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

Yusaku Hontani, Jennifer Mehlhorn, Tatiana Domratcheva, Sebastian Beck, Miroslav Kloz, Peter Hegemann, Tilo Mathes, John T.M. Kennis*

*Corresponding author for this work

Research output: Contribution to JournalArticleAcademicpeer-review

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 FADS1 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. Excited-state 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 13C labeling of FAD and 15N 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.

Original languageEnglish
Pages (from-to)1040-1052
Number of pages13
JournalJournal of the American Chemical Society
Volume145
Issue number2
Early online date6 Jan 2023
DOIs
Publication statusPublished - 18 Jan 2023

Bibliographical note

Funding Information:
Y.H., M.K., T.M., and J.T.M.K. were supported by NWO through a VICI grant to J.T.M.K. This work was supported by a NWO Middelgroot investment grant to J.T.M.K. J.M. and this research were supported by the German Research Organisation (DFG) (HE3824/24-1) and the Netherlands Organization for Scientific Research (NWO)-DFG Bilateral Cooperation program (HE3824/25-1). M.K. was supported by the Czech Science Foundation (21-404 09692M). P.H. is Hertie Professor for neuroscience and supported by the Hertie Foundation. This work has been adapted from doctoral theses by Y.H. and J.M.

Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.

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