Probing of carotenoid-tryptophan hydrogen bonding dynamics in the single-tryptophan photoactive Orange Carotenoid Protein

Eugene G Maksimov,Alexander Jelzow,Evgeny A Shirshin,Anton I Maydykovskiy,Elena A Protasova,Igor A Yaroshevich,Marcus Moldenhauer,Yury B Slonimskiy,Thomas Friedrich,Nikolai N Sluchanko,Georgy V Tsoraev,Timofey S Gostev

doi:10.1038/s41598-020-68463-8

Abstract

The photoactive Orange Carotenoid Protein (OCP) plays a key role in cyanobacterial photoprotection. In OCP, a single non-covalently bound keto-carotenoid molecule acts as a light intensity sensor, while the protein is responsible for forming molecular contacts with the light-harvesting antenna, the fluorescence of which is quenched by OCP. Activation of this physiological interaction requires signal transduction from the photoexcited carotenoid to the protein matrix. Recent works revealed an asynchrony between conformational transitions of the carotenoid and the protein. Intrinsic tryptophan (Trp) fluorescence has provided valuable information about the protein part of OCP during its photocycle. However, wild-type OCP contains five Trp residues, which makes extraction of site-specific information impossible. In this work, we overcame this problem by characterizing the photocycle of a fully photoactive OCP variant (OCP-3FH) with only the most critical tryptophan residue (Trp-288) in place. Trp-288 is of special interest because it forms a hydrogen bond to the carotenoid’s keto-oxygen to keep OCP in its dark-adapted state. Using femtosecond pump-probe fluorescence spectroscopy we analyzed the photocycle of OCP-3FH and determined the formation rate of the very first intermediate suggesting that generation of the recently discovered S* state of the carotenoid in OCP precedes the breakage of the hydrogen bonds. Therefore, following Trp fluorescence of the unique photoactive OCP-3FH variant, we identified the rate of the H-bond breakage and provided novel insights into early events accompanying photoactivation of wild-type OCP.

Highlights

The regulation of excitation energy flows in photosynthetic organisms plays a crucial role for improving biomass production in changing environmental conditions[1]
Following the fluorescence readout of a fully photoactive Orange Carotenoid Protein (OCP) variant with only the critical Trp-288 left in place, we identified the rate of the hydrogen bond breakage which allowed us to associate the formation of the first intermediate of the OCP photocycle with a specific carotenoid excited state
Since the hydrodynamic properties of OCP-3FH and wild-type OCP are identical, we conclude that the dark-adapted 3FH protein stays in a compact state, which is not disturbed by the introduced mutations

Summary

Introduction

The regulation of excitation energy flows in photosynthetic organisms plays a crucial role for improving biomass production in changing environmental conditions[1]. As the carotenoid molecule is insoluble in water and cannot discriminate between normal and high light conditions, a special 35 kDa watersoluble protein is required to modulate its quenching abilities and to deliver the quencher to the phycobilisome core in order to control primary photosynthetic processes. This pigment–protein complex is called the Orange Carotenoid Protein (OCP)[23,24,25,26,27]. The involvement of the enigmatic S* state in the primary stages of OCP photoactivation appears very appealing, additional experimental approaches are necessary to prove or disprove a correlation with the breaking of hydrogen bonds on the same timescale (tens of picoseconds)

Methods

Results

Conclusion