Revealing the Functional States in the Active Site of BLUF Photoreceptors from Electrochromic Shift Calculations

Abstract

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 tautomerization of the glutamine at position 63, which was proposed as molecular mechanism for the transition between the dark and the light-adapted state, explains the measured 10 to 15 nm red shift in flavin absorption between these two states of the protein. We believe that the accurateness of our results provides evidence that the BLUF photoreceptors absorption is fine-tuned through electrostatic interactions between the chromophore and the protein matrix, and finally that the simplicity of our theoretical model is advantageous as regards easy reproducibility and further extensions.

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.…”

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase

“…The flavin receives a proton as well as an electron when excited, and Gln-48 Ne1 is found to move slightly from the N5 atom of FMN on photoactivation of OaPAC (dark state 3.16 Å, active 3.53 Å). This shift suggests that the glutamine receives a proton from the tyrosine and loses one to the flavin, giving the tautomeric (imidic) form suggested by spectroscopic and computational analyses, but requiring only very modest sidechain rotation (15,18,27,28,(33)(34)(35). The hydrogen bonding pattern around the flavin may change as shown in Fig.…”

“…This ambiguity has led to models in which photoactivation involves a tautomerization or flip of the glutamine sidechain (27), so that in one state it donates a hydrogen bond to the tyrosine, but in the other, it donates a hydrogen bond to the C4 = O carbonyl of the flavin. Both orientations of the glutamine sidechain are found in the known crystal structures of BLUF domains, but neither has been convincingly demonstrated using structural data alone (19,27,28). 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.…”

The quest for energy traps in the CP43 antenna of photosystem II

“…[39][40][41] and V mn for all Chls and Chl pairs, which ultimately yields an answer to the second question. Our electrostatic approach to this problem [36][37][38] is approximate, but turned out to give reasonable results, when applied to the Fenna-Matthews-Olson (FMO) protein of green sulfur bacteria [42][43][44][45], photosystem I [46], the antenna system of plant photosystem II [47][48][49], and the BLUF photoreceptor [50]. In the present paper, we apply it to the CP43 core antenna of PSIIcc, updating earlier work [51].…”