EFFECTS OF FOLDING ON METALLOPROTEIN ACTIVE-SITES

Experimental data for the unfolding of cytochrome c and azurin by quan idinium chloride (GuHCl) are used to construct free-energy diagrams fo r the folding of the oxidized and reduced proteins. With cytochrome c, the driving force for folding the reduced protein is larger than that for the oxidized form. Both the oxidized and the reduced folded forms of yeast cytochrome c are less stable than the corresponding states o f the horse protein. Due to the covalent attachment of the heme and is fixed tetragonal coordination geometry, cytochrome c folding can be d escribed by a two-state model. A thermodynamic cycle leads to an expre ssion for the difference in self-exchange reorganization energies for the folded and unfolded proteins, The reorganization energy for electr on exchange in the folded protein is approximately 0.5 eV smaller than that for a heme in aqueous solution. The finding that reduced azurin unfolds at lower GuHCl concentrations than the oxidized protein sugges ts that the coordination structure of copper is different in oxidized and reduced unfolded states: it is likely that the geometry of Cu-I in the unfolded protein is linear or trigonal, whereas Cu-II prefers to be tetragonal. The evidence indicates that protein folding lowers the azurin reorganization energy by roughly 1.7 eV relative to an aqueous Cu(1,10-phenanthroline)(2)(2+/+) reference system.