Hydrogen Bonding Environments in the Photocycle Process around the Flavin Chromophore of the AppA-BLUF domain

Abstract

Three kinds of photochemical reactions are known in flavins as chromophores of photosensor proteins, reflecting the various catalytic reactions of the flavin in flavoenzymes. Sensor of blue light using the flavin FAD (BLUF) domains exhibit a unique photoreaction compared with other flavin-binding photoreceptors in that the chromophore does not change its chemical structure between unphotolyzed and intermediate states. Rather, the hydrogen bonding environment is altered, whereby the conserved Gln and Tyr residues near FAD play a crucial role. One proposal for this behavior is that the conserved Gln changes its chemical structure from a keto to an enol. We applied light-induced difference Fourier transform infrared (FTIR) spectroscopy to AppA-BLUF. The spectra of AppA-BLUF exhibited a different feature upon N-Gln labeling compared with the previously reported spectra from BlrB, a different BLUF domain. The FTIR signals were interpreted from quantum mechanics/molecular mechanics (QM/MM) calculation as the keto-enol tautomerization and rotation of the Gln63 side chain in the AppA-BLUF domain. The former was consistent with the result from BlrB, but the latter was not uniquely determined by the previous study. QM/MM calculation also indicated that the infrared signal shape is influenced depending on whether a Trp side chain forms a hydrogen bond with the Gln side chain. FTIR spectra and QM/MM simulations concluded that Trp104 does not flip out but is maintained in the intermediate state. In contrast, our data revealed that the Trp residue at the corresponding position in BlrB faces outward in both states.

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

“…The TRIR data measured on the light-adapted W64F and Y21FY56FW64F mutant show a downshift of frequency of C4=O carbonyl which can be explained based on the model suggested by Iwata et al 65 Model 7which assumes a hydrogen bond formed between the enol form of Q63 and W104.…”

The explicit role of electron exchange in the hydrogen bonded molecular complexes

“…Intermolecular hydrogen bonds (H‐bonds) are of paramount importance in supramolecular chemistry, 1,2 crystal engineering 3 and biochemistry 4 . These noncovalent interactions have been intensively studied by various theoretical and experimental techniques 5–8 : ab initio calculations, 9,10 molecular dynamic modeling, 11 quantum mechanics/molecular mechanics calculations, 12 NMR and IR spectroscopy, 13–15 X‐Ray and neutron diffraction 16–18 and thermochemical experiments 19 . Among theoretical approaches, the electron density 7,20–23 and energy decomposition analyses 7,24 are gaining considerable popularity in the studies of H‐bonds in the isolated state, 25,26 solution 27 and solid state 28,29 .…”

Site-Specific Protein Dynamics Probed by Ultrafast Infrared Spectroscopy of a Noncanonical Amino Acid

“…2,5 Only a ca 15 nm red shift in the electronic spectrum of the fully oxidized flavin co-factor is observed, which develops in < 1 ns and is associated with a change in H-bonding interactions between the isoalloxazine and its protein matrix. [6][7][8][9][10][11][12][13][14][15][16] This local structure change results in reorganization of the secondary structure leading to signalling state formation on the microsecond timescale. Structures of the flavin binding region for two BLUF domains proteins, PixD and AppA, are shown in Figure 1 (the latter, isolated by truncation of AppA, is hereafter labelled AppABLUF); secondary structures are shown in Supporting Information (S1).…”