Thermodynamics of Metal Ions Binding to Peptides with MXCXXC Sequences from Proteins in a Mercury Detoxification Pathway

The thermodynamics of Hg2+, Cd2+, and Zn2+ binding to three peptides, two containing the MXCXXC sequence from proteins in a mercury detoxification pathway and one with longer bis-Cys spacing, were quantified using isothermal titration calorimetry. Measurements in multiple buffers were used to determine the condition-independent binding thermodynamics and to quantify the number of protons displaced from the peptide upon metal binding, which indicates the Cys coordination. Competition with thiol ligands was necessary to determine the very high affinity of the MXCXXC peptides for Hg2+ (log K > 40), which originates from equal enthalpic and entropic contributions. The bis-Cys peptide has a significantly lower affinity due to smaller enthalpic and entropic contributions. These results provide an estimate of the Hg2+-thiolate bond enthalpy (20 kcal/mol) and reveal a ~7 kcal/mol enthalpic contribution from the conserved MXCXXC sequence. Under the experimental ITC conditions, the peptides bind Zn2+ and Cd2+ with variable stoichiometries (1:1, 1:2, 1:4) and coordination, depending on the X residues in the sequence, but with similar entropically-driven affinities for each metal ion. The results are compared to metal affinities from previous studies of MXCXXC peptides and proteins and preliminary thermodynamic results for MXCXXC proteins from mercury and copper pathways.