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Photoactive yellow protein (PYP) is a blue-light photoreceptor responsible for the negative phototaxis of the purple sulfur bacterium Ectothiorhodospira halophila . Photoexcitation of PYP with a blue light triggers a photocycle that involves several intermediate states. High-resolution gas-phase fluorescence excitation experiments performed on the neutral form of PYP's p CA chromophore involved excitation wavelength-dependent photochemistry. This is explained by the existence of a low-lying second electronic state ( n → π * origin). Based on steady state and transient absorption measurements, it is concluded that upon excitation, simultaneous population of those states may occur and the complex photophysics of PYP arise due to competing relaxation pathways from those states. This chapter uses femtosecond time-resolved fluorescence up-conversion spectroscopy to elucidate the quasi-degenerate nature of the PYP's excited state. Detailed fluorescence anisotropy measurements are carried out on native PYP, 3,4-dihydroxy cinnamic acid analogue, and several mutant PYP systems.

Ultrafast photoreactions in PNS of PYP have been studied by means of fs fluorescence up conversion method. Conclusions obtained are: (a) Photoreaction in PNS (chromophore twisting) occurs from vibrationally unrelaxed fluorescence state and coherent oscillations in the fluorescence decay curves have been observed for the first time. (b) Comparative studies on fluorescence dynamics of mutants and w.-t. PYP have proved that the w.-t. PYP is best engineered for the ultrafast reaction. (c) The coherent oscillations in the fluorescence decay completely disappeared and the reaction was much slower in the denatured state, demonstrating the supremely important role of PNS for the photoreaction.

Among various factors regulating the photo-induced ET reactions, the energy gap law dependence is a diagnostic tool in testing ET theories. Recently attempts were made to utilize a large energy gap for charge separation (CS) from S2 state of Zinc-porphyrin to directly linked electron acceptor (a series of imide compounds abbreviated as I) to confirm unambiguously the existence of the inverted region. By means of the detailed femtosecond fluorescence studies on the series of ZP-I compounds in polar solvents, such as, tetrahydrofurane (THE), triacetin (GTA) and acetonitrile (ACN), experts succeeded in demonstrating unequivocally as the first example for the photo-induced CS reaction the bell-shaped energy gap law (EGL) with a distinct inverted region. In addition, these results for EGL observed in polar solvents THE, GTA, ACN were reproduced satisfactorily by means of the Ulstrup-Jortner equation assuming the nonadiabatic mechanism, and the averaged single frequency hfm (intramolecular high frequency mode) coupled with CS reaction. This chapter confirms the modification of the EGL for the CS from S 2 induced by the change of solvent polarity by comparing the EGL in toluene (Tol) and methylcyclohexane (MCH) solution with that in THE. Further, this chapter compares the rate constants (λ p ) of the S 1 state formation by S 2 excitation with the decay rate constants (λ 1 ) of S 2 state in ZP-I series and examines solvent polarity effects on these rate constants comparing THE with Tol or MCH solutions.