The experimentally obtained time-resolved fluorescence spectra of photosystem II (PS II) core complexes, purified from a thermophilic cyanobacterium Thermosynechococcus vulcanus, at 5–180 K are compared with simulations. Dynamic localization effects of excitons are treated implicitly by introducing exciton domains of strongly coupled pigments. Exciton relaxations within a domain and exciton transfers between domains are treated on the basis of Redfield theory and generalized Förster theory, respectively. The excitonic couplings between the pigments are calculated by a quantum chemical/electrostatic method (Poisson-TrEsp). Starting with previously published values, a refined set of site energies of the pigments is obtained through optimization cycles of the fits of stationary optical spectra of PS II. Satisfactorily agreement between the experimental and simulated spectra is obtained for the absorption spectrum including its temperature dependence and the linear dichroism spectrum of PS II core complexes (PS II-CC). Furthermore, the refined site energies well reproduce the temperature dependence of the time-resolved fluorescence spectrum of PS II-CC, which is characterized by the emergence of a 695 nm fluorescence peak upon cooling down to 77 K and the decrease of its relative intensity upon further cooling below 77 K. The blue shift of the fluorescence band upon cooling below 77 K is explained by the existence of two red-shifted chlorophyll pools emitting at around 685 and 695 nm. The former pool is assigned to Chl45 or Chl43 in CP43 (Chl numbering according to the nomenclature of Loll et al. Nature2005, 438, 1040) while the latter is assigned to Chl29 in CP47. The 695 nm emitting chlorophyll is suggested to attract excitations from the peripheral light-harvesting complexes and might also be involved in photoprotection.

To elucidate the dynamics and mechanisms of radiationless transitions from higher excited electronic states as well as the ultrafast intramolecular vibronic relaxation in porphyrin derivatives, we have studied the fluorescence dynamics of Zn-tetraphenylporphyrin (ZnTPP) and Zn-diphenylporphyrin derivatives (ZnDPP) in fs-ps time regimes by means of fluorescence up-conversion technique. Detailed measurements on ZnTPP in ethanol have demonstrated fluorescence dynamics over the whole spectral range from 430 to 620 nm when excited to the S 2 state. The time constant (∼2.3 ps) of the single-exponential decay of S 2 fluorescence around 430 nm agreed with that of the single-exponential rise of S 1 fluorescence around 600 nm (wavelength of 0-0 transition in the stationary spectrum), indicating that the relaxation by the ultrafast vibronic redistribution immediately after S 2 f S 1 internal conversion mainly gives lower vibronic states near the bottom of the S 1 state. However, we have observed the dynamics of weak hot fluorescence probably from the nonrelaxed vibronic state immediately after internal conversion and also higher vibronic states in S 1 formed in competition with the main product of the vibronic redistribution, all over the wavelength region between S 2 and S 1 . Preliminary results of our studies on ZnDPP were very similar to those of ZnTPP.

We have studied excited-state dynamics of “nonfluorescent” flavoproteins including riboflavin binding protein (RBP), d-amino acid oxidase benzoate complex (DAOB), and others by means of femtosecond fluorescence up-conversion method and have observed ultrafast fluorescence quenching dynamics for the first time. We have interpreted the fluorescence quenching mechanisms of these flavoproteins as due to the ultrafast electron transfer (ET) to flavin chromophore (F) in the excited electronic state from nearby tryptophan (Trp . NH) or tyrosine (Tyr . OH) residues placed in the protein nanospace (PNS), on the basis of their X-ray structures. Extremely fast fluorescence quenching in RBP (τf ∼ 90−100 fs) could be attributed to the compact stacked arrangement, Trp . NH.....F.....Tyr . OH, supremely favorable for the ultrafast ET reaction dynamics. Comparisons of fluorescence time profiles and spectral characteristics of F in solution with those in PNS have indicated the existence of extremely fast FC (Franck−Condon) → Fl (fluorescence) state conversion in PNS within the time resolution of the apparatus. The ultrafast FC → Fl conversion may be a coherent process coupled with intra-chromophore high-frequency modes leading to formation of vibrationally nonrelaxed or only partially relaxed Fl state, from which barrierless ET seems to occur. Fluorescence dynamics of DAOB have indicated faster initial decay in both blue and red sides of the spectrum contrary to other flavoproteins which showed practically wavelength-independent fluorescence dynamics. This result of DAOB is similar to those of photoactive yellow protein and visual rhodopsin although their reaction mechanism (twisting) is different from DAOB (ET). We have proposed a possible mechanism for this fluorescence dynamics of DAOB on the basis of an extremely compact stacked configuration of F...benzoate-...Tyr . OH which seems to undergo moderate frequency intermolecular vibration coupled with intra-chromophore high-frequency modes of F in the course of ET from Tyr . OH to excited F.

BLUF (a sensor of Blue-Light Using FAD) is a novel putative photoreceptor domain that is found in many bacteria and some eukaryotic algae. As found on genome analysis, certain cyanobacteria have BLUF proteins with a short C-terminal extension. As typical examples, Tll0078 from thermophilic Thermosynechococcus elongatus BP-1 and Slr1694 from mesophilic Synechocystis sp. PCC 6803 were comparatively studied. FAD of both proteins was hardly reduced by exogenous reductants or mediators except methylviologen but showed a typical spectral shift to a longer wavelength upon excitation with blue light. In particular, freshly prepared Tll0078 protein showed slow but reversible aggregation, indicative of light-induced conformational changes in the protein structure. Tll0078 is far more stable as to heat treatment than Slr1694, as judged from flavin fluorescence. The slr1694-disruptant showed phototactic motility away from the light source (negative phototaxis), while the wild type Synechocystis showed positive phototaxis toward the source. Yeast two-hybrid screening with slr1694 showed self-interaction of Slr1694 (PixD) with itself and interaction with a novel PatA-like response regulator, Slr1693 (PixE). These results were discussed in relation to the signaling mechanism of the "short" BLUF proteins in the regulation of cyanobacterial phototaxis.

Proteins with a BLUF (sensor of blue light using flavin adenine dinucleotide) domain represent a newly recognized class of photoreceptors that is widely distributed in the genomes of photosynthetic bacteria, cyanobacteria, and Euglena. Recently, Okajima et al. [Okajima, K., Yoshihara, S., Geng, X., Katayama, M. and Ikeuchi, M. (2003) Plant Cell Physiol. 44 (Suppl), 162] purified BLUF protein Tll0078 encoded in the genome of thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 by expressing the protein in Escherichia coli. We investigated the photocycle of Tll0078 by measuring the picosecond fluorescence kinetics, transient absorption changes, and the UV-visible absorption spectra at 10 to 330 K. The absorption spectrum of the FAD moiety of Tll0078 showed a 10-nm red shift upon illumination at 278-330 K. The quantum efficiency of the formation of the red-shifted form was 29%. Illumination at 10 K, on the other hand, caused only a 5-nm red shift in about one-half of the protein population. The 5-nm-shifted form was stable at 10 K. The 5-nm red-shifted form was converted into the 10-nm red-shifted form at 50-240 K upon warming in the dark. At room temperature, the 10-nm red-shifted final product appeared within 10 ns after laser flash excitation. The lifetime of the fluorescence of FAD was found to be 120 ps at room temperature. These results reveal a fast and efficient photoconversion process from the singlet-excited state to the final product at room temperature. A photocycle of BLUF protein is proposed that includes the 5-nm red-shifted intermediate form as the precursor for the 10-nm red-shifted final product. The temperature dependence of each step of the photocycle is also discussed.