A pulse fluorometry study of lipoamide dehydrogenase. Evidence for non-equivalent FAD centers.

The time dependence of the fluorescence of tryptophanyl and flavin residues in lipoamide dehydrogenase has been investigated with single-photon decay spectroscopy. When the two FAD molecules in the enzyme were directly excited the decay could only be analyzed in a sum of two exponentials with equal amplitudes. This phenomenon was observed at 4 degrees C (tau-1 = 0.8 ns, tau-2 = 4.7 ns) and at 20 degrees C (tau-1 = 0.8 ns, tau-2 = 3.4 ns) irrespective of the emission and excitation wavelengths. This result reveals a difference in the nature of the two FAD centers. By excitation at 290 nm the fluorescence decay curves of tryptophan and FAD were obtained. The decays are analyzed in terms of energy transfer from tryptophanyl to flavin residues. The results, which are in good agreement with those obtained previously with static fluorescence methods, show that one of the two tryptophanyl residues within the subunit transfers its excitation energy to the flavin located at a distance of 1.5 nm.