Electrochemistry of unfolded cytochrome c in neutral and acidic urea solutions.

The present investigation reports the first experimental measurements of the reorganization energy of unfolded metalloprotein in urea solution. Horse heart cytochrome c (cyt c) has been found to undergo reversible one-electron transfer reactions at pH 2 in the presence of 9 M urea. In contrast, the protein is electrochemically inactive at pH 2 under low-ionic strength conditions in the absence of urea. Urea is shown to induce ligation changes at the heme iron and lead to practically complete loss of the R-helical content of the protein. Despite being unfolded, the electron-transfer (ET) kinetics of cyt con a 2-mercaptoethanolmodified Ag(111) electrode remain unusually fast and diffusion controlled. Acid titration of ferric cyt cin 9M urea down to pH 2 is accompanied by protonation of one of the axial ligands, water binding to the heme iron (p Ka ) 5.2), and a sudden protein collapse (pH < 4). The formal redox potential of the urea-unfolded six-coordinate His18-Fe(III)-H2O/five-coordinate His18-Fe(II) couple at pH 2 is estimated to be -0.083 V vs NHE, about 130 mV more positive than seen for bis-His-ligated urea-denatured cyt c at pH 7. The unusually fast ET kinetics are assigned to low reorganization energy of acid/urea-unfolded cyt c at pH 2 (0.41 ( 0.01 eV), which is actually lower than that of the native cyt cat pH 7 (0.6 ( 0.02 eV), but closer to that of native bis-His-ligated cyt b5 (0.44 ( 0.02 eV). The roles of electronic coupling and heme-flattening on the rate of heterogeneous ET reactions are discussed.