Electrochemistry of Unfolded Cytochrome c in Neutral and Acidic Urea Solutions

Abstract

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 alpha-helical content of the protein. Despite being unfolded, the electron-transfer (ET) kinetics of cyt c on a 2-mercaptoethanol-modified Ag(111) electrode remain unusually fast and diffusion controlled. Acid titration of ferric cyt c in 9 M urea down to pH 2 is accompanied by protonation of one of the axial ligands, water binding to the heme iron (pK(a) = 5.2), and a sudden protein collapse (pH < 4). The formal redox potential of the urea-unfolded six-coordinate His18-Fe(III)-H(2)O/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 c at pH 7 (0.6 +/- 0.02 eV), but closer to that of native bis-His-ligated cyt b(5) (0.44 +/- 0.02 eV). The roles of electronic coupling and heme-flattening on the rate of heterogeneous ET reactions are discussed.

Redox thermodynamics of cytochromes c subjected to urea induced unfolding

“…The poor reversibility of wave II (Fig. 1) has already been observed [23,44] and was attributed to the effects of the cathodic reduction of water occurring in the same potential range corresponding to the reduction of the bishistidinate form of the cytochromes [23]. At high urea concentrations ([urea] C 8) the anodic counterpart of wave II appears and the shape of the CV curves becomes similar to that of a reversible process.…”

“…The appearance of a new wave (wave II) at potentials approximately 0.5 V lower than that of wave I (Fig. 1, Tables 2, 3 and 4) most likely corresponds to the formation of a new conformer with an altered heme iron axial coordination [14,[22][23][24].…”

“…The properties of wave II suggest its assignment to a species subjected to substitution of the axial ligand Met80 by a harder base such as a nitrogen atom of a histidine or lysine residue. At high urea concentrations ([urea] C 8 M) it is well established that a bishistidinate form of the cytochrome c is the prevailing species [14,22,23] while at low urea concentrations (4 \ [urea] \ 8) an equilibrium between a His-Lys form and the bis-histidinate one has been proposed [14]. The more basic character of the nitrogen atom(s) of the histidine (or lysine) ligand compared to the thioether sulfur of the native methionine stabilizes the ferriheme and is likely to be mainly responsible for the dramatic decrease in E°0 value of wave II as compared to wave I.…”

“…The small decrease in E°0 value observed increasing the urea concentration is consistent, however, with the progressive substitution of the lysine with the histidine. At [urea] C 8 M the His-Lys form is no more present and the bishistidinate species is the only form present in the solution [14,[22][23][24]. The poor reversibility of wave II (Fig.…”

“…For this reason, several studies have been devoted to the characterization of ET processes of cytochrome c in solution in the presence of denaturant agents [22][23][24], and recently the structural perturbation in the heme cavity due to the unfolding processes was investigated through analysis of the reorganization energy k [25].…”