Hydrogen Bonding Environment of the N3-H Group of Flavin Mononucleotide in the Light Oxygen Voltage Domains of Phototropins

The light oxygen voltage (LOV) domain is a flavin-binding blue-light receptor domain, originally found in a plant photoreceptor phototropin (phot). Recently, LOV domains have been used in optogenetics as the photosensory domain of fusion proteins. Therefore, it is important to understand how LOV domains exhibit light -induced structural changes for the kinase domain regulation, which enables the design of LOV-containing optogenetics tools with higher photoactivation efficiency. In this study, the hydrogen bonding environment of the N3 H group of flavin mononucleotide (FMN) of the LOV2 domain from Adiantum neochrome (neo) 1 was investigated by low -temperature Fourier transform infrared spectroscopy. Using specifically N-15-labeled FMN, [1,3-N-15(2)]FMN, the N3-H stretch was identified at 2831 cm(-1) for the unphotolyzed state at 150 K, indicating that the N3-H group forms a fairly strong hydrogen bond. The N3 H stretch showed temperature dependence, with a shift to lower frequencies at <= 200 K and to higher frequencies at >= 250 K from the unphotolyzed to the intermediate states. Similar trends were observed in the LOV2 domains from Arabidopsis photl and phot2. By contrast, the N3-H stretch of the Q1029L mutant of neol-LOV2 and neol-LOV1 was not temperature dependent in the intermediate state. These results seemed correlated with our previous finding that the LOV2 domains show the structural changes in the beta-sheet region and/or the adjacent Ja helix of LOV2 domain, but that such structural changes do not take place in the Q1029L mutant or neol-LOV1 domain. The environment around the N3-H group was also investigated.