The overall objective of the proposed work is to gain a fundamental understanding of how enzymes work by exploring single-molecule enzymology. The ubiquitous flavoproteins are responsible for a variety of redox reactions in metabolism. The natural fluorescence of flavin allows us to conduct single-molecule fluorescence measurements. Real-time observations of enzymatic turnovers of single cholesterol oxidase (COx) molecules have been made by monitoring the emission from the enzyme's fluorescent active site, flavin adenine dinucleotide (FAD). Statistical analyses revealed a significant slow fluctuation in the rate of cholesterol oxidation by FAD. The rate fluctuation originates from a slow conformational fluctuation. The proposed experiments will provide microscopic understanding and test the generality of the phenomena: (a) Spontaneous conformational fluctuations as well as substrate-induced conformational changes of COx will be probed by recording trajectories of fluorescence lifetimes of FAD.(b) Enzymatic turnovers of single COx on a cholesterol-containing membrane will be monitored so one can understand the detailed enzymatic mechanisms under physiological conditions. (c) The intersubunit interactions and cooperativity of dimeric flavoenzymes, such as glutathione reductase and glucose oxidase, will be studied. Although the proposed work is focused on flavoenzymes, the methodology will be useful for other enzymatic systems. This work and its variations will significantly enhance the knowledge of how protein dynamics influence enzymatic functions.
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