Autoreduction of horse heart ferricytochrome c: Kinetic and equilibrium studies of the over-all reaction

Abstract

The kinetics of the autoreduction, i.e. a slow reduction in the absence of a chemical reducing agent, of native horse heart ferricytochrome c in aqueous solution have been studied. Alkaline conditions were found to be necessary for the reaction to proceed at a measurable rate; the initial rate exhibited an apparent pH optimum of about 9.9 (100 m m-glycine-NaOH buffer, 25 °C). At this pH the reaction proceeds almost to completion (70% reduction at equilibrium), and the product thus obtained revealed a visible spectrum identical to that of ferrocytochrome c obtained by dithionite reduction. The generation of reducing equivalents has also been demonstrated and followed kinetically by observing the ability of ferricytochrome c to catalyze the reduction of an exteral one-electron acceptor (K 3Fe(CN) 6). A comparison between the kinetics of the autoreduction of ferricytochrome c and its reduction of ferricyanide revealed that the iron in the two compounds is reduced at rates which are of the same order of magnitude. This fact supports the conclusion that the rate-limiting process in the over-all reactions is the generation of the reducing equivalents. A comparison between the pH-dependence of the autoreduction and the ferricyanide reduction, as well as spectrophotometric studies of ferricytochrome c in alkaline solutions, supports the conclusion that the decrease in rate of autoreduction at pH values > 9.9 derives from a pH-induced conformational transition with p K′ of 9.30. This eonformational change results in a displacement of the polypeptide chain from close proximity to the haem moiety i.e. an opening up of the “closed” crevice structure, making the electron transfer from the polypeptide chain to the haem iron more difficult. The effect of urea adds further support to this view as well as to the idea that the polypeptide chain has a primary role in the reaction. Dissolved molecular oxygen apparently has no effect on the autoreduction except at pH ≳ 10 where ferrocytochrome c becomes autoxidizable. The results are discussed in the context of electronic energy transfer in cytcochrome c under physiological conditions.