Enzymatic reduction of oxygen by small laccase. A rapid freeze-quench EPR study

Since the discovery of enzymes as biological catalysts, the mechanism of enzymatic reactions has been a key question to enzymologists. Elucidating the reaction kinetics and the nature of enzymatic intermediates are necessary to understand such reactions. An important challenge in these studies is the limited lifetime of such intermediates, usually on the time scale of milliseconds to seconds. Therefore, a suited trapping method is required. Rapid freeze-quench (RFQ) is a proven technique to trap the intermediates on the time scale of milliseconds. The RFQ technique has been mostly combined with electron paramagnetic resonance (EPR) spectroscopy to reveal the nature of the paramagnetic intermediates involved in enzymatic reactions. In this thesis, we improve and extend the combination of conventional RFQ technique with EPR spectroscopy up to a microwave frequency of 275 GHz to trap and characterize the intermediates involved in the enzymatic reduction of O2 by small laccase (SLAC).NWOBiological and Soft Matter Physic