The kinetics of the aerobic oxidation of ferrocytochrome c by cytochrome c oxidase in solvents of increased viscosity are partially diffusion controlled.

The steady-state spectrophotometrically determined initial velocity kinetics of the aerobic oxidation of ferrocytochrome c by cytochrome c oxidase were examined for effects of diffusion control in solvents of increased viscosity. Both glycerol/water and sucrose/water proved unsatisfactory as viscosogens due to weak competitive inhibition (Ki values of 2.6 M and 1.6 M, respectively). However, polyethylene glycol (PEG) was satisfactory as a viscosogen. The measured diffusion coefficient of ferrocytochrome c in PEG/water was shown to follow closely the Stokes-Einstein equation. In PEG/water mixtures at high ionic strength the minimum association rate constant (kmin = Vmax/(Km[EO]) is partly diffusion controlled with contributions from diffusion control and chemical activation control being about equal at 5 mPa X s, a viscosity that may be physiologically relevant. This finding can be interpreted to mean that cytochrome c oxidase is an enzyme that has evolved to approach its maximum efficiency. The steady-state kinetics were also examined at low ionic strength where multiphasic kinetics are exhibited. The effect of increased viscosity was exhibited over the whole experimentally accessible region indicating that there are effects due to diffusion control on both the high-affinity and low-affinity binding of ferrocytochrome c. Several models for diffusion control were examined and a comparison is made with other diffusion-controlled reactions of proteins.