## 3 Results and Discussion
### Modular Diversity of BLUF Domains  
In the present communication, we have selected 34 different modular architectures for the BLUF domain in combinations with various effector domains (Table 1; Figure 1). The annotated BLUF domains in our database sequences range from 80 to 100 amino acids, situated mostly at the N-terminal region of the effector domains. However, the predicted BLUF sequences in our database show a high sequence similarity across the BLUF region, with E-values in the range of \(\mathrm{e}^{-40}\) to \(\mathrm{e}^{-120}\). The residues show an upper conservation pattern around the flavin binding region (Figure 4). The secondary structural analysis indicates that the BLUF sequences are composed mostly of five \(\beta\) strands and two \(\alpha\) helices. The E-value, sequence length and sequence conservation details are summarized in Table 1. Three-dimensional models of the BLUF domains were constructed using the Phyre2 modeling software [53]. The BLUF domains reveal conserved \(\beta\alpha\beta\beta\alpha\beta\beta\) fold conformations in which two \(\alpha\)-helices surround a five-stranded antiparallel \(\beta\)-sheet platform (Figure 4). Figure 3: Phylogenetic analysis by the Maximum Likelihood (ML) method. The analysis was done using 34 amino acid sequences of the modular BLUF domain containing proteins. All positions containing gaps and missing data were eliminated. There were a total of 17 positions in the final dataset. A solid red circle represents protein sequences of prokaryotic origin, and a solid red square represents protein sequences of eukaryotic origin. Evolutionary analyses were conducted using MEGA6 [51]. We modeled the binding modes of the flavin chromophore based on the observations in the known BLUF domain structures [33; 57]. The isoalloxazine ring of flavin can be readily accommodated in the pocket in the models sandwiched between two \(\alpha\) helices, suggesting that the active site is appropriately formed in BLUF models (Figures 4 and 5). Spectroscopic analyses and photochemistry  
Figure 4: Models of the BLUF domains and details of its flavin binding pocket. (**A**) Superposition of the modelled BLUF domains using the Phyre server with a crystal structure of _Rhodobacter sphaeroides_ BlrB (PDB: 2 BYC) (magenta). The homology models include the annotated BLUF domains from WP_045444510.1 (_Psychroucher sp._) (Green tone); WP_014148160.1 (_Methylomicrobium alcaliphulum_) (Brown tone); XP_025342216.1 (_Pseudomicrostroma glucosiphulum_) (Blue tone); AFL74487.1 (_Thicocystis violascens_) (Violet tone); EHQ08139.1 (_Leptonema illini_) (Yellow tone); ABP71929.1 _Rhodobacter sphaeroides_ ATCC 17025 (Pink tone); WP_0229622806.1 (_Pseudomonas pelagic_) (Skyblue tone); and ORY86082.1 (_Protonyces inouye_) (Smudge tone). The domain boundaries of the modelled BLUFs are mentioned in Table 1. (**B**) The BLUF photocycle scheme shows the protein/FAD interactions through the hydrogen bonding pattern of the flavin moiety with conserved glutamine upon illumination. (**C**) Superposition of the flavin binding pocket in the BLUF models in comparison to _Rhodobacter sphaeroides_ BlrB (PDB: 2 BYC) and the BLUF domain of AppA (PDB: 1 YRX). The side chains of residues with potentially important roles in catalysis and/or substrate binding are shown as stick models and are labelled. The selected regions of the same are shown in a reduced multiple sequence alignment. of BLUF proteins have shown that flavin chromophore facilitates blue light-induced electron transfers by a hydrogen bond rearrangement between flavin N5 and O4 and conserved tyrosine, glutamine, and tryptophan or methionine of the BLUF [14; 17], leading to a spectral shift from blue to red and the transmission of the signal further downstream.