Elucidation of Irreversible Reactions in Light Receptors and Enzymes by the Combination of an Infrared Quantum Cascade Laser with a Flow Cell System

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Time-resolved infrared spectroscopy on proteins in H2O with a resolution better than milliseconds is currently challenging because of the need for very large sample quantities or highly efficient reactions. We will employ a quantum cascade laser as an advanced probe light source with high emission power and broad tuning range to monitor time traces of reactions at single wavenumbers. The combination of the small focal area of the laser with a flow cell system for H2O established in our laboratory will allow us to gain access to irreversible photoreactions in proteins with low yields and with low sample consumption. The performance of the new setup will be thoroughly characterized after assembly in comparison to that of step-scan spectroscopy. The limits of the setup with respect to time resolution and noise level will be reached using the photoreactions of bacteriorhodopsin and free flavin as model reactions. The approach will be utilized to elucidate the mechanisms of two cryptochrome light receptors and a homologous DNA repair enzyme, the (6-4) photolyase. Cryptochromes regulate plant development, govern the daily rhythm of plants and insects, and act as magnetoreceptors. We will study the structural response of a plant cryptochrome to blue light under the influence of ATP binding. For direct comparison, the recently found animal-like cryptochrome aCRY will be characterized with respect to its remarkable response to red light. Furthermore, the repair activity of aCRY on (6-4) lesions in DNA under UVA light consumption will be exploited to elucidate the late steps in the repair mechanism. These reactions of homologous flavoproteins in three different oxidation states all have in common that they are irreversible and inefficient. Therefore, they have not been addressed previously by time-resolved infrared spectroscopy. The comprehensive study will significantly improve our understanding of the signaling pathways of cryptochromes and repair mechanisms of (6-4) photolyases. In particular, signal progression in different cryptochromes will be evaluated with respect to the concept of charge formation causing conformational changes versus the model of a rigid redox cascade.