RESUMO
TePixD is a blue light using flavin (BLUF) protein of a thermophilic cyanobacterium, Thermosynechococcus elongatus. The fluorescence dynamics of TePixD was observed for the first time in both its dark-adapted and signaling (red-shifted) forms with a 200-fs time resolution. The fluorescence up-conversion setup was used in the time region up to 60 ps, and the streak-camera setup was used in the time region up to 1 ns. To avoid the accumulation of the red-shifted form by the exciting laser irradiation, the sample solution was circulated using a diaphragm pump. A handmade flow cuvette with a small cross section was used to achieve a fast flow of the solution in the excited region. The fluorescence decay times were unequivocally determined to be 13.6 and 114 ps for the dark-adapted form and 1.37 ps for the red-shifted form. The double-exponential fluorescence decay in the dark-adapted form suggested the coexistence of two conformations that have the 13.6- and 114-ps decay components, respectively. The single-exponential fluorescence decay in the red-shifted form suggested the elimination of heterogeneity in the conformation upon the light-induced conversion. The fast fluorescence-quenching components were almost eliminated in the mutant in which the conserved tyrosine Tyr8 is replaced by phenylalanine. Thus, the fluorescence quench was concluded to arise from the electron transfer from Tyr8, to the excited flavin chromophore. The 10-fold-faster quenching in the red-shifted form suggested the acceleration of the electron transfer. The faster decay time of 13.6 ps for the dark-adapted form was found to be almost temperature independent in the region from 10 to 40 degrees C. This suggested that the energy gap, DeltaG, in Marcus's electron-transfer theory is optimized to give the fastest rate. The acceleration of the electron transfer in the red-shifted form is interpreted to be due to the enhancement of the electronic-coupling factor between the donor and acceptor. A shortening of the Tyr8-flavin distance by 1.0-1.5 A was suggested if we adopt the empirical formula for the donor-acceptor distance dependence of the electron transfer rate.
