ELECTROSTATIC FORCES AND DIELECTRIC POLARIZABILITY OF MULTIPLY PROTONATED GAS-PHASE CYTOCHROME-C IONS PROBED BY ION MOLECULE CHEMISTRY/

We demonstrate a method to quantitatively determine both Coulomb energ y and the intrinsic dielectric polarizability of large, multiply proto nated gas-phase protein ions. Information about the conformation and m aximum charge state of these ions in the gas phase is also obtained. T he apparent gas-phase basicities (GB(app)) of individual charge states are measured; these values are compared to those calculated from a re latively simple model in which charges are assigned to sites in an ion such that the overall ion free energy is minimized. For cytochrome c, we find our calculations can be fit to measured values of GB(app) of the 3+ to 15+ ions using a fully denatured ion conformation and an eps ilon(r) = 2.0+/-0.2. This value is substantially higher than that of t he small cyclic decapeptide gramicidin s, but below that predicted by theory for the interior of a protein. We find that the intrinsic basic ity of individual basic charge sites, estimated by GB measurements of small peptides, is 13-18 kcal/mol higher than those of the correspondi ng individual amino acid, consistent with independent intramolecular i nteraction (self-solvation) of the charge site occurring in these larg e multiply protonated ions. For the 21+ ion in a denatured conformatio n, we find that the minimum Coulomb contribution to ion zero-point ene rgy is 24 eV. This substantial Coulomb energy accounts for the increas ed reactivity and decreased stability of these highly charged ions. Ou r calculations indicate that the maximum charge state observed in elec trospray mass spectra is determined by the relative apparent gas-phase basicity of the ion/solvent combination. Finally, we find that the ga s-phase conformation of cytochrome c ions is consistent with a denatur ed form, although our calculations indicate that cytochrome c electros prayed from an aqueous solution is initially in its native conformatio n subsequent to its desorption into the gas phase.