Evidence for Tautomerisation of Glutamine in BLUF Blue Light Receptors by Vibrational Spectroscopy and Computational Chemistry

Tatiana Domratcheva,Ilme Schlichting,Tilman Kottke,Elisabeth Hartmann

doi:10.1038/srep22669

Tatiana Domratcheva, Ilme Schlichting + Show 2 more

Open Access

https://doi.org/10.1038/srep22669

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Abstract

BLUF (blue light sensor using flavin) domains regulate the activity of various enzymatic effector domains in bacteria and euglenids. BLUF features a unique photoactivation through restructuring of the hydrogen-bonding network as opposed to a redox reaction or an isomerization of the chromophore. A conserved glutamine residue close to the flavin chromophore plays a central role in the light response, but the underlying modification is still unclear. We labelled this glutamine with 15N in two representative BLUF domains and performed time-resolved infrared double difference spectroscopy. The assignment of the signals was conducted by extensive quantum chemical calculations on large models with 187 atoms reproducing the UV-vis and infrared signatures of BLUF photoactivation. In the dark state, the comparatively low frequency of 1,667 cm−1 is assigned to the glutamine C=O accepting a hydrogen bond from tyrosine. In the light state, the signature of a tautomerised glutamine was extracted with the C=N stretch at ~1,691 cm−1 exhibiting the characteristic strong downshift by 15N labelling. Moreover, an indirect isotope effect on the flavin C4=O stretch was found. We conclude that photoactivation of the BLUF receptor does not only involve a rearrangement of hydrogen bonds but includes a change in covalent bonds of the protein.

Highlights

The blue light sensor using flavin (BLUF)[3] is a photosensory protein domain with a unique feature of preserving the chemical structure and, the UV-vis spectrum of its flavin chromophore in the two functional states[4,5]
Formation of the imidic acid of glutamine in the BLUF domain can in principle be detected experimentally by the strong effect of the specific 15N isotopic labelling
The dark state model postulating this glutamine orientation persists in the literature, because the Trp-in protein conformation of the dark state is in line with the influence of the tryptophan on the decay rate of the flavin excited state[32]

Summary

Introduction

The blue light sensor using flavin (BLUF)[3] is a photosensory protein domain with a unique feature of preserving the chemical structure and, the UV-vis spectrum of its flavin chromophore in the two functional states[4,5]. On the second to minutes time scale, depending on the particular BLUF protein, the red-shifted state undergoes thermal conversion back to the dark state. Initial interpretation of these light-induced spectral shifts was attempted on the basis of the crystal structures. The results of quantum chemical calculations linking experimental and theoretical spectra to reveal the glutamine structure in the dark and light states provided support for the opposing glutamine rotation and tautomerisation photoreactions[18,20,21,26,27,28,29]. We identified the spectral signature of the glutamine imidic acid tautomer in the light state

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