The passage of electrons through cytochrome c oxidase is directly related to the activity of water. Reducing the activity in a system containing reductant, oxygen, and cytochrome oxidase blocks electron transfer between reduced cytochrome a and oxidized cytochrome a3. The extent of the block is directly related to the osmotic pressure of the system, implying that the protein shell of the oxidase acts as a semipermeable membrane that excludes osmotic perturbants but not water. It appears that approximately 10 water molecules must enter and leave the oxidase in order for internal electron transfer to occur.

Oxidized cytochrome c oxidase exists in two reasonably well-defined conformations, a high-spin conformation with maximal absorption at 418 nm and a low-spin conformation with maximal absorption at 428 nm. The equilibrium between these two conformations has been studied as a function of pH, pressure, and temperature. pH effects the equilibrium between the two conformations, the maximum fraction of the 418-nm form being found at about pH 6.8. Increasing pressure displaced the equilibrium toward the 428-nm form; the molar volume changes found are independent of pH but strongly dependent on temperature. Increasing temperature over the range -20 to 25 degrees C displaces the equilibrium toward the 428-nm form; the van't Hoff plots that result show a discontinuity at about 10 degrees C. Above 10 degrees C, delta H is relatively constant as a function of pH; below 10 degrees C, delta H is strongly pH dependent. Delta G, delta H, delta S, and delta V have been evaluated for the equilibrium.