Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction

Abstract

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS 1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excitedstate model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13 C labeling of FAD and 15 N labeling of glutamine, identified the glutamine imidic acid C�N stretch vibration in the LA state and the Gln C�O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction.

Difference in the Charge-Separation Energetics between Distinct Conformers in the PixD Photoreceptor

“…The calculated absorption wavelengths for states 1 and 6 are highly correlated with the experimentally measured absorption wavelengths 378 nm for state 1 and 382 nm for state 6 in the second lowest excitation (S 0 –S 2 ) and 443 nm for state 1 and 450 nm for state 6 in the first lowest excitation (S 0 –S 1 ) (Figure ). The results indicate the relevance of the present QM/MM/PCM-optimized geometries to the dark-adapted and signaling states observed in the spectroscopic study …”

“…In the absence of bulk water, the energy level of the charge-separated state after reorganization is ∼500 meV higher than that of the photoexcited state before reorganization (Figure S6). This result indicates that the initial charge separation does not occur in the absence of bulk water, which is not consistent with the observation of FMN •– in the transient absorption and fluorescence studies. ,− Therefore, consideration of the solvation effects is required in the calculation of energy levels.…”

“…The results indicate the relevance of the present QM/MM/PCMoptimized geometries to the dark-adapted and signaling states observed in the spectroscopic study. 10 Notably, the difference in the calculated absorption wavelength between the W91 out and W91 in conformations are significantly small for the observed correlation (Figure 6). It seems likely that the W91 out conformation is not distinct from the W91 in conformation in the spectroscopic studies.…”

“…Spectroscopic studies ,− and theoretical studies suggested that photoexcitation of FMN leads to electron transfer from Tyr8 to FMN, forming the initial charge-separated Tyr8-OH •+ /Gln50/FMN •– state . The release of the proton from Tyr8-OH •+ toward FMN •– also occurs via Gln50, forming the charge-neutral biradical Tyr8-O • /[HO-Gln50-NH]/FMNH • state, where HO-Gln50-NH is the charge-neutral enol form .…”

“…Finally, charge recombination leads to reorientation of the [HO-Gln50-NH] side chain , and formation of the light-adapted signaling state with absorption peaks at 450 nm (S 0 –S 1 transition for the lowest electronic excitation) and 382 nm (S 0 –S 2 transition for the second lowest electronic excitation) . Using Fourier transform infrared (FTIR) spectroscopy, it was suggested that the enol group of Gln50 donates an H-bond to the O4 site of FMN (Figure b) in the signaling state . Remarkably, spectroscopic studies suggested that the fast and slow phases coexist in the charge-separation process. ,, The fast phase of 7–14 ps was interpreted as the faster formation of FMN •– and FMNH • . , The dark-adapted state that occurs the charge separation with the fast phase forms the signaling state.…”