Thema der Dissertation:
Fluorescence and Infrared Spectroscopic Studies of Surface Protonation Dynamics at the K-channel Entrance and Redox-Linked Electrostatic Alterations at the Binuclear Centre of Cytochrome c Oxidase
Fluorescence and Infrared Spectroscopic Studies of Surface Protonation Dynamics at the K-channel Entrance and Redox-Linked Electrostatic Alterations at the Binuclear Centre of Cytochrome c Oxidase
Abstract: This thesis investigates surface protonation dynamics and electrostatic coupling in cytochrome c oxidase (CcO), the terminal enzyme of the electron transport chain and a key component of oxidative phosphorylation. Despite decades of research, critical aspects of proton transfer and its regulation remain unresolved.
The first part of the thesis focuses on the K-channel entrance to determine whether surface protonation dynamics at the K-channel entrance are governed by individual amino acids or by a cooperative network of residues forming a proton-collecting antenna (PCA). To achieve this, site-directed mutagenesis was employed to substitute key residues (histidine 526, histidine 73, and glutamate 78) with alanine, and proline 301 with cysteine, which was used for site-specific fluorescent labeling. Steady-state UV–Vis and fluorescence spectroscopy were then employed to monitor redox-dependent protonation dynamics and conformational changes. The results provide experimental evidence supporting the existence of a PCA, indicating an interconnected surface network that modulates proton affinity and facilitates proton delivery toward the K-channel.
The second part of the thesis examines the role of residue arginine 473, located near the binuclear center (BNC). This residue is proposed to be critical for coupling electron transfer to proton pumping through electrostatic interactions. Based on electrochemical infrared spectroscopy, the measurements show that arginine 473 is not essential for the overall function of the CcO protein, though it is necessary for maintaining optimal redox equilibrium and electron transport in the BNC. This implies that the effects are primarily due to local electrostatic changes.
Overall, this thesis elucidates how specific amino acids at the protein surface and in the BNC contribute to redox-coupled proton and electron transfer in CcO.
The first part of the thesis focuses on the K-channel entrance to determine whether surface protonation dynamics at the K-channel entrance are governed by individual amino acids or by a cooperative network of residues forming a proton-collecting antenna (PCA). To achieve this, site-directed mutagenesis was employed to substitute key residues (histidine 526, histidine 73, and glutamate 78) with alanine, and proline 301 with cysteine, which was used for site-specific fluorescent labeling. Steady-state UV–Vis and fluorescence spectroscopy were then employed to monitor redox-dependent protonation dynamics and conformational changes. The results provide experimental evidence supporting the existence of a PCA, indicating an interconnected surface network that modulates proton affinity and facilitates proton delivery toward the K-channel.
The second part of the thesis examines the role of residue arginine 473, located near the binuclear center (BNC). This residue is proposed to be critical for coupling electron transfer to proton pumping through electrostatic interactions. Based on electrochemical infrared spectroscopy, the measurements show that arginine 473 is not essential for the overall function of the CcO protein, though it is necessary for maintaining optimal redox equilibrium and electron transport in the BNC. This implies that the effects are primarily due to local electrostatic changes.
Overall, this thesis elucidates how specific amino acids at the protein surface and in the BNC contribute to redox-coupled proton and electron transfer in CcO.
Zeit & Ort
10.04.2026 | 16:00
Hörsaal A (1.3.14)
(Fachbereich Physik, Arnimallee 14, 14195 Berlin)