Date of Award
Doctor of Philosophy (PhD)
In the work described in this thesis we undertook studies of the electron transfer between cytochromes c and iron hexacyanides, a reaction which serves as a simple, well defined model for electron transfer in vivo. In particular, we were interested in the following aspects of electron transfer: the mechanism of the oxidation of ferrocytochrome c by ferricyanide, the mechanism of reduction of ferricytochrome c by ferrocyanide, the effect of charges on the protein and the ionic strength of the medium on electron transfer, and the effect of ions which bind to cytochrome c on oxidation and reduction. Since ion binding to cytochrome c has been observed by several techniques, cytochrome c preparations in this study were electrodialyzed, whenever possible, in order to remove contaminating bound ions.
The results of our work indicate that the oxidation of ferrocytochrome c by ferricyanide in nonbinding (tris-cacodylate) buffer follows irreversible second-order kinetics. The reduction of ferricytochrome by ferrocyanide was found consistent with the following reaction scheme:
Our data suggests that there are different pathways for reduction and oxidation of cytochrome c when iron hexacyanides are used.
The effect of the ionic strength on the bimolecular rate for the oxidation of horse heart and P. aeruginosa ferrocytochromes in non-binding buffer was analyzed using Debye-Huckel theory, and suggested that the oxidation of horse heart ferrocytochrome c proceeds at nearly the diffusion-controlled rate. From this analysis an effective charge of +7.8 units for horse heart cytochrome c was obtained. The oxidation of P. aeruginosa cytochrome c was much slower and was not consistent with a diffusion-controlled reaction. The ionic strength dependence of the rate of oxidation for M. denitrificans ferrocytochrome c could not be fitted to Debye-auckel theory.
The ionic strength was also found to affect the reduction of ferricytochrome c by ferrocyanide. The measurements revealed that the dissociation constant Kd (= k₁/k₂) and the rate constant k₄ in the above mechanism are ionic strength dependent.
Analysis of the effect of binding ions on the rate of oxidation for horse heart ferrocytochrome c indicated that more than one chloride, phosphate, potassium and picrate ion were bound to horse heart ferrocytochrome c. The bound ion slowed the rate of oxidation.
The reduction of ferricytochrome c by ferrocyanide was approximated by a reversible second-order reaction, and the overall reduction and oxidation rates were derived. The ratios of these rate constants, measured in various buffers, were used to estimate the number of ions bound to ferricytochrome c and their respective binding constants. Approximately one chloride ion and almost two picrate ions were bound to horse heart ferricytochrome c. The rate of reduction of ferricytochrome c having bound ion(s) was also slower than normal.
Chemical modification (guanidination) of all the lysine residues of cytochrome c showed that positive charges and not lysines are necessary for efficient electron transfer between cytochrome c and iron hexacyanides.
Peterman, Branko F., "Electron Transfer Reactions of Cytochromes c" (1976). Open Access Dissertations and Theses. Paper 3765.