Reactivity of the two essential cysteine residues of the periplasmic mercuric ion-binding protein, MerP

Reactivities of the two essential cysteine residues in the heavy metal bind ing motif, MTC(14)AAC(17), of the periplasmic Hg2+-binding protein, MerP, h ave been examined. While Cys-14 and Cys-17 have previously been shown to be Hg2+-binding residues, MerP is readily isolated in an inactive Cys-14-Cys- 17 disulfide form, In vivo results demonstrated that these cysteine residue s are reduced in the periplasm of Hg2+-resistant Escherichia coli. Denatura tion and redox equilibrium studies revealed that reduced MerP is thermodyna mically favored over the oxidized form. The relative stability of reduced M erP appears to be related to the lowered thiol pK(a) (5.5) of the Cys-17 si de chain. Despite its much lower pK(a), the Cys-17 thiol is far less access ible 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 cont ain a similar C-X-X-C motif, except in those cases the more exposed thiol h as the lowered pK(a). In terms of MerP function, electrostatic attraction b etween Hg2+ and the buried Cys-17 thiolate may be important for triggering the structural change that MerP has been reported to undergo upon Hg2+ bind ing. Control of cysteine residue reactivity in heavy metal binding motifs m ay generally be important in influencing specific metal-binding properties of proteins containing them.