The membrane modulates internal proton transfer in cytochrome c oxidase.

The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O(2) reduction. Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pK(a) in the pH dependence profile of the proton-uptake rate. This pK(a) change resulted in decreased proton-uptake rates in the pH range of ~6.5-9.5, which is explained in terms of lowering of the pK(a) of an internal proton donor within CytcO. At pH 7.5, the rate decreased to the same extent when vesicles were prepared from the pure zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho(1-rac-glycerol) (DOPG). In addition, a small change in the internal Cu(A)-heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.