FTIR study on the hydrogen bond structure of a key tyrosine residue in the flavin-binding blue light sensor TePixD from Thermosynechococcus elongatus.

The BLUF (sensor of blue light using FAD) domain is a blue light receptor possessing a flavin molecule as an active cofactor. A conserved Tyr residue located adjacent to flavin has been proposed to be a key amino acid in the mechanism of the photoreaction of the BLUF domain. We have studied the structure of this key Tyr residue and the relevance to the photoreaction in the BLUF protein of the cyanobacterium Thermosynechococcus elongatus, TePixD, by means of Fourier transform infrared (FTIR) difference spectroscopy and density functional theory (DFT) calculations. Light-induced FTIR difference spectra of unlabeled and [4-13C]Tyr-labeled TePixD in H2O and D2O revealed that the nuCO/deltaCOH vibrations of a photosensitive Tyr side chain are located at 1265/1242 cm-1 in the dark-adapted state and at 1273/1235 cm-1 in the light-induced signaling state. These signals were assigned to the vibrations of Tyr8 near flavin from the absence of the effect of [4-13C]Tyr labeling in the Tyr8Phe mutant. DFT calculations of H-bonded complexes of p-cresol with amides as models of the Tyr8-Gln50 interactions showed that Tyr8 acts as a H-bond donor to the Gln50 in both of the dark and light states. Further DFT analysis suggested that this H-bond is strengthened upon photoconversion to the light state accompanied with a change in the H-bond angle. The change in the H-bond structure of Tyr8 is coupled to the flavin photoreaction probably through the Tyr8-Gln50-flavin H-bond network, suggesting a significant role of Tyr8 in the photoreaction mechanism of TePixD.