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 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.
Fedurco
Milan
M
Department of Chemistry, University of Geneva, Switzerland.
Augustynski
Jan
J
Indiani
Chiara
C
Smulevich
Giulietta
G
Antalík
Marián
M
Bánó
Mikulás
M
Sedlák
Erik
E
Glascock
Mary C
MC
Dawson
John H
JH
D000818:Animals; D045304:Cytochromes c / Q000737:chemistry*; D004563:Electrochemistry; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D012996:Solutions; D014508:Urea / Q000737:chemistry*; D014783:Viscosity
D012996:Solutions; D014508:Urea; D045304:Cytochromes c