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

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 (15)N 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 (15)N labelling. Moreover, an indirect isotope effect on the flavin C(4)=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.