Multicopper oxidase, laccase, can efficiently reduce oxygen to water and are mostly used in the enzymatic biofuel cells. However, they suffer from low stability when functionalized over an... Show moreMulticopper oxidase, laccase, can efficiently reduce oxygen to water and are mostly used in the enzymatic biofuel cells. However, they suffer from low stability when functionalized over an electrode. This can be overcome by designing artificial catalysts for the oxygen reduction reaction based on the active site of laccase which requires a detailed understanding of the active site. The current research is aimed at characterizing the active site of small laccase from Streptomyces coelicolor using a combination of paramagnetic NMR spectroscopy, EPR spectroscopy, mutagenesis and quantum mechanical (QM) calculations. The presence of chemical exchange at the active site of laccase attributed to the coordinating histidines is reported. QM calculations showed the importance of the orientation of the coordinating water derived ligand. Mutagenesis study showed the importance of second shell residue in stabilizing intermediates during the oxygen reduction reaction. It is also reported that by changing the pH, a new intermediate could be experimentally observed however, further research is needed to characterize this. The resonance assignment shown in the current research can be used as spies to characterize the active site of laccase. This might in future provide insight into the catalytic mechanism of oxygen reduction reaction by laccase. Show less
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... Show moreSince 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). Show less
