Applying Ion Mobility-Mass Spectrometry Techniques for Explicitly Identifying the Products of Cu(II) Reactions of 2His-2Cys Motif Peptides.

The Cu(II) and pH titrations of four structurally similar 2His-2Cys motif peptides were investigated by electrospray ionization-ion mobility-mass spectrometry. The results provided insight into the pH dependent redox processes that took place in solution and identified the number of inter- or intramolecular disulfide bridges, the number of Cu(I) or Cu(II) ions, the deprotonation sites, and likely Cu(I/II) coordination of the various products. Competitive Cu(II) titrations of binary peptide mixtures at pH 5 indicated which species would preferably bind Cu(I) ions over forming the intramolecular disulfide bridge. Moreover, these reactions were pH dependent and included the formation of various multimers and multiple Cu(I/II) binding. For example, for the mildly acidic solution (pH ∼ 3-6) each monomer (whether it was free or in a multimer) primarily bound up to 3 Cu(I) ions, whereas at pH ∼ 8-11 the fully oxidized monomer or multimer (where all Cys formed a disulfide bond) primarily bound up to 2 Cu(II) ions. This behavior was indicative of linear bridging of Cu(I) by Cys thiolate and His imidazole groups, whereas the coordination of Cu(II) involved His and the nitrogens of deprotonated backbone amide groups, resulting in either distorted T-shaped or square planar geometries.