Alkaline Isomerization of Oxidized Cytochrome c

Abstract

Equilibriumand kinetic studies of the isomerization of horse heart ferricytochrome c at alkaline pH have been carried out using difference spectroscopy and stopped flow techniques.The minimal reaction scheme is: (NC) Cyt-c-Fen' 0.05 s-1 II 6.0 s-' (C) Hf + Cyt-c-Fern 11 pKn = 11 (CH) HCyt-c-Fern + [Feri(CN)B1-a , 2.6 X lo4 M-l s-l 8 X lo6 M-1 s-1 HCyt-c-Fen + [Fern(CN)6l-3 where CH and C are species of ferricytochrome c which exhibit a 69.5 nm absorbance band, and NC (no color) is a form without this band; the oxidation-reduction potentials of CH and NC differ significantly.The values of the constants in this scheme were determined at 25" except for the oxidation and reduction rate constants, which were determined at 22".The pH dependence of the observed rate constant for the conformational changes was measured as the change in the 695 nm band absorbance and as the release and uptake of hydrogen ions.From this it was calculated that the ionizing group in the scheme above has an apparent pK of 11.0 f 0.1 at 25".The conformation equilibrium constant, K,, defined as kfjkb, was found to be 125 + 36 at 25".An over-all pK of 9.0 was calculated from the product of the equilibrium constants, in agreement with the over-all pK of 9.0 determined from the pH dependence of the 695 nm band at equilibrium.Spectrophotometric measurements at 243 nm indicate that tyrosine is not the ionizing group