Glutamine Rotamers in BLUF Photoreceptors: A Mechanistic Reappraisal

Abstract

The blue light using FAD (BLUF) photosensory protein domain is activated by a unique photoreaction that results in a hydrogen-bond rearrangement around the flavin chromophore. The chemical structure of the hydrogen bond switch is a long-standing debate: The two main hypotheses postulate rotation as opposed to tautomerization of a conserved glutamine residue. Attempts to resolve the debate were inconclusive so far, despite numerous experimental and computational studies. Here we propose physical criteria for the dark and light state structures as well as for the light-activation process to evaluate existing models of BLUF using quantum-chemical calculations. The glutamine rotamer assignment of the crystal structure with the pdb code 1YRX does not satisfy our criteria because after equilibrating the intermolecular forces the glutamine rotamer in 1YRX is incompatible with the experimental density. We identified the root of the mechanistic controversy in the incorrect glutamine rotamer assignment of 1YRX . Furthermore, we show that the glutamine side chain may rotate without light activation in the BLUF dark state. Finally, we demonstrate that the tautomerized glutamine is consistent with our criteria and observations of the BLUF light state.

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.…”

“…Conserved tyrosine, glutamine, and methionine residues are found at the flavin binding site of all BLUF domains, equivalent to Tyr-6, Gln-48, and Met-92 in OaPAC. Numerous studies have highlighted the importance of the tyrosine for initiating the photocycle (12)(13)(14) and the glutamine for signal transduction (15)(16)(17)(18)(19). Some BLUF models show a very different position of the methionine (20), displaced by a partly conserved tryptophan residue (Trp-90 in OaPAC) found near the start of the fifth β-strand.…”

Role of active site conformational changes in photocycle activation of the AppA BLUF photoreceptor

“…The tautomerization of Gln63 has been proposed to be involved in the BLUF photocycle in several studies in the literature (13,15,32). Our QM/MM free-energy calculations (SI Appendix, Figs.…”

“…Quantum mechanical/molecular mechanical (QM/MM) geometry optimizations and vertical excitation energy calculations provided insight into the conformations consistent with experimental absorption data for the dark-adapted and signaling states (25). Other studies mostly used either conventional molecular dynamics trajectories or potential energy scans that were often conducted in gas-phase models of the active site (13)(14)(15)27). The reliability of such approaches is somewhat limited by insufficient or no conformational sampling, especially given the complicated conformational landscapes of these proteins.…”

“…Despite numerous experimental and computational studies (8,(13)(14)(15)(16)(17), the exact nature of the signaling state and the mechanism of long-range signaling in BLUF proteins are not well understood. The active site conformation in even the dark-adapted state is still under debate because of discrepancies in the orientations of the key active site Trp, Met, and Gln residues among different experimental crystal and solution structures of BLUF domains (18)(19)(20)(21)(22)(23)(24)(25)(26).…”