Primary Photoprocesses Involved in the Sensory Protein for the Photophobic Response ofBlepharisma japonicum

Abstract

We present new femtosecond transient-absorption and picosecond fluorescence experiments performed on OBIP, the oxyblepharismin-binding protein believed to trigger the photophobic response of the ciliate Blepharisma japonicum. The formerly identified heterogeneity of the sample is confirmed and rationalized in terms of two independent populations, called rOBIP and nrOBIP. The rOBIP population undergoes a fast photocycle restoring the initial ground state in less than 500 ps. Intermolecular electron transfer followed by electron recombination is identified as the excited-state decay route. The experimental results support the coexistence of the oxyblepharismin (OxyBP) radical cation signature with a stimulated-emission signal at all times of the evolution of the transient-absorption spectra. This observation is interpreted by an equilibrium being reached between the locally excited state and a charge-transfer state on the ground of a theory developed by Mataga and co-workers to explain the fluorescence quenching of aromatic hydrogen-bonded donor-acceptor pairs in nonpolar solvents. OxyBP is supposed to bind to an as yet unknown electron acceptor by a hydrogen-bond (HB) and the coordinate along which forward and backward electron transfer proceed is assumed to be the shift of the HB proton. The observed kinetic isotope effect supports this interpretation. Protein relaxation is finally proposed to accompany the whole process and give rise to the highly multiexponential observed dynamics. As previously reported, the fast photocycle of rOBIP can be interpreted as an efficient sunscreen mechanism that protects Blepharisma japonicum from continuous irradiation. The nrOBIP population, the transient-absorption of which strongly reminds that of free OxyBP in solution, might be proposed to actually trigger the photophobic response of the organism through excited-state deprotonation of the chromophore occurring in the nanosecond regime. Additional femtosecond transient-absorption spectra of OxyBP and peri-deprotonated OxyBP are also reported and used as a comparison basis to interpret the results on OBIP.

Ultrafast photoinduced flavin dynamics in the unusual active site of the tRNA methyltransferase TrmFO

“…These are typical features of excited FAD ox (FAD ox *) already reported for other oxidized flavoproteins 26,36,40,47,48 and free FAD in solution. 39,49 From 0.3 to 1.8 ps (Figure 1A) the intensity of all bands decrease with the notable exception of the TA 2 band that increases until 0.9 ps before starting to decrease (Figure 1A). Simultaneously the TA 1 band decreases, more rapidly on its blue side (365 nm) than on its red side (400 nm), thereby acquiring a broader shape.…”

“…14,16 The steady-state absorption spectra of the samples used for transient absorption spectroscopy are provided in ESI, Section S2 (Figure S2). All BsTrmFO mutants essentially exhibit the characteristic bands of FAD ox , 36,[38][39][40] here situated around 375 and 445 nm. The WT spectrum additionally contains a contribution of the methylene-FAD adduct, around 360 nm.…”

“…Complementary recordings were made under excitation at 405 nm, obtained by frequency doubling of the laser source beam in a BBO crystal. 39 It was checked by comparison with a reference sample of [Ru(bpy) 3 ] 2+ complex 41,42 that the fraction of excited molecules in the probed volume was about 4%, ensuring excitation in the linear regime. Experiments under 475-nm excitation were performed with polarized probe, 36 thereby allowing access to transient anisotropy data in addition to the standard isotropic transient absorption spectra (see below).…”

“…The experiments under 405-nm excitation were performed at the magic angle configuration of pump and probe polarizations, which only provides isotropic spectra. 39 The samples were contained in 1-mm optical path quartz cells (width: 2 mm, containing ~60 µL)…”

Squarylium‐Triazine Dyad as a Highly Sensitive Photoradical Generator for Red Light

“…The kinetic analysis of the spectro‐temporal data was performed by singular value decomposition 30–32. The best fit of the four first weighted orthogonal kinetics leads to three time constants and one step function.…”