posted on 2023-01-06, 14:08authored byYusaku Hontani, Jennifer Mehlhorn, Tatiana Domratcheva, Sebastian Beck, Miroslav Kloz, Peter Hegemann, Tilo Mathes, John T. M. Kennis
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 CN stretch vibration in the LA state and the Gln
CO in the DA state. Hence, our study provides support for
glutamine tautomerization and side-chain rotation in the BLUF photoreaction.