Conformational Transitions Associated with Electrochemically-Induced Redox Processes Through the Cytochrome C Oxidase Followed by Time-Resolved 2d-Surface-Enhanced Infrared Absorption Spectroscopy (tr-2d-Seiras)

Electrochemically-induced redox processes of Cytochrome c oxidase (CcO) from R. sphaeroides were investigated using Surface-Enhanced ATR-FTIR-Spectroscopy. The CcO with the his-tag attached to subunit II (SU II) was immobilized in a strict orientation on a two-layer gold film deposited on the ATR crystal of the IR spectrometer using the his-tag technology. A lipid bilayer was subsequently reconstituted by in-situ dialysis around the protein to yield a protein-tethered bilayer lipid membrane.This system enabled us to observe the sequential electron transfer (eT) within the multi-redox-site membrane protein induced by electronic wiring to the gold surface using time-resolved (tr)-SEIRAS. Conformational transitions concerning a large number of single amino acids and also of secondary structures as a consequence of eT could be seen in a wide range of frequencies from 0.7 Hz to 2 kHz. A high resolution of the spectra was achieved by a combination of Two-Dimensional Infrared (2D IR) Spectroscopy and phase-sensitive detection. Kinetic constants were obtained by applying periodic potential pulses and recording spectral changes as a function of time. Methods were developed to separate these kinetic constants from the contribution due to charging currents.