Radical Decay Kinetics in Ferrocytochrome c Model Membranes. A Spin Label Study

The radical decay kinetics of the interaction of the small nitroxide spin label Tempol with model membranes composed of ferrocytochrome c, various phospholipids, and water, and with the complementary binary systems composed of protein-H₂O and lipid-H₂O have been investigated by electron spin resonance. Studies involving ferricytochrome c, porphyrin cytochrome c, and pure and impure lipids have served as controls and show that in pure systems an electron from the ferro iron atom is the source of the radical decay. The observed reactions in both the model membrane and binary ferrocytochrome C-H₂O systems are second order and yield rate constants (k = 1.9 ⨯ 10^-2 and 1.7 ⨯ 10^-2 M^-1 sec^-1, respectively) and activation energies (7.2 and 7.9 kcal mol^-1, respectively) which are equal within experimental error for each system, suggesting that the mechanism of oxidation of ferrocytochrome c is the same in both cases. In addition, the results of a study involving the radical decay kinetics of Tempol with ferrous phenanthroline suggest that the electron transfer reaction occurs by an outer-sphere process. From a consideration of the structural and chemical characteristics of cytochrome c our results favor a proposed mechanism of oxidation of ferrocytochrome c in which the electron leaves the protein through the heme crevice area. To the extent that these results apply as well in mitochondrial membranes the same mechanism is favored for the in vivo mechanism of this important protein.