The investigation of interactions in the excited state of flavins using time-resolved spectroscopy

In paper I the results obtained with a very short (3 nsec) intense laser pulse as excitation source are described. This pulse excites such a large amount of flavin molecules into higher excited singlet and triplet states that changes in absorption of these higher excited states can be analyzed with a second less intense light flash of longer duration (5 μsec) fired synchronously with the laser pulse. The time-resolved absorption spectra obtained in this way were analyzed very carefully. When the pH was varied it was concluded that at 50 nsec after the laser pulse at least two species were present. One species could be identified as a flavin triplet. From photochemical and other data a structure for the second species was proposed, assuming the presence of flavin dimers. In such a flavin complex a very fast photoreaction occurs resulting in a photodimer, which is stable only in the excited state.In paper II another pulse technique (single photon counting) was applied to the free nucleotides FMN and FAD. These flavins were excited with a repetitive cycle of short (2 nsec) weak light flashes and the fluorescence response was measured as a function of both wavelength and time. From these time-correlated fluorescence spectra several conclusions regarding the interaction of adenine and flavin moieties (in FAD) were drawn. The most important outcome was that only the open conformation of FAD is fluorescent. An appendix (1) is added, which treats the theory and method of the time-resolved fluorescence relevant to the FAD-system.Paper III describes the results obtained with a flavoprotein (lipoamide dehydrogenase). Especially energy transfer experiments from tryptophan residues to the flavin indicate a crude distance and relative orientation between the 2 chromophores. In this study the time dependence of the tryptophan (donor) fluorescence was important. This was obtained from the phase delay of the fluorescence response on sinusoidally modulated exciting light.Paper IV gives independent evidence from the single photon counting method that the distance between a tryptophan-flavin couple is in the same order of magnitude as calculated from static analysis (paper III). Furthermore the experiments indicate that not all the tryptophans are involved in energy transfer, a result also found in paper III. The formulas describing the fluorescence decays of donor and acceptor molecules are summarized in appendix 2. A close examination of the flavin fluorescence decay demonstrates that the two FAD's occupy different sites in the dimeric protein. Several experimental tests for establishing this inequality are treated in appendix 1. The most interesting observations and significant results are discussed at the end of this thesis