Intrinsic Luminescence Studies on the Apoenzyme and Holoenzyme of Lipoamide Dehydrogenase.

The following fluorescence parameters have been investigated: emission and excitation spectra, relative quantum yields, polarization values and lifetimes of the apo- and holoenzyme of lipoamide dehydrogenase. Furthermore, fluorescence perturbation techniques have been used to characterize qualitatively the tryptophan and flavin environment in the protein. Also emission spectra at low temperature have been recorded to examine changes occurring on strong immobilization of the enzyme. From lifetimes and quantum yields of the protein fluorescence it can be concluded that the molecular structures of the two apoenzymes, which can be prepared from this enzyme, are different. The quenched protein fluorescence of the holoenzyme is characterized by a very short fluorescence lifetime due to radiationless energy transfer from tryptophan to flavin. Excitation polarization spectra of flavin and tryptophan fluorescences show that the chromophores are rigidly bound to the protein. Luminescence studies at liquid nitrogen temperatures reveal that the triplet state of the tryptophanyl-residues is not involved in energy transfer to the flavin. Collisional fluorescence quenching with KI shows in the dimeric holoenzyme, in contrast with the free molecules, only a small reduction in fluorescence intensity of tryptophanyl and flavin residues. Charge effects might be responsible for these observations. It can be derived from the experimental results together with the indication that only one of the two tryptophans is transferring its excitation energy to the flavin, that the distance between donor and acceptor is 1.3-1.6 nm.