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Fluorescence Correlation Spectroscopy on Electron Transfer Reaction: Probing Inter- and Intramolecular Redox Processes

Promotores: G.W. Canters, T.J. Aartsma

S. Sen
30 juni 2016
Thesis in Leiden Repository

We developed a new FRET-based technique, “Fluredox”, which allows fluorescence readout of the redox state of oxido-reductases at single molecule level. Commercially available red-absorbing fluorophore ATTO655 was selected for labeling Azurin, a small blue mononuclear copper protein. Single molecule fluorescence correlation spectroscopy (FCS) of the fluorescently labeled Copper azurin in solution reveals how the position of the label in the 3-D structure of the protein affects the redox kinetics of the redox center as well as the label. Under certain redox conditions, we have been able to observe a microsecond dynamics for intramolecular ET reaction between the label and the metal center in azurin. Our results show that this FRET technique can be profitably used to study the enzyme activity of dye-labeled oxidoreductases.