It is confirmed by measurements of fluorescence spectra and quantum yields that the fluorescence in tolan originates from the same state that causes the absorption band at lowest energy. The temperature dependence of the fluorescence quantum yield shows that this state is thermally deactivated with an activation energy of Ea = 14.0 kJ/mol. Geometry optimizations of the states SO, SI, and TI of tolan with the semiempirical AM1 method lead to planar structures with Dzh symmetry. Potential energy curves along the triple-bond stretching coordinate have been calculated for several low-lying excited states with a combination of the AM1 and the INDO/S methods. It is found that for large triple-bond lengths, the llA,-state with UT* character becomes thelowest excited singlet state. It is proposed that thermal deactivation of SI( llBlu) leads to this state. Nonvertical excitation of 11A, could explain the weak lines found in supersonic jet experiments below the onset of the llA, -l1B1,, transition.

LOV domains are the light sensitive parts of phototropins and many other light-activated enzymes that regulate the response to blue light in plants and algae as well as some fungi and bacteria. Unlike all other biological photoreceptors known so far, the photocycle of LOV domains involves the excited triplet state of the chromophore. This chromophore is flavin mononucleotide (FMN) which forms a covalent adduct with a cysteine residue in the signaling state. Since the formation of this adduct from the triplet state involves breaking and forming of two bonds as well as a change from the triplet to the singlet spin state, various intermediates have been proposed, e.g. a protonated triplet state (3)FMNH(+), the radical anion (2)FMN˙(-), or the neutral semiquinone radical (2)FMNH˙. We performed an extensive search for these intermediates by two-dimensional transient absorption (2D-TA) with a streak camera. However, no transient with a rate constant between the decay of fluorescence and the decay of the triplet state could be detected. Analysis of the decay associated difference spectra results in quantum yields for the formation of the adduct from the triplet of ΦA(LOV1) ≈ 0.75 and ΦA(LOV2) ≈ 0.80. This is lower than the values ΦA(LOV1) ≈ 0.95 and ΦA(LOV2) ≈ 0.99 calculated from the rate constants, giving indirect evidence of an intermediate that reacts either to form the adduct or to decay back to the ground state. Since there is no measurable delay between the decay of the triplet and the formation of the adduct, we conclude that this intermediate reacts much faster than it is formed. The LOV1-C57S mutant shows a weak and slowly decaying (τ > 100 μs) transient whose decay associated spectrum has bands at 375 and 500 nm, with a shoulder at 400 nm. This transient is insensitive to the pH change in the range 6.5-10.0 but increases on addition of β-mercaptoethanol as the reducing agent. We assign this intermediate to the radical anion which is protected from protonation by the protein. We propose that the adduct is formed via the same intermediate by combination of the radical ion pair.

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A new data acquisition mode has been implemented to a velocity-map ion-imaging setup, which records the velocity distributions of molecular photofragments with vibrational and rotational resolution. Compared to conventional velocity-map ion-imaging, the acquired data are of remarkable brilliance. This allows for unambiguous assignment of the fragment quantum states and the analysis of all rotational bands apparent in the electronic transition of the molecular fragment. The acquisition time is the same as required for recording a REMPI spectrum of the photofragments. The method is illustrated by the measurement of the rotational state distribution of NO created in the photolytical decomposition of NO(2) at 225 nm. Different rotational distributions were observed for each vibrational state and for each of the four energetically accessible electronic channels.