Lipid Composition Affects the Efficiency in the Functional Reconstitution of the Cytochrome c Oxidase.

The transmembrane protein cytochrome c oxidase (CcO) is the terminal oxidase in the respiratory chain of many aerobic organisms and catalyzes the reduction of dioxygen to water. This process maintains an electrochemical proton gradient across the membrane hosting the oxidase. CcO is a well-established model enzyme in bioenergetics to study the proton-coupled electron transfer reactions and protonation dynamics involved in these processes. Its catalytic mechanism is subject to ongoing intense research. Previous research, however, was mainly focused on the turnover of oxygen and electrons in CcO, while studies reporting proton turnover rates of CcO, that is the rate of proton uptake by the enzyme, are scarce. Here, we reconstitute CcO from R. sphaeroides into liposomes containing a pH sensitive dye and probe changes of the pH value inside single proteoliposomes using fluorescence microscopy. CcO proton turnover rates are quantified at the single-enzyme level. In addition, we recorded the distribution of the number of functionally reconstituted CcOs across the proteoliposome population. Studies are performed using proteoliposomes made of native lipid sources, such as a crude extract of soybean lipids and the polar lipid extract of E. coli, as well as purified lipid fractions, such as phosphatidylcholine extracted from soybean lipids. It is shown that these lipid compositions have only minor effects on the CcO proton turnover rate, but can have a strong impact on the reconstitution efficiency of functionally active CcOs. In particular, our experiments indicate that efficient functional reconstitution of CcO is strongly promoted by the addition of anionic lipids like phosphatidylglycerol and cardiolipin.

Surface Enhanced Resonance Raman Spectroscopy Reveals Potential Induced Redox and Conformational Changes of Cytochrome c Oxidase on Electrodes.

Immobilization of Cytochrome c oxidase (CcO) on electrodes makes voltage-driven reduction of oxygen to water possible. Efficient catalytic turnover in CcO/electrode systems is, however, often observed at large overpotentials that cannot be rationalized by the redox properties of the enzyme itself. To understand the structural basis for this observation, CcO was electrostatically adsorbed on amino-functionalized Ag electrodes, and the redox transitions of heme a and a3 were monitored via surface enhanced resonance Raman spectroscopy (SERRS) as a function of applied potential. Under completely anaerobic conditions, the reduction of heme a3 could be seen at potentials close to those measured in solution indicating an intact catalytic center. However, in the immobilized state, a new non-native heme species was observed that exhibited a redox potential much more negative than measured for the native hemes. Analysis of the high and low frequency SERR spectra indicated that this new species is formed from heme a upon axial loss of one histidine ligand. It is concluded that the formation of the non-native heme a species alters the potential-dependent electron supply to the catalytic reaction and, thus, can have a impact on the applicability of this enzyme in biofuel cells.