Even though the structures of cytochrome c oxidase (CcO) from different sources have been determined by X-ray crystallography in both the reduced and oxidized redox states, information about redox-induced structure-function relationships is still very limited. In the current work, redox-dependent structural changes are determined for CcO reconstituted in a protein-tethered bilayer lipid membrane by surface-enhanced infrared absorption spectroscopy in the ATR mode. Significantly, the redox changes in the enzyme are attained by direct wiring of CcO to a gold electrode, ensuring that sequential intra-protein electron transfer occurs by a directed pathway that is natural to the system. The characteristics of CcO were observed to be dramatically altered after the reconstituted enzyme was allowed to turn over in the presence of O2. The data suggest that the enzyme is initially in an "inactive" state, but that direct electron transfer in the presence of O2 converts the enzyme to an "activated" form which returns to the inactive conformation when the enzyme remains idle under anaerobic conditions. Potentiometric titrations are performed and reduced-minus-fully oxidized and oxidized-minus-fully reduced absorbance spectra are recorded at decreasing and increasing potentials, respectively, applied to the electrode in a regular succession. The two sets of difference spectra show mirror symmetry, however, they markedly differ from those measured in the presence of redox mediators. Plots of band area of individual bands obtained by Fourier self deconvolution vs. applied potential show a sigmoid dependence as expected for a redox process. However, the sigmoid curves do not coincide but are displaced depending on the direction of the potential change. In other words, these curves show hysteresis, which is an indication of cooperativity and non-equilibrium states for electron transfer and/or conformational changes of the protein. This is discussed in terms of known concepts of molecular hysteresis.
ASJC Scopus subject areas
- Condensed Matter Physics