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

Joachim Kübel,Saik Ann Ooi,Michał Maj,Valentyna Kuznetsova,Linnéa Isaksson,Sebastian Westenhoff,Janne A Ihalainen,Manoop Chenchiliyan,Emil Gustavsson

doi:10.1039/c9cp06995j

Abstract

Phytochromes are photosensory proteins in plants, fungi, and bacteria, which detect red- and far-red light. They undergo a transition between the resting (Pr) and photoactivated (Pfr) states. In bacterial phytochromes, the Pr-to-Pfr transition is facilitated by two intermediate states, called Lumi-R and Meta-R. The molecular structures of the protein in these states are not known and the molecular mechanism of photoconversion is not understood. Here, we apply transient infrared absorption spectroscopy to study the photocycle of the wild-type and Y263F mutant of the phytochrome from Deinococcus radiodurans (DrBphP) from nano- to milliseconds. We identify two sequentially forming Lumi-R states which differ in the local structure surrounding the carbonyl group of the biliverdin D-ring. We also find that the tyrosine at position 263 alters local structure and dynamics around the D-ring and causes an increased rate of Pfr formation. The results shed new light on the mechanism of light-signalling in phytochrome proteins.

Highlights

Triggers the reversible photochromic switch between the Pr and Pfr states.[9,10] by cycling between Pr and Pfr states, phytochromes act as a light-activated molecular switch that coordinates key biological processes, essential for the growth and sustainability of many organisms on Earth
We apply transient infrared absorption spectroscopy to study the photocycle of the wild-type and Y263F mutant of the phytochrome from Deinococcus radiodurans (DrBphP) from nanoto milliseconds
The dark-adapted Pr state of the Y263F mutant becomes red-shifted by about 2 nm, whereas the light-induced Pfr spectrum is blue-shifted by B10 nm with respect to the wild type (WT) spectrum

Summary

Introduction

Triggers the reversible photochromic switch between the Pr and Pfr states.[9,10] by cycling between Pr and Pfr states, phytochromes act as a light-activated molecular switch that coordinates key biological processes, essential for the growth and sustainability of many organisms on Earth. The D-ring in the chromophore undergoes Z to E isomerization that triggers a series of subsequent changes in the protein structure.[11] Previous studies show that, in bacterial phytochromes, the photocycle involves three intermediate states named Lumi-R, Meta-Ra and Meta-Rc.[12] The first intermediate, Lumi-R, forms from a short-lived excited state within hundreds of picoseconds.[13,14] In tens of microseconds, the Lumi-R thermally decays to the Meta-Ra state. By combining steady-state FTIR and fluorescence spectroscopy with the X-ray scattering results, we further proposed the possible existence of an alternative signaling routes to the tongue to accomplish the photocycle. In another recent report, we documented the photocycle pathways of the wild type (WT) DrBphP phytochrome using step-scan FTIR spectroscopy.[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]

Methods

Results

Discussion

Conclusion