Phototropin (Phot) is a blue-light receptor in plants. The molecule has two FMN (flavin mononucleotide) binding domains named LOV (light-, oxygen-, and voltage-sensing), which is a subset of the PAS (Per-Arnt-Sim) superfamily. Illumination of the phot-LOV domains in the dark state (D447) produces a covalent C(4a) flavin-cysteinyl adduct (S390) via a triplet excited state (L660), which reverts to D447 in the dark. In this work, we studied the light-induced structural changes in the LOV2 domain of Adiantum phytochrome3 (phy3), which is a fusion protein of phot containing the phytochrome chromophoric domain, by low-temperature UV-visible and FTIR spectroscopy. UV-visible spectroscopy detected only one intermediate state, S390, in the temperature range from 77 to 295 K, indicating that the adduct is produced even at temperatures as low as 77 K, although a portion of D447 cannot be converted to S390 at low temperatures possibly because of motional freezing. In the whole temperature range, FTIR spectra in the S-H stretching frequency region showed that Cys966 of phy3-LOV2 is protonated in D447 and unprotonated on illumination, supporting adduct formation. The pK(a) of the S-H group in D447 is estimated to be >10. FTIR spectra also showed the light-induced appearance of a positive peak around 3621 cm(-1) in the whole temperature range, indicating that adduct formation accompanies rearrangement of a hydrogen bond of a water molecule(s), which can be either water25, water45, or both, near the chromophore. In contrast to the weak temperature dependence of the spectral changes in the UV-visible absorption and the FTIR of both S-H and O-H stretching bands, light-induced changes in the amide I vibration that probes protein backbone structure vary significantly with the increase in temperature. The spectral changes suggest that light excitation of FMN loosens the local structure around it, particularly in turns, in the early stages and that another change subsequently takes place to tighten it, mainly in beta-structure, but some occur in the alpha-helical structure of the protein moiety as well. Interestingly, these changes proceed without altering the shape of UV-visible spectra, suggesting the presence of multiple conformation states in S390.

Phototropin (phot) is a blue-light receptor in plants. The molecule has two FMN (flavin mononucleotide)-binding domains named the LOV (light-oxygen-voltage) domain, that is a subset of a PAS (per-arnt-sim) superfamily. Illumination of phot-LOV domains produces a covalent C(4a) flavin-cysteinyl adduct, which is called the S390 intermediate state. According to the crystal structures of the LOV2 domain of Adiantum phytochrome3 (phy3), a fusion protein of phot containing the phytochrome chromophoric domain, in the unphotolyzed and S390 states, and the side chain of Gln1029 switches hydrogen bonds with the FMN chromophore. Gln1029 is the hydrogen-bonding donor of the C(4)=O group of FMN in the unphotolyzed state, whereas Gln1029 is the hydrogen-bonding acceptor of the N(5)-H group of FMN in S390. In this paper, we measured the light-induced structural changes in the Q1029L mutant protein of phy3-LOV2 by means of low-temperature FTIR spectroscopy, and the obtained spectra are compared with those of the wild type. Low-temperature UV-visible spectroscopy of Q1029L detected only one intermediate state, S390, at 77-295 K, as well as the wild type. The C(4)=O stretch of FMN at 1710 cm(-1) is shifted to 1723 cm(-1) in Q1029L, presumably because of the lack of hydrogen bonds between Gln1029 and FMN. Upon formation of S390, the C(4)=O group hydrogen bond is weakened in both wild type and Q1029L. These observations are fully consistent with the X-ray crystal structures of the unphotolyzed and S390 states. On the other hand, the C(4)=O stretch of FMN and amide-I vibrations are temperature-independent in Q1029L, in contrast to wild type, in which highly temperature-dependent FTIR spectra are detected. Amide-I vibrations of Q1029L at room temperature are similar to those of the wild type at 77-150 K but not at room temperature. These facts imply that the Q1029L mutant protein lacks progressive protein structural changes following flavin-cysteinyl adduct formation in the wild type, which eventually alter structures of beta sheet and alpha helix in the protein moiety. Hydrogen-bonding interaction of Gln1029 with the FMN chromophore likely plays an important role in the protein structural changes of phy3-LOV2.