Pigment-protein interactions in phytochromes probed by fluorescence line narrowing spectroscopy.

Fluorescence line narrowing (FLN) spectroscopy was used to study bacteriophytochromes and variants from various species in their red-absorbing Pr ground state, including phytochromes Agp1 from Agrobacterium tumefaciens , DrBphP from Deinococcus radiodurans , and RpBphP2 and RpBphP3 from Rhodopseudomonas palustris . A species-dependent narrowing of the fluorescence emission bands is observed. The results suggest varied pigment-protein interactions, possibly connected to chromophore mobility or extended water pyrrole networks inside of the differing binding pockets. Solvent water isotope exchange from H2O-based buffer to D2O-based buffer solutions was used to identify specific vibrational modes of the chromophore. In addition to the expected frequency shifts upon isotope exchange, the line narrowing efficiency is increased in deuterated compared to protonated surroundings. We conclude that proton dynamics inside of the protein binding pocket are a dominant source of spectral diffusion at low temperatures, which possibly relates to the previous observation that the electronic transition is directly coupled to proton transfer. The FLN spectra of Agp1 reconstituted with a synthesized pigment shows strong line narrowing efficiency even in protonated buffer solution. The FLN spectra of a point mutant of RpBphP3 highlight the involvement of aspartate 216 in a hydrogen bond network around the chromophore. On the basis of similar FLN characteristics in RpBphP2 and RpBphP3, we propose a similarly extended hydrogen bond network around their chromophores despite the different photoreactions leading to red- or blue-shifted absorption relative to the respective photoreceptors' ground-state absorption.