatic attraction between Hg(2+) and the buried Cys-17 thiolate may be important for triggering the structural change that MerP has been reported to undergo upon Hg(2+) binding. Control of cysteine residue reactivity in heavy metal binding motifs may generally be important in influencing specific metal-binding properties of proteins containing them.
authors.affiliation: Department of Chemistry, Concordia University, Montreal, Quebec H3G 1M8, Canada. powlow@vax2.concordia.ca
authors.forename: J
authors.initials: J
authors.lastname: Powlowski
authors.forename: L
authors.initials: L
authors.lastname: Sahlman
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007460:Iodoacetamide / Q000737:chemistry; D007700:Kinetics; D008628:Mercury / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D011506:Proteins
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; C086060:MerP protein, Bacteria; D011506:Proteins; D008628:Mercury; D003545:Cysteine; D007460:Iodoacetamide