Direct voltammetric observation of redox driven changes in axial coordination and intramolecular rearrangement of the phenylalanine-82-histidine variant of yeast iso-1-cytochrome c

Direct square-wave and cyclic voltammetric electrochemical examination of the yeast iso-1-cytochrome c Phe82His/Cys102Ser variant revealed the intricacies of redox driven changes in axial coordination, concomitant with intramolecular rearrangement. Electrochemical methods are ideally suited for such a redox study, since they provide a direct and quantitative visualization of specific dynamic events. For the iso-1-cytochrome c Phe82His/Cys102Ser variant, square-wave voltammetry showed that the primary species in the reduced state is the Met(80)-Fe2+-His(18) coordination form, while in the oxidized state the His(82)-Fe3+-His(18) form predominates. The addition or removal of an electron to the appropriate form of this variant serves as a switch to a new molecular form of the cytochrome. Using the 2 x 2 electrochemical mechanism, simulations were done for the cyclic voltammetry experiments at different scan rates. These, in turn, provided relative rate constants for the intramolecular rearrangement/ligand exchange and the equilibrium redox potentials of the participating coordination forms: k(b,AC) = 17 s(-1) for Met(80)-Fe3+-His(18) --> His(82)-Fe3+-His(18) and k(f,BD) > 10 s(-1) for His(82)-Fe2+-His(18) --> Met(80)-Fe2+-His(18); E-0' = 247 mV for Met(80)-Fe3+/2+-His(18) couple, E-0' = 47 mV for His(82)-Fe3+/2+-His(18) couple, and E-0' = 176 mV for the cross-reaction couple, His(82)-Fe3+-His(18) + e(-) --> Met(80)-Fe2+-His(18). Thermodynamic parameters, including the entropy of reaction, Delta S-Rxn(0)', were determined for the net reduction/rearrangement reaction, His(82)-Fe3+-His(18) + e(-) --> Met(80)-Fe2+-His(18), and compared to those for wild-type cytochrome, Met(80)-Fe3+-His(18) + e(-) --> Met(80)-Fe2+-His(18). For the Phe82His variant mixed redox couple, Delta S-Rxn(0)' = -80 J/mol.K compared to Delta S-Rxn(0)' = -52 J/mol.K for the wild-type cyt c couple without rearrangement. Comparison of these entropies indicates that the oxidized His(82)-Fe3+-His(18) form is highly disordered. It is proposed that this high level of disorder facilitates rapid rearrangement to Met(80)-Fe2+-His(18) upon reduction.