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 t he intricacies of redox driven changes in axial coordination, concomit ant with intramolecular rearrangement. Electrochemical methods are ide ally suited for such a redox study, since they provide a direct and qu antitative visualization of specific dynamic events. For the iso-1-cyt ochrome c Phe82His/Cys102Ser variant, square-wave voltammetry showed t hat the primary species in the reduced state is the Met(80)-Fe2+-His(1 8) 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, si mulations were done for the cyclic voltammetry experiments at differen t scan rates. These, in turn, provided relative rate constants for the intramolecular rearrangement/ligand exchange and the equilibrium redo x 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(8 2)-Fe3+-His(18) + e(-) --> Met(80)-Fe2+-His(18). Thermodynamic paramet ers, including the entropy of reaction, Delta S-Rxn(0)', were determin ed for the net reduction/rearrangement reaction, His(82)-Fe3+-His(18) + e(-) --> Met(80)-Fe2+-His(18), and compared to those for wild-type c ytochrome, Met(80)-Fe3+-His(18) + e(-) --> Met(80)-Fe2+-His(18). For t he 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 coup le without rearrangement. Comparison of these entropies indicates that the oxidized His(82)-Fe3+-His(18) form is highly disordered. It is pr oposed that this high level of disorder facilitates rapid rearrangemen t to Met(80)-Fe2+-His(18) upon reduction.