Factors influencing intermolecular and intramolecular electron transfer in the cytochrome c: Cytochrome c peroxidase complex.

• Main Author: Hazzard, James Taylor.
• Other Authors: Tollin, Gordon
• Language: en
• Published: The University of Arizona. 1989
• Subjects: Oxidation-reduction reaction > Mathematical models; Cytochrome c; Chemical kinetics.
• Online Access: http://hdl.handle.net/10150/184957

The kinetics of reduction by free flavin semiquinones of the individual components of 1:1 complexes of yeast cytochrome c peroxidase and the cytochrome c from horse, tuna, and yeast, including several site-specific mutants of either the cytochrome c or cytochrome c peroxidase, have been studied. The orientations of the various cytochromes c within electrostatically-stabilized complexes with the peroxidase are not equivalent. This is shown by differential decreases in the rate constants for cytochrome reduction by neutral flavin semiquinones upon complexation which are in the order: tuna ≫ horse > yeast iso-2 > yeast iso-1. We have also directly measured the physiologically-significant intracomplex one-electron transfer rate constants from the ferrous cytochromes c to the peroxide-oxidized species of the peroxidase at several ionic strengths. The rate constants at low ionic strength are highly species dependent, again consistent with the contention that the orientations of the various cytochromes within the complex with CcP are not the same. Increasing the ionic strength in all cases resulted in an increase in the rate constant for the first-order process which controls electron transfer from cytochrome c to the peroxidase Compound I species of the peroxidase. When the two proteins are immobilized by covalent cross-linking, no such rate enhancement is observed, suggesting that the ionic strength effect is manifested by an increase in the number of geometric orientations between the two proteins which results in more rapid electron transfer. Similar rate enhancing effects are observed when positively charged residues on the surface of cytochrome c are converted to electrostatically neutral amino acids by site-specific mutagenesis. The effect of site-specific mutagenesis of two residues of cytochrome c peroxidase have also been studied. His-181, when converted to a glycine has little effect on the electron transfer rate constant, whereas when Trp-191 is converted to a phenylalanine no intracomplex electron transfer could be observed, indicating an obligatory role of this residue in the electron transfer process.