Catalytic activity of cytochrome c oxidase in a biomimetic surface platform investigated by spectro-electrochemistry

The immobilization of membrane proteins in biomimetic architectures on metal electrodes allows for the functional characterization of energy-transducing membrane proteins using a combination of electrochemistry and spectroscopy techniques. Here cytochrome c oxidase (CcO) is immobilized onto gold surfaces using his-tag technology and reconstituted in-situ into a protein-tethered bilayer lipid membrane (ptBLM). The strictly oriented and functional reconstitution is a prerequisite for the investigation of the electron-transfer process through the multi-redox site membrane protein. When the enzyme is immobilized with the cytochrome c binding site directed towards the electrode, electrochemical investigations show that under aerobic and reducing conditions the enzyme undergoes a gradual transition into an activated state. It is only in this state that proton pumping and catalytic currents can be observed. The potential of the catalytic current, however, is shifted in the negative range with respect to the standard redox potential of CuA, whereas standard redox potentials of all the centers in the positive potential range can be observed if the enzyme is kept under anaerobic conditions. This conformational state of CcO is characterized by a lack of proton pumping activity and is therefore addressed as a non-activated state. On the other hand, if the enzyme is immobilized with the cytochrome c binding site directed away from the electrode, it can only be activated under aerobic conditions after addition of cytochrome c. In this case, simulations are used to identify the electron pathway between cytochrome c bound to the enzyme and the electrode.