Elucidation of Irreversible Reactions in Light Receptors and Enzymes by the Combination of an Infrared Quantum Cascade Laser with a Flow Cell System
Final Report Abstract
Time-resolved infrared spectroscopy provides valuable information on the mechanisms of proteins in solution. Irreversible reactions in photoreceptors and enzymes are challenging to study because a manifold repeatability for averaging is not possible. To access these reactions, we developed a setup using a modern quantum cascade laser (QCL) with broad tunability and high power as a probe light. Sample consumption was minimized by focusing the laser into a flow cell and using a micrometer stage for moving the sample perpendicular to the flow. We recorded spectra and kinetics of the irreversible photoreduction of flavin in H2O in the time range of 20 nanoseconds to 1 second with a sample consumption of only 12 microliters. These included a reference spectrum for the flavin anion radical as a common intermediate in enzymatic reactions. In an experimental comparison of the QCL setup to a dual frequency comb spectrometer, we show the complementarity of the two modern approaches. The latter allowed us for the first time to characterize a protein reaction in a single shot, whereas the former achieves a higher time resolution and accesses a much broader spectral range. The QCL setup was then successfully applied to the photoreceptors plant cryptochrome and animal- like cryptochrome in H2O, which bind flavin as a chromophore and regulate central responses of green algae to light. We demonstrate that key changes in secondary structure in the plant cryptochrome occur only later than 100 ns and are therefore decoupled from the femtosecond electron transfer. In conclusion, the developed setup using a quantum cascade laser as probe light strongly facilitates the elucidation of irreversible processes in photoreceptors and cofactors in H2O by strongly reducing sample consumption.
Publications
- (2018) Single-Shot Sub-microsecond Mid-infrared Spectroscopy on Protein Reactions with Quantum Cascade Laser Frequency Combs, Anal. Chem. 90, 10494-10500
J. L. Klocke, M. Mangold, P. Allmendinger, A. Hugi, M. Geiser, P. Jouy, J. Faist and T. Kottke
(See online at https://doi.org/10.1021/acs.analchem.8b02531) - (2020) A Quantum Cascade Laser Setup for Studying Irreversible Photoreactions in H2O with Nanosecond Resolution and Microlitre Consumption, Phys. Chem. Chem. Phys. 22, 26459-26467
J. L. Klocke and T. Kottke
(See online at https://doi.org/10.1039/D0CP03164J)
