The vibrational dynamics of the retinal chromophore all-trans–to–13-cis photoisomerization in bacteriorhodopsin has been studied with mid-infrared absorption spectroscopy at high time resolution (about 200 femtoseconds). After photoexcitation of light-adapted bacteriorhodopsin, the transient infrared absorption was probed in a broad spectral region, including vibrations with dominant C–C, C=C, and C=NH stretching mode amplitude. All photoproduct modes, especially those around 1190 reciprocal-centimeters that are indicative for a 13-cis configuration of the chromophore, rise with a time constant of ∼0.5 picosecond. The results presented give direct vibrational-spectroscopic evidence for the isomerization taking place within 0.5 picosecond, as has been suggested by previous optical femtosecond time-resolved experiments but questioned recently by picosecond time-resolved vibrational spectroscopy experiments.

The photoinduced low-spin (S = 0) to high-spin (S = 2) transition of the iron(II) spin-crossover systems [Fe(btpa)](PF₆)₂ and [Fe(b(bdpa))](PF₆)₂ in solution have been studied for the first time by means of ultrafast transient infrared spectroscopy at room temperature. Negative and positive infrared difference bands between 1000 and 1065 cm⁻¹ that appear within the instrumental system response time of 350 fs after excitation at 387 nm display the formation of the vibrationally unrelaxed and hot high-spin ⁵T₂ state. Vibrational relaxation is observed and characterized by the time constants 9.4 ± 0.7 ps for [Fe(btpa)](PF₆)₂/acetone and 12.7 ± 0.7 ps for both [Fe(btpa)](PF₆)₂/acetonitrile and [Fe(b(bdpa)](PF₆)₂/acetonitrile. Vibrational analysis has been performed via DFT calculations of the low-spin and high-spin state normal modes of both compounds as well as their respective infrared absorption cross sections. The simulated infrared difference spectra are dominated by an increase of the absorption cross section upon high-spin state formation in accordance with the experimental infrared spectra.

Phytochromes are widely distributed photoreceptors with a bilin chromophore that undergo a typical reversible photoconversion between the two spectrally different forms, Pr and Pfr. The phytochrome Agp2 from Agrobacterium tumefaciens belongs to the group of bathy phytochromes that have a Pfr ground state as a result of the Pr to Pfr dark conversion. Agp2 has untypical spectral properties in the Pr form reminiscent of a deprotonated chromophore as confirmed by resonance Raman spectroscopy. UV/visible absorption spectroscopy showed that the pK_a is >11 in the Pfr form and ∼7.6 in the Pr form. Unlike other phytochromes, photoconversion thus results in a pK_a shift of more than 3 units. The Pr/Pfr ratio after saturating irradiation with monochromatic light is strongly pH-dependent. This is partially due to a back-reaction of the deprotonated Pr chromophore at pH 9 after photoexcitation as found by flash photolysis. The chromophore protonation and dark conversion were affected by domain swapping and site-directed mutagenesis. A replacement of the PAS or GAF domain by the respective domain of the prototypical phytochrome Agp1 resulted in a protonated Pr chromophore; the GAF domain replacement afforded an inversion of the dark conversion. A reversion was also obtained with the triple mutant N12S/Q190L/H248Q, whereas each single point mutant is characterized by decelerated Pr to Pfr dark conversion.