Redox-driven changes in axial coordination in the phenylalanine-82-histidine mutant of yeast iso-1-cytochrome c: A direct voltammetric study

Using square-wave and cyclic voltammetric methods, the direct electrochemistry of a novel yeast iso-1-cytochrome c Phe82His/Cys102Ser mutant revealed the intricacies of the redox driven changes in axial coordination, concomitant with intramolecular rearrangement. The equilibrium redox potentials of Met80-Fe{dollar}sp{lcub}3+/2+{rcub}{dollar}-His18 and His82-Fe{dollar}sp{lcub}3+/2+{rcub}{dollar}-His18 redox couples were +229 mV and {dollar}-{dollar}148 mV versus Standard Hydrogen Electrode at 25{dollar}spcirc{dollar}C, respectively. Electrochemical methods are ideally suited for such a redox study, since they provide a direct and quantitative visualization of dynamic events. For the iso-1-cytochrome c Phe82His/Cys102Ser mutant, square-wave voltammetry showed that the primary specie in the reduced state is the Met80-Fe{dollar}sp{lcub}2+{rcub}{dollar}-His18 hemochrome, while in the oxidized state the His82-Fe{dollar}sp{lcub}3+{rcub}{dollar}-His18 hemochrome predominated. Cyclic voltammetry, in contrast, showed that even when the cytochrome is in a predominate form, there is an equilibrium between the two hemochrome forms in both redox states. This was clearly observable for the oxidized state: His82-Fe{dollar}sp{lcub}3+{rcub}{dollar}His18 {dollar}sbsp{lcub}gets{rcub}{lcub}to{rcub}{dollar} Met80-Fe{dollar}sp{lcub}3+{rcub}{dollar}-His18. Through the digital simulation of cyclic voltammetry at different scan rates, rate constants for the intramolecular rearrangement and ligand exchange were determined: k{dollar}sb{lcub}rm b,Ox{rcub}{dollar} = 32.5 sec{dollar}sp{lcub}-1{rcub}{dollar} for Met80-Fe{dollar}sp{lcub}3+{rcub}{dollar}-His18 {dollar}to{dollar} His82-Fe{dollar}sp{lcub}3+{rcub}{dollar}-His18 and k{dollar}sb{lcub}rm f,Red{rcub}{dollar} = 660 sec{dollar}sp{lcub}-1{rcub}{dollar} for His82-Fe{dollar}sp{lcub}2+{rcub}{dollar}-His18 {dollar}to{dollar} Met80-Fe{dollar}sp{lcub}2+{rcub}{dollar}-His18. A complete 2 x 2 cyclic mechanism is presented and discussed. Entropy changes were determined for the His82-Fe{dollar}sp{lcub}3+/2+{rcub}{dollar}-His 18 and Met80-Fe{dollar}sp{lcub}3+/2+{rcub}{dollar}-His18 redox couples for the reaction: Cyt c{dollar}sb{lcub}rm 0x{rcub}{dollar} + e{dollar}sp-to{dollar} Cyt c{dollar}sb{lcub}rm Red{rcub}{dollar}, via a non-isothermal electrochemical cell arrangement. For the Met80-Fe{dollar}sp{lcub}3+/2+{rcub}{dollar}-His18 couple, {dollar}rm Delta Ssbsp{lcub}Rxn{rcub}{lcub}0prime{rcub}{dollar} = {dollar}-{dollar}80 J/mol{dollar}cdot{dollar}K compared to {dollar}rm Delta Ssbsp{lcub}Rxn{rcub}{lcub}0prime{rcub}{dollar} = {dollar}-{dollar}52 J/mole{dollar}cdot{dollar}K for the near wild type Cys102Ser mutant. An entropy map shows that the {dollar}rm Ssp{lcub}0prime{rcub}sb{lcub}Ox{rcub}{dollar} is unusually high for the oxidized Phe82His mutant and it is proposed that the rearrangement Met80-Fe{dollar}sp{lcub}3+{rcub}{dollar}-His18 {dollar}to{dollar} His82-Fe{dollar}sp{lcub}3+{rcub}{dollar}-His18 is facilitated by this large negative entropy change of the reaction. Other thermodynamic contributions are also discussed.