Unusual ring D fixation by three crucial residues promotes phycoviolobilin formation in 1 the DXCF-type cyanobacteriochrome without the second Cys 2

Cyanobacteriochromes are linear tetrapyrrole-binding photoreceptors produced by 13 cyanobacteria. Their chromophore-binding GAF domains are categorized into many lineages. 14 Among them, dual Cys-type cyanobacteriochrome GAF domains possessing not only a 15 highly conserved “first Cys” but also a “second Cys” are found from multiple lineages. The 16 first Cys stably attaches to C3 1 of the A-ring, while the second Cys mostly shows reversible 17 ligation to the C10 of the chromophore. Notably, position of the second Cys in the primary 18 sequence is diversified, and the most abundant dual Cys-type GAF domains have a “second 19 Cys” within the DXCF motif, which are called DXCF GAF domains. It has been long known 20 that the second Cys in the DXCF GAF domains not only shows the reversible ligation but 21 also is involved in isomerization activity (reduction of C4=C5 double bond) from the initially 22 incorporated phycocyanobilin to phycoviolobilin. However, comprehensive site-directed 23 mutagenesis on the DXCF GAF domains, AM1_6305g1 and AM1_1499g1, revealed that the 24 second Cys is dispensable for isomerization activity, in which three residues participate by 25 fixing the C- and D-rings. Fixation of the chromophore on both sides of the C5 bridge is 26 necessary, even though one side of the fixation site is far from this bridge, with the other side 27 at C3 1 fixed by the first Cys.


Introduction
isomerization activity) [4,20]. The second Cys contributes to this isomerization activity. In

Results and discussion
78 Second Cys residue is dispensable for PCB-to-PVB isomerization in the AM1_6305g1 79 scaffold 80 We have previously reported engineering of the CBCR GAF domains, AM1_1499g1 and 81 AM1_6305g1, within one of the DXCF lineages, AM1_1499g1/AM1_6305g1 lineage [24]. engineering on AM1_6305g1 for loss of the isomerization activity. 88 We thus replaced the second Cys of AM1_6305g1 with Ser, the original residue in 89 AM1_1499g1 (AM1_6305g1_C 99 S) (Fig. S2A-i). Unexpectedly, AM1_6305g1_C 99 S showed 90 a green/teal photocycle, which is nearly the same as that of the wild-type (Fig. 1A, Fig. 2A, 91 and Table 1). Furthermore, the chromophore incorporated into AM1_6305g1_C 99 S was 92 assigned not to PCB but to PVB by comparing the denatured difference absorption spectrum 93 (dark state-photoproduct state) of the C 99 S variant with those of the PCB-and PVB-binding 94 CBCR GAF domains ( Fig. 3A and Table 1). These results indicate that the second Cys 95 residue is dispensable for the isomerization activity in the AM1_6305g1 scaffold. Ser and 96 Cys have a common skeleton with distinctive functional groups at the end of the side-chains; 97 a hydroxy group for Ser and a thiol group for Cys. We speculate that the hydroxy group of 98 Ser can complement the functionality of the thiol group of Cys in AM1_6305g1. 99 We next replaced these polar amino acids with Ala (AM1_6305g1_C 99 A), which has 100 a nonpolar methyl group smaller than that of Ser and Cys to verify this assumption ( Fig.   101 S2A-ii). Spectral analysis revealed two photoconvertible components for 6 AM1_6305g1_C 99 A; green/teal and orange/green (Fig. 4A). We were able to separately 103 excite a single component and independently characterize the photoconversion properties and 104 the binding chromophore species based on the absorption spectra of the native and denatured 105 molecules using various monochromic light sources (Fig. 4B, C and Table 1). The green/teal 106 component bound PVB ( Fig. 4C and Table 1, shown by a cyan line) and showed reversible 107 photoconversion between a green-absorbing dark state (~560 nm) and a teal-absorbing 108 photoproduct state (~490 nm) (Fig. 4A, shown by cyan arrowheads). The orange/green 109 component bound PCB ( Fig. 4C and Table 1, shown by a magenta line) and showed 110 reversible photoconversion between an orange-absorbing dark state (~620 nm) and a green-  122 We have found that the AM1_6305g1 scaffold is clearly distinct from the AM1_1499g1 123 scaffold in the context of isomerization activity, despite their close homologous relationship.

124
Namely, the second Cys residue is dispensable for isomerization activity in the AM1_6305g1 125 scaffold but not in the AM1_1499g1 scaffold, indicating that the distinct residues between 126 these two molecules are determinants for this divergence. We have already determined in a 7 previous study that such amino acid alterations are crucial for color-tuning of the dark state 128 via a D-ring twist; Leu 132 /Asn 140 in AM1_6305g1 and Tyr 151 /Thr 159 in AM1_1499g1 ( Fig.   129 S1C-E) [24]. We hypothesize that the alterations of these two residues are key not only for 130 the dark-state color-tuning but also for isomerization activity. Although replacement of 131 Leu 132 /Asn 140 with Tyr/Thr based on the AM1_6305g1 wild-type background 132 (AM1_6305g1_L 132 Y/N 140 T) resulted in a red shift of the dark state, no effects on the 133 isomerization activity have been detected ( Fig. 1B-i, Fig. 2B-i, Fig. 3B the wild-type. In this context, these two residues may be crucial for the isomerization activity 137 without the second Cys residue.

138
To verify this hypothesis, we replaced these two residues (Leu 132 residue and that the Leu and Asn residues, but not the Tyr and Thr   155 residues, could support the Ser residue for full isomerization of PCB to PVB (Fig. 1A, B, Fig.   156 2A, B, Fig. 3A, B, and Table 1). Thus, these two amino acid positions are involved not only 157 in dark-state color-tuning but also in isomerization activity. 158 We constructed singly mutated variant molecules based on the C 99 S background  Table 1). This finding could be explained by the previously proposed  Table 1).

174
In conclusion, the Ser residue can fully complement the isomerization function of the    Table 1). In conclusion, the replacement of a total of four residues (C 99 S/Q 112 R/L 132 Y/N 140 T) 201 resulted in almost complete inactivation of the isomerization activity.  Table 1). Namely, AM1_6305g1 has constructed a robust system for isomerization activity,  3A-ii to D-ii, and Table 1). It is of note that replacements of these residues do not largely  Table 1). The  Table 1). The Leu 132 /Asn 140 residues have been 300 elucidated to fix the D-ring into a twisted geometry, which would assist the Ser 99 in the 301 distortion of the C4=C5 double bond. Gln 112 is predicted to be positioned near the C-ring propionate and contribute to the stabilization of the chromophore conformation. However, 303 this prediction is based on the TePixJg structure with the "bent" chromophore, whose 304 conformation would be largely different from that of "unbent" one ( Fig. S1B, D). Therefore, 305 it is difficult to elucidate the precise role of Gln 112 in AM1_6305g1. It is of note that not only Evolutionary trace of the AM1_1499g1/AM1_6305g1 lineage 323 We constructed a phylogenetic tree of the CBCR GAF domains with the phytochrome GAF 324 domains as the outgroup for the evolutionary trace ( Fig. 8B-i) the isomerization activity without the second Cys residue (Fig. S1E). We consider that, activity. In fact, AM1_1499g1 has lost not only the second Cys but also the Leu/Asn residues 346 (Fig. S1E). AM1_1499g1 has experienced multiple amino acid replacements after 347 diversification to establish a longer wavelength perception (Fig. 8A-v and B). On the other 348 hand, AM1_6305g1 has specifically acquired Gln 112 , which supports isomerization activity 349 for a more robust isomerization system (Fig. 8B-ii and Fig. S1E)

Identification of the chromophore incorporated into the AM1_6305g1 and AM1_1499g1
433 variant molecules containing two photoconvertible components 434 We established the experimental protocol in this study to determine the binding chromophore In this study, we did not obtain any sequence and structural data to deposit in community-451 approved public repositories. We used sequence and structural information of  Table 1.  orange/green one in the long-wavelength region, magenta) were assigned by colored triangles.

637
The normalized difference spectra are shown in Figure 6.