Ascorbate reduction of horse heart cytochrome c. A zero-energy reduction reaction.

The ascorbate reduction of horse heart ferricytochrome c in 0.05 M phosphate + 0.25 M sodium sulfate, at pH 7.3, as a function of temperature, 12-36 degrees C, and at alkaline pH 8.4 using stopped flow technique has been examined. The data have been analyzed in terms of a two-step mechanism, binding followed by reduction (Myer, Y.P., Thallam, K.K., and Pande, A. (1980) J. Biol. Chem. 255, 9666-9673). At neutral pH and up to about 26 degrees C, the first order reduction constant is independent of temperature, i.e. with zero or near-zero activation energy. At higher temperatures, it becomes temperature-dependent, increasing with increasing temperature with an activation energy of about 35 kcal/mol. The stability of the cytochrome c-ascorbate complex is enhanced in the range 12-26 degrees C, with an enthalpy and an entropy change of about 3 kcal/mol and 32 e.u., respectively. Above 26 degrees C, the stability of the complex decreases. At pH 8.4, the reduction reaction is biphasic, and the behavior of the rapid, ascorbate-dependent component is consistent with the proposed two-step mechanism. A pH change of 1.1 units increases the first order reduction constant by a factor of 6, while the stability constant of the complex decreases to about one-fourth its value. The slow component at pH 8.4 is ascorbate-independent, with a rate constant of 0.043 +/- 0.006 s-1. The zero or near-zero activation energy for the reduction reaction below 26 degrees C and the development of temperature dependence at higher temperatures constitute the bases for concluding that the reduction reaction occurs via tunneling at temperatures below 26 degrees C. The observed reduction constant is consistent with tunneling from a distance of about 16 A, with an energy barrier of about 35 kcal/mol.