Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects

Fluorescence Correlation Spectroscopy (FCS) was used to investigate the exc ited-state properties of flavins and flavoproteins in solution at the singl e molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flav in cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in exper iments with very low light intensity ( < 10 kW/cm(2)), FMN and FAD displaye d fluorescence properties equivalent to those found with conventional fluor escence detection methods. Due to the high triplet quantum yield of FMN, th e system very soon starts to build up a population of non-fluorescent molec ules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photoblea ching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of F MN with the quenching agent potassium iodide at higher concentrations ( > 5 0 mM of I-) resulted in quenched flavin fluorescence as expected, low conce ntrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state., thereby improving the fluorescence signal. F CS experiments on FAD exhibited an improved photostability of FAD as compar ed to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoa mide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysic al relaxation or photochemical destruction of the enzyme. As the time const ant of the process induced at higher light intensities resembles the diffus ion time constant of free flavin, photodestruction with the concomitant rel ease of the cofactor offers a reasonable explanation. (C) 2001 Elsevier Sci ence B.V. All rights reserved.