Fluorescence and optical characteristics of reduced flavines and flavoproteins

Abstract

The fluorescence and absorption properties of a series of reduced flavoproteins have been measured and compared with the properties of suitable model compounds.<br />Contrary to common belief, a number of reduced flavoproteins have been found to exhibit appreciable fluorescence emission with maxima in the range 500-530 nm. In keeping with common observation, the reduced model flavines are devoid of fluorescence in solution at room temperature, but show marked fluorescence emission in the range 476-512 nm at 77°K in rigid glasses. The fluorescence quantum yield of reduced lactate oxidase (emission λmax 507 nm) is increased 4.7 times upon formation of covalent N5 adducts and the emission maximum is shifted to 476 nm. In the case of the nonfluorescent reduced D-amino acid oxidase, formation of covalent adducts leads to appearance of a fluorescence emission maximal at 520 nm. Among the enzymes investigated the absorption spectra of the reduced forms vary markedly. The chromophore of the reduced flavine was found to be very sensitive to variations of the polarity, pH, or viscosity of the solvent and to substitution. An attempt is made to interpret the absorption and fluorescence emission spectra and to correlate the mentioned effects. Thus valid information about the protein environment at the flavine binding site may be obtained. In particular, in the case of fluorescent-reduced enzymes, the vibrational mobility of the reduced flavocoenzyme is restricted by the protein.