Direct electron transfer (ET) to multi-center membrane proteins was achieved by electronic wiring of cytochrome c oxidase, CcO, from R. sphaeroides, when the enzyme was immobilized in a strict orientation on the gold film of an ATR crystal, followed by reconstitution in a protein-tethered bilayer lipid membrane (ptBLM). Proton translocation could be detected. This system allows the application of surface-enhanced resonance Raman (SERR) and IR absorption (SEIRA) spectroscopy triggered by electrochemical modulation. Moreover, direct ET could be studied by electrochemical measurements alone, for example fast scan voltammetry, analyzed by model calculations. A sequential four-electron transfer model was established, according to which direct ET is coupled to protonation-dependent chemical reaction kinetics. It has been concluded that direct ET mimics the natural ET pathway through the CcO. Time-resolved (tr-SEIRAS) triggered by electrochemical modulation was performed under anaerobic and aerobic conditions. Conformational changes of the protein backbone were detected in the ms time scale. tr-SEIRA studies were then expanded into the catalytic regime when the enzyme works in the presence of oxygen. Single bands isolated from a broad band in the amide I region using phase sensitive detection were attributed to different redox centers. A fitting routine was established to fit the model mentioned above to the time course of these band absorbances. Thereby -sheets were shown to undergo more substantial conformational changes than -helices, which is well in agreement with recent MD calculations of the CcO.
|Titel||EC COST Thematic Workshop|
|Publikationsstatus||Veröffentlicht - 2013|
|Veranstaltung||EC COST Thematic Workshop - |
Dauer: 8 Okt. 2013 → 9 Okt. 2013
|Konferenz||EC COST Thematic Workshop|
|Zeitraum||8/10/13 → 9/10/13|