FTIR Study on the Hydrogen Bond Structure of a Key Tyrosine Residue in the Flavin-Binding Blue Light Sensor TePixD fromThermosynechococcus elongatus

Abstract

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.

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

“…The X-ray datasets of OaPAC (dark-state PDB 4YUS, and this work PDB 5X4T) allow the atomic temperature factors to be refined and imply that the Oe1 atom of Gln-48 receives a hydrogen bond from the Tyr-6 hydroxyl group throughout the photocycle. This inference matches the conclusion from FTIR studies of TePixD (29) and AppA (30), which show that a hydrogen bond donated by the tyrosine to the glutamine oxygen becomes stronger on photoactivation. The oxygen-oxygen distance in the OaPAC X-ray models falls from 2.82 Å in the dark state to 2.69 Å after illumination, but the difference is barely significant, given the atomic positional error.…”

Seeing the light with BLUF proteins

“…Anderson and colleagues (Anderson et al 2005) noted that such a flip might explain the spectroscopically detected changes in hydrogen bonding to the flavin Unno et al 2006). However, Fourier transform infrared (FTIR) spectroscopy studies of the blue-light photoreceptor TePixD (Takahashi et al 2007) and AppA (Iwata et al 2011) indicate that the hydrogen bond donated by the tyrosine to the glutamine oxygen becomes stronger upon photo-activation, which is consistent with the proposal that the glutamine residue forms an imidic tautomer as the hydrogen bond pattern around the flavin changes (Collette et al 2014;Domratcheva et al 2016;Khrenova et al 2013). It remains unclear how the imidic tautomer is maintained for any length of time, given the roughly 40 kJ/mol energy cost over the amidic form, but the stronger hydrogen bonds formed around the flavin must provide some compensation (Fig.…”

Structural Refinement of a Key Tryptophan Residue in the BLUF Photoreceptor AppA by Ultraviolet Resonance Raman Spectroscopy

“…In various x-ray crystal and NMR solution structures (14)(15)(16)(17)(18)28), as well as spectroscopic (13,22,(39)(40)(41) and theoretical (42)(43)(44) studies, the dark-state orientation of Gln-63 has remained ambiguous. As illustrated in Fig.…”