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

• Published in: Biochemistry (Easton) 1998-09, Vol.37 (38), p.13091-13101
• Main Authors: Feinberg, Benjamin A, Liu, Xiangjun, Ryan, Michael D, Schejter, Abel, Zhang, Chongyao, Margoliash, Emanuel
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - genetics; Animals; Cysteine - genetics; Cytochrome c Group - chemistry; Cytochrome c Group - genetics; Cytochrome c Group - metabolism; Cytochromes c; Electrochemistry - methods; Histidine - chemistry; Histidine - genetics; Horses; Imidazoles - pharmacology; iso-1-cytochrome c; Mutagenesis, Site-Directed; Oxidation-Reduction; Phenylalanine - chemistry; Phenylalanine - genetics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins; Serine - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi981037n

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 Met80-Fe2+-His18 coordination form, while in the oxidized state the His82-Fe3+-His18 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 × 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 Met80-Fe3+-His18 → His82-Fe3+-His18 and k f,BD > 10 s-1 for His82-Fe2+-His18 → Met80-Fe2+-His18; E 0‘ = 247 mV for Met80-Fe3+/2+-His18 couple, E 0‘ = 47 mV for His82-Fe3+/2+-His18 couple, and E 0‘ = 176 mV for the cross-reaction couple, His82-Fe3+-His18 + e- → Met80-Fe2+-His18. Thermodynamic parameters, including the entropy of reaction, ΔS 0‘Rxn, were determined for the net reduction/rearrangement reaction, His82-Fe3+-His18 + e- → Met80-Fe2+-His18, and compared to those for wild-type cytochrome, Met80-Fe3+-His18 + e- → Met80-Fe2+-His18. For the Phe82His variant mixed redox couple, ΔS 0‘Rxn = −80 J/mol·K compared to ΔS 0‘Rxn = −52 J/mol·K for the wild-type cyt c couple without rearrangement. Comparison of these entropies indicates that the oxidized His82-Fe3+-His18 form is highly disordered. It is proposed that this high level of disorder facilitates rapid rearrangement to Met80-Fe2+-His18 upon reduction.