Chromophore–Protein Interplay during the Phytochrome Photocycle Revealed by Step-Scan FTIR Spectroscopy

Abstract

Phytochrome proteins regulate many photoresponses of plants and microorganisms. Light absorption causes isomerization of the biliverdin chromophore, which triggers a series of structural changes to activate the signaling domains of the protein. However, the structural changes are elusive, and therefore the molecular mechanism of signal transduction remains poorly understood. Here, we apply two-color step-scan infrared spectroscopy to the bacteriophytochrome from Deinococcus radiodurans. We show by recordings in HO and DO that the hydrogen bonds to the biliverdin D-ring carbonyl become disordered in the first intermediate (Lumi-R) forming a dynamic microenvironment, then completely detach in the second intermediate (Meta-R), and finally reform in the signaling state (Pfr). The spectra reveal via isotope labeling that the refolding of the conserved "PHY-tongue" region occurs with the last transition between Meta-R and Pfr. Additional changes in the protein backbone are detected already within microseconds in Lumi-R. Aided by molecular dynamics simulations, we find that a strictly conserved salt bridge between an arginine of the PHY tongue and an aspartate of the chromophore binding domains is broken in Lumi-R and the arginine is recruited to the D-ring C═O. This rationalizes how isomerization of the chromophore is linked to the global structural rearrangement in the sensory receptor. Our findings advance the structural understanding of phytochrome photoactivation.

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

“…The lifetimes of the remaining SADS components, corresponding to the subsequent states in the photocycle, are in good agreement with the earlier reports. 6,16,28 We confirm that these three SADS represent the transitions between Lumi-R, Meta-R and Pfr states, respectively. The newly resolved extra state has spectral features similar to those of the Lumi-R state except the characteristic chromophore absorption above 1700 cm À1 .…”

“…The SADS corresponding to the Pfr state ( Fig. 5A) matches closely with the steady-state FTIR difference spectrum (Pfr minus Pr, 28 and Fig. S3, ESI †), which indicates that the complete photocycle has been resolved in the time window of the experiment.…”

“…In another recent report, we documented the photocycle pathways of the wild type (WT) DrBphP phytochrome using step-scan FTIR spectroscopy. 28 To unravel the exact photocycle routes of the DrBphP and its Y263F mutant, we employed ns Vis pump -fs mid-IR probe spectroscopy that follows the structural changes on the nanosecond to millisecond timescales. There are many studies on phytochromes in which transient IR techniques are applied; however, to the best of our knowledge, none of these achieved to follow the complete photocycle routes from Pr to Pfr states.…”

“…14,[29][30][31][32] The majority of the previous spectroscopic studies on phytochromes were carried out using either femtosecond (fs) laser spectroscopy, 13,14,30,33 (resonance) Raman, 10,11,34,35 or step-scan FTIR methods. 28 These techniques each have a different set of advantages, but the common problem is their rather limited experimental time window. In fs experiments, the delay between the visible and infra-red pulses is usually controlled by a mechanical translation stage, which allows reaching the relative delay of several nanoseconds, at most.…”

“…The higher resolution in our study allows us resolve the spectral features of each intermediate in the photocycle. 28 All the spectra were recorded at room temperature, which is fundamentally important for understanding the biologically significant dynamics within the intermediate states of the photocycle. 35,38 The experimental data identify a new state, which forms before the previously identified Lumi-R state in the photocycle.…”