Tyrosine 263 in Cyanobacterial Phytochrome Cph1 Optimizes Photochemistry at the prelumi‐R→lumi‐R Step

Abstract

We report a low-temperature fluorescence spectroscopy study of the PAS-GAF-PHY sensory module of Cph1 phytochrome, its Y263F mutant (both with known 3D structures) as well as Y263H and Y263S to connect their photochemical parameters with intramolecular interactions. None of the holoproteins showed photochemical activity at low temperature, and the activation barriers for the Pr→lumi-R photoreaction (2.5-3.1 kJ mol(-1)) and fluorescence quantum yields (0.29-0.42) were similar. The effect of the mutations on Pr→Pfr photoconversion efficiency (ΦPr→Pfr) was observed primarily at the prelumi-R S0 bifurcation point corresponding to the conical intersection of the energy surfaces at which the molecule relaxes to form lumi-R or Pr, lowering ΦPr→Pfr from 0.13 in the wild type to 0.05-0.07 in the mutants. We suggest that the Ea activation barrier in the Pr* S1 excited state might correspond to the D-ring (C19) carbonyl - H290 hydrogen bond or possibly to the hindrance caused by the C13(1) /C17(1) methyl groups of the C and D rings. The critical role of the tyrosine hydroxyl group can be at the prelumi-R bifurcation point to optimize the yield of the photoprocess and energy storage in the form of lumi-R for subsequent rearrangement processes culminating in Pfr formation.

Transient IR spectroscopy identifies key interactions and unravels new intermediates in the photocycle of a bacterial phytochrome

“…The presence of Pfr-like secondary structure in the Pr state of the mutant reduces the degree of structural reorganization needed for the photoconversion. As suggested for the Y263F mutant of DrBphP, 27 and previously for the Y263F mutant of Cph1, 51 we observe a reduced quantum yield for the Lumi-R formation. Noteworthy, the scaling factor of 2.5, which was applied to the mutant spectra shown in Fig.…”

“…5, is consistent with the relative quantum yield determined in the case of the related Cph1 mutant. 51 Nonetheless, our data show that similar dynamics occur up to the meta-R state. This can be interpreted in terms of redundant signalling components in DrBphP: despite the decoupling of a specific signaling route, such as tongue refolding, the protein remains photo-switchable.…”

Influence of Heterogeneity on the Ultrafast Photoisomerization Dynamics of Pfr in Cph1 Phytochrome

“…A loss of the additional hydrogen bonds should result in a disappearance of the two ground states. Up to now, only the photodynamics of the Pr form of Cph1 mutants at position 263 were investigated . The signals at 1635 cm −1 (−)/1660 cm −1 (+) were observed with a time constant of 5 ps only.…”

Fast Photochemistry of Prototypical Phytochromes—A Species vs. Subunit Specific Comparison

“…With NMR-studies (which probes solely the electronic ground states), Song et al ( 2011 ) stated the presence of multiple Pr states in the Cph1 system, whereas the Pfr state is homogenous. Also low temperature single-molecule spectroscopic and site-specific fluorescence experiments indicate heterogeneity in the Pr state in several species (Nieder et al, 2009 , 2013 ; Sineshchekov et al, 2014 ; Yang et al, 2014 ). In fact, Nieder et al revealed, in addition to the heterogeneity between individual particles, spectral diffusion among single particles (Nieder et al, 2009 ).…”