T-CYCLE EPR Development at 275 GHz for the study of reaction kinetics & intermediates

A difficulty of studies on chemical kinetics are the reaction time scales and detection of their intermediates. Rapid Freeze-Quench (RFQ) is one of the most common techniques to investigate chemical kinetics. Since the intermediates of many reactions are paramagnetic, coupling RFQ to Electron Paramagnetic Resonance (EPR) is a desirable goal, especially at high-frequency (HF-EPR). HF-EPR offers high resolution and better spectral definition. However, collection of RFQ samples for HF-EPR is troublesome. In Chapter 2, the successful coupling of RFQ to HF-EPR at 275 GHz is described. Chapter 3 describes the development of Temperature-Cycle EPR (T-Cycle EPR), a novel high-frequency EPR technique that couples laser-induced T-jumps of the sample to a high-frequency 275 GHz EPR spectrometer, to detect short-lived paramagnetic intermediates and kinetics of chemical reactions in aqueous solutions. Chapter 4 discusses the application of T-Cycle EPR on a model reaction unfolding over hundreds of milliseconds, proving the technique is suitable to study many (bio)chemical systems. Chapter 5 shows an attempt to apply T-Cycle EPR to an enzymatic system on the sub-second time. T-Cycle EPR experiments at 275 GHz are performed on the reoxidation of a mutant of Small Laccase in the sub-second time regime, without making use of RFQ.