The concept of high- and low-affinity reactions in bovine cytochrome c oxidase steady-state kinetics

(1) Analysis of the data from steady-state kinetic studies shows that two reactions between cytochrome c and cytochrome c oxidase sufficed to describe the concave Eadie-Hofstee plots ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 1 · 10}{}^{\mathrm{?8}}\text{M}$ and $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 2 · 10}{}^{\mathrm{?5}}\text{M}$). It is not necessary to postulate a third reaction of $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{? 10}{}^{\mathrm{?6}}\text{M}$. (2) Change of temperature, type of detergent and type of cytochrome c affected both reactions to the same extent. The presence of only a single catalytic cytochrome c interaction site on the oxidase could explain the kinetic data. (3) Our experiments support the notion that, at least under our conditions (pH 7.8, low-ionic strength), the dissociation of ferricytochrome c from cytochrome c oxidase is the rate-limiting step in the steady-state kinetics. (4) A series of models, proposed to describe the observed steady-state kinetics, is discussed.