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 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 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: kb,AC = 17 s-1 for Met80-Fe3+-His18 --> His82-Fe3+-His18 and kf,BD > 10 s-1 for His82-Fe2+-His18 --> Met80-Fe2+-His18; E0' = 247 mV for Met80-Fe3+/2+-His18 couple, E0' = 47 mV for His82-Fe3+/2+-His18 couple, and E0' = 176 mV for the cross-reaction couple, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18. Thermodynamic parameters, including the entropy of reaction, DeltaS0'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, DeltaS0'Rxn = -80 J/mol.K compared to DeltaS0'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.
Feinberg
B A
BA
Department of Chemistry, University of Wisconsin-Milwaukee 53211, USA. feinberg@csd.uwm.edu
Liu
X
X
Ryan
M D
MD
Schejter
A
A
Zhang
C
C
Margoliash
E
E
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D003545:Cysteine / Q000235:genetics; D003574:Cytochrome c Group / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D045304:Cytochromes c; D004563:Electrochemistry / Q000379:methods; D006639:Histidine / Q000737:chemistry / Q000235:genetics*; D006736:Horses; D007093:Imidazoles / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine / Q000235:genetics
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D007093:Imidazoles; D029701:Saccharomyces cerevisiae Proteins; D012694:Serine; D010649:Phenylalanine; D006639:Histidine; C029899:imidazole; D045304:Cytochromes c; D003545:Cysteine