Electron transfer between horse heart and Candida krusei cytochromes c in the free and bound states

Electron transfer between horse heart and Candida krusei cytochromes c in the free and phosvitin-bound states was examined by difference spectrum and stopped-flow methods. The difference spectra in the wavelength range of 540–560 nm demonstrated that electrons are exchangeable between the cytochromes c of the two species. The equilibrium constants of the electron transfer reaction for the free and phosvitin-bound forms, estimated from these difference spectra, were close to unity at 20°C in 20 mM Tris-HCl buffer (pH 7.4). The electron transfer rate for free cytochrome c was (2–3) · 10⁴ M^?1 · s^?1 under the same conditions. The transfer rate for the bound form increased with increase in the binding ratio at ratios below half the maximum, and was almost constant at higher ratios up to the maximum. The maximum electron exchange rate was about 2 · 10⁶ M^?1 · s^?1, which is 60–70 times that for the free form at a given concentration of cytochrome c. The activation energy of the reaction for the bound cytochrome c was equal to that for the free form, being about 10 kcal/mol. The dependence of the exchange rate on temperature, cytochrome c concentration and solvent viscosity suggests that enhancement of the electron transfer rate between cytochromes c on binding to phosvitin is due to increase in the collision frequency between cytochromes c concentrated on the phosvitin molecule.