The Appearance of Transient Species of Cytochrome c upon Rapid Oxidation or Reduction at Alkaline pH

Abstract

Abstract The rapid reduction and oxidation of horse heart cytochrome c has been studied in the pH range 7 to 12 by stopped flow spectrophotometry. Above pH 8 both the oxidation of ferrocytochrome c by ferricyanide, and the reduction of ferricytochrome c by dithionite, show biphasic kinetics in which electron transfer occurs rapidly in the first phase with a rate dependent on oxidant or reductant concentration. The rate of the second phase is independent of the reductant or oxidant concentration, and the amplitude of the absorbance changes is pH-dependent with an apparent pK near 9.3. Reduction of ferricytochrome c by dithionite at pH 10.7 produces a transient form of ferrocytochrome c characterized by a sharper and more intense Soret peak centered at 418 nm (em ≥ 173,000), a more intense α-band centered at 550 nm (em ≥ 31,000) and a reduced β-band near 520 nm (em ≤ 14,500). During the second phase, this transient form of ferrocytochrome c changes to the classical stable form with a rate constant of 2 to 8 s-1, depending upon the pH. Rapid oxidation of ferrocytochrome c by ferricyanide at pH 10.5 produces a transient species of ferricytochrome c with a 695-nm band absorption and an EPR spectrum characteristic of ferricytochrome c at neutral pH. With time, both the EPR spectrum and the 695-nm band absorption change to the forms normally observed at pH 10.5, and the rate constant of this change is 0.8 s-1. A model is proposed which features a change in the sixth ligand of cytochrome c as being responsible for the changes observed during the second phase.