Abstract:
In this study, an attempt has been made to determine the stoichiometry of the electrostatic
complex between horse heart ferricytochrome c and cytochrome c oxidase using gel filtration
chromatography. The conditions under which this complex may be formed or dissociated were studied.
The oxidase was, however, first isolated and purified from bovine heart according to the method of
Yonetani (1961).
Being the terminal enzyme if' the electron transport chain, cytochrome c oxidase accepts four
electrons from the single electron donor, ferrocytochrome c, to effect the four electron reduction of
dioxygen to water in a catalytic cycle. A requisite step for the proton translocation and mitochondrial
energy conservation carried out by this enzyme is, therefore, the formation of a complex (if only transient
under some conditions) between the two proteins, that is competent to transfer electrons.
The experiments reported in this study indicate that under conditions of low ionic strength, the enzyme
formed a tight 1:1 electrostatic complex with cytochrome c (Kd - 10 nM) (i.e. one molecule of
cytochrome c per functional unit of cytochrome c oxidase). This contrasts with the findings of other
workers that each functional unit of cytochrome c oxidase contains twc binding sites for cytochrome c.
The electrostatic complex between cytochrome c and oxidase is a tight and stable one over a fairly
wide pH range, at low ionic strength « 20 mM) which is far lower than that which is reported as
physiological (- 100 mM). It may be that extremely small local variations in ionic strength, in the area of
the binding sites on one or both proteins, brought about for example during proton translocation by
cytochrome c oxidase, or exclusion of surface water molecules during 'docking" may have dramatic
effects in vivo on the affinity of cytochrome c for cytochrome c oxidase.
