Revealingthe Functional States in the Active Siteof BLUF Photoreceptors from Electrochromic Shift Calculations

摘要

Photoexcitation with blue light of the flavin chromophore in BLUF photoreceptors induces a switch into a metastable signaling state that is characterized by a red-shifted absorption maximum. The red shift is due to a rearrangement in the hydrogen bond pattern around Gln63 located in the immediate proximity of the isoalloxazine ring system of the chromophore. There is a long-lasting controversy between two structural models, named Q63A and Q63J in the literature, on the local conformation of the residues Gln63 and Tyr21 in the dark state of the photoreceptor. As regards the mechanistic details of the light-activation mechanism, rotation of Gln63 is opposed by tautomerism in the Q63A and Q63J models, respectively. We provide a structure-based simulation of electrochromic shifts of the flavin chromophore in the wild type and in various site-directed mutants. The excellent overall agreement between experimental and computed data allows us to evaluate the two structural models. Compelling evidence is obtained that the Q63A model is incorrect,whereas the Q63J is fully consistent with the present computations.Finally, we confirm independently that a keto–enol tautomerizationof the glutamine at position 63, which was proposed as molecular mechanismfor the transition between the dark and the light-adapted state, explainsthe measured 10 to 15 nm red shift in flavin absorption between thesetwo states of the protein. We believe that the accurateness of ourresults provides evidence that the BLUF photoreceptors absorptionis fine-tuned through electrostatic interactions between the chromophoreand the protein matrix, and finally that the simplicity of our theoreticalmodel is advantageous as regards easy reproducibility and furtherextensions.