Thema der Dissertation:
Infrared Spectroscopic Studies of Experimentally Accessible Intermediate States in Cytochrome c Oxidase
Infrared Spectroscopic Studies of Experimentally Accessible Intermediate States in Cytochrome c Oxidase
Abstract: In living cells, the controlled translocation of protons across biological membranes is essential for energy conversion. Infrared (IR) spectroscopy is one of the few techniques capable of detecting protonation reactions.
Protonation dynamics often occur alongside electron transfer in a process called proton-coupled electron transfer, which drives many key biochemical reactions. Among these is the final step of aerobic respiration in the mitochondrial respiratory chain, where cytochrome c oxidase (CcO) reduces molecular oxygen to water while pumping protons across the inner mitochondrial membrane. This dual action removes harmful oxygen radicals and generates the proton motive force that drives ATP synthesis.
These reactions occur on the scale of individual amino acids and cofactors, and within nanoseconds to milliseconds, making them difficult to track in real time. In this thesis, I applied reaction-induced IR difference spectroscopy to monitor protonation dynamics in CcO with high molecular specificity. Experiments were conducted primarily using a newly developed time-resolved IR spectroscopy setup based on external cavity quantum cascade lasers with high brilliance, enabling nanosecond probing of liquid samples. Complementary measurements employed a homebuilt in situ setup with commercial FTIR spectrometers, allowing controlled reactant delivery and monitoring under turnover conditions.
Two ground states of CcO, namely its fully reduced form and its two-electron reduced mixed-valence form, were used to target different mechanistic questions. Carbon monoxide served as a sensitive vibrational probe of the enzyme’s active center. By photolyzing CO-bound states, I captured the earliest molecular events in the catalytic cycle, linking vibrational changes to redox reactions and directional proton transport. The results show that the protonation of a key glutamic acid residue (E286) is governed by the redox state of heme a, supporting its role as a molecular valve that directs proton flow at critical steps.
These findings advance our understanding of how CcO coordinates proton transfer during its catalytic cycle and establish methodological approaches that combine CO vibrational probing, in situ reaction control, and time-resolved IR spectroscopy, which can be applied to other complex membrane proteins.
Protonation dynamics often occur alongside electron transfer in a process called proton-coupled electron transfer, which drives many key biochemical reactions. Among these is the final step of aerobic respiration in the mitochondrial respiratory chain, where cytochrome c oxidase (CcO) reduces molecular oxygen to water while pumping protons across the inner mitochondrial membrane. This dual action removes harmful oxygen radicals and generates the proton motive force that drives ATP synthesis.
These reactions occur on the scale of individual amino acids and cofactors, and within nanoseconds to milliseconds, making them difficult to track in real time. In this thesis, I applied reaction-induced IR difference spectroscopy to monitor protonation dynamics in CcO with high molecular specificity. Experiments were conducted primarily using a newly developed time-resolved IR spectroscopy setup based on external cavity quantum cascade lasers with high brilliance, enabling nanosecond probing of liquid samples. Complementary measurements employed a homebuilt in situ setup with commercial FTIR spectrometers, allowing controlled reactant delivery and monitoring under turnover conditions.
Two ground states of CcO, namely its fully reduced form and its two-electron reduced mixed-valence form, were used to target different mechanistic questions. Carbon monoxide served as a sensitive vibrational probe of the enzyme’s active center. By photolyzing CO-bound states, I captured the earliest molecular events in the catalytic cycle, linking vibrational changes to redox reactions and directional proton transport. The results show that the protonation of a key glutamic acid residue (E286) is governed by the redox state of heme a, supporting its role as a molecular valve that directs proton flow at critical steps.
These findings advance our understanding of how CcO coordinates proton transfer during its catalytic cycle and establish methodological approaches that combine CO vibrational probing, in situ reaction control, and time-resolved IR spectroscopy, which can be applied to other complex membrane proteins.
Time & Location
Sep 22, 2025 | 02:00 PM
Hörsaal A (1.3.14)
(Fachbereich Physik, Arnimallee 14, 14195 Berlin)