Conserved histidine and tyrosine determine spectral responses through the water network in Deinococcus radiodurans phytochrome

This is a self-archived version of an original article. This version may differ from the original in pagination and typographic details. Author(s): Title: Year: Version: Copyright: Rights: Rights url: Please cite the original version: CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Conserved histidine and tyrosine determine spectral responses through the water network in Deinococcus radiodurans phytochrome © The Author(s) 2022 Published version Lehtivuori, Heli; Rumfeldt, Jessica; Mustalahti, Satu; Kurkinen, Sami; Takala, Heikki Lehtivuori, H., Rumfeldt, J., Mustalahti, S., Kurkinen, S., & Takala, H. (2022). Conserved histidine and tyrosine determine spectral responses through the water network in Deinococcus radiodurans phytochrome. Photochemical and Photobiological Sciences, 21(11), 1975-1989. https://doi.org/10.1007/s43630-022-00272-6 2022 Vol.:(0123456789) 1 3 Photochemical & Photobiological Sciences https://doi.org/10.1007/s43630-022-00272-6 ORIGINAL PAPERS Conserved histidine andtyrosine determine spectral responses throughthewater network inDeinococcus radiodurans phytochrome HeliLehtivuori1,2 · JessicaRumfeldt3 · SatuMustalahti4 · SamiKurkinen3,5 · HeikkiTakala3,6 Received: 29 April 2022 / Accepted: 12 July 2022 © The Author(s) 2022 Abstract Phytochromes are red light-sensing photoreceptor proteins that bind a bilin chromophore. Here, we investigate the role of a conserved histidine (H260) and tyrosine (Y263) in the chromophore-binding domain (CBD) of Deinococcus radiodurans phytochrome (DrBphP). Using crystallography, we show that in the H260A variant, the missing imidazole side chain leads to increased water content in the binding pocket. On the other hand, Y263F mutation reduces the water occupancy around the chromophore. Together, these changes in water coordination alter the protonation and spectroscopic properties of the biliverdin. These results pinpoint the importance of this conserved histidine and tyrosine, and the related water network, for the function and applications of phytochromes. Graphical abstract Keywords Spectral responses· Water network· Biliverdin protonation· Phytochrome structure 1 Introduction Phytochromes are red light-sensing photoreceptors originally discovered in plants [1] but can also be found in bacteria and fungi [2]. They bind a linear tetrapyrrole (bilin) chromophore and cycle between a red light-absorbing Pr state and a far-red light-absorbing Pfr state in response to This publication is dedicated to Prof. Silvia E. Braslavsky, a pioneer in photobiology and photobiophysics, on the occasion of her 80th birthday. * Heikki Takala [email protected] Extended author information available on the last page of the article Photochemical & Photobiological Sciences 1 3 red/far-red irradiation [3]. Like many other photoreceptors, phytochromes consist of a photosensory module and an effector module. The photosensory module consists of a PAS (Per-ARNT-Sim), a GAF (cGMP phosphodiesteraseadenylate cyclase FhlA), and a PHY (phytochrome-specific) domain. In bacteriophytochromes, a biliverdin (BV) chromophore is covalently bound to a conserved cysteine in the PAS domain but physically embedded within the GAF domain (Fig.1A). This PAS-GAF entity is often referred to as a chromophore-binding domain (CBD) [4]. As demonstrated first for a model bacteriophytochrome from Deinococcus radiodurans (DrBphP), red/far-red light induces large structural changes in the PSM [5]. These changes are then relayed to the effector module, regulating its activity [6, 7]. The photoreactions of bacteriophytochromes start with the photon absorption by the BV. Upon light absorption, phytochrome converts to the Pfr state, which absorbs far-red light (λ ≈ 750nm). This Pr-to-Pfr transition requires both CBD and PHY domain [8], has several intermediate states, and involves Z-to-E isomerization of the biliverdin D-ring with concomitant rotation of the C15-C16 methine bridge between the C- and D-ring [9–11]. The intermediate states include Lumi- and Meta-states; their kinetic and structural properties differ slightly between phytochrome species. The yield of reaching the first intermediate state, Lumi-R, is relatively low (< 15%) [12–17], and alternative reaction pathways, such as fluorescence and non-radiative relaxation (Fig.1B), are generally competitive. The intricate network of interactions formed by amino acid side chains and water molecules in the chromophorebinding pocket plays an important role in guiding the photoresponses of the BV (Fig.1A). For example, residues D207, H260, Y263, and H290 (numbering according to DrBphP) have been shown to play a role in the spectral fine-tuning of the photoresponses. In particular, D207 and Y263 play a critical role in the normal photocycle [19–21]. The two histidines, H260 and H290, are conserved in phytochromes and are centrally involved in the Pr-state structural heterogeneity [10, 22–26]. In addition, H260 is closely associated with the chromophore, placing its imidazole ring along with the inner pyrrole rings B and C. The most central water molecule in the pocket, called pyrrole water (W1 in Fig.1A), is coordinated between D207, H260, and the pyrrole nitrogens of the BV. It has been shown that initial photoreactions include its structural de-localiza- tion, accompanied by additional structural rearrangements about the chromophore [27]. Although the positions of several other coordinated waters in the chromophore-binding pocket are highly conserved in phytochromes, their role remains poorly understood. Lenngren etal. have shown that replacement of H290 side chain with threonine paves a way for two additional waters that affect the spectral responses of the BV [28]. In addition, Kraskov etal. have noticed that the histidine/threonine-dependent structural differences mainly refer to hydrogen bonding interactions with the ring D carbonyl group and, as a consequence, the torsion of the C–D methine bridge [26]. The water content in the vicinity of the chromophore has been traditionally linked to reduced fluorescence as the water network promotes vibrational relaxation pathways [29–31]. During the last decades, much effort has been made to modify the fluorescent properties of microbial fluorescent BA C Pr hν Lumi-R fluorescence relaxation Pfr abs. (dark) abs. (red) flour. emission ].u.a[ ecnabrosbA 0 1 400 600 800 Wavelength nm[ ] ].u.a[ ytisnetnI 0 1 biliverdin D207 H290 H260 Y263 P P A A S S G G A A F F W1 C24 Fig. 1 Structure and photocycle of phytochromes. A Chromophore-binding pocket structure in wild-type CBD (CBD-WT) of DrBphP, PDB code 5K5B [18]. Biliverdin (orange sticks), key residues (blue sticks), and the pyrrole water (W1, red sphere) are indicated. B Three alternative reaction pathways induced by red light. Bacteriophytochrome can enter the photocycle via Lumi-R state. Alternatively, its biliverdin can emit a photon (fluorescence) or relax to the ground state by other means (e.g., through vibrations or heat). C Normalized absorption and fluorescence spectra of a wild-type CBD fragment from D. radiodurans phytochrome. For absorption spectra, the sample was illuminated either with 785nm light (“dark”) or 655nm light (“red”) and normalized to the maximum of “dark” data. Emission spectrum was excited at 550nm and normalized to its maximum. All spectra were measured at pH 8.0 Photochemical & Photobiological Sciences 1 3 protein markers from DrBphP [31–35], and phytochromes from Rhodopseudomonas palustris [36–39] and cyanobacteria [40–42]. Fluorescent phytochromes are a rapidly increasing area of current bioimaging research and will have an enormous impact on biological imaging strategies [43]. The wild-type CBD (CBD-WT) from DrBphP has a low fluorescence quantum yield (QY) of (3.5 ± 0.5)% at pH 7.0 [44]. To improve this, protein modifications have been made to slow competing processes that occur after excitation, in particular non-radiative relaxation (Fig.1B). The challenge in this kind of development is that improving the fluorescence QY typically results in emission in the orange part of the spectrum (∼ 690nm). Yet in DrBphP, the fluorescence QY of CBD has been increased up to almost 10% by site-selective mutations, while still maintaining the emission maximum at around 720nm [31, 35]. In prototypical bacteriophytochromes, the absorption spectrum of the resting Pr state has two bands at around 400nm (Soret band) and 700nm (Q band) due to the covalently bound bilin chromophore (Fig.1C). Experimental and theoretical studies indicate that in most phytochromes, the bilin chromophore exists in the fully protonated form at physiological pH. In this form, each of the four pyrrole nitrogens binds one hydrogen giving the molecule an overall positive charge [45–49]. The protonation state of bilin has been shown to influence its spectroscopic properties significantly [9, 23, 50–52]. As the pH increases, one hydrogen is released, most likely from the C-ring, which reduces the absorbance of the Q band [53]. Light- and X-ray-induced deprotonation of the C-ring has also been observed [47] reinforcing the notion that one of the pyrrole protons is labile. In this study, we concentrate on the CBD fragment of DrBphP. We reveal how H260 together with Y263, plays an important role in the phytochrome photoresponses and protonation in the CBD. The crystal structures of the H260A and H260A/Y263F variants reveal that removal of the imidazole side chain clears the way for additional water molecules in the chromophore-binding pocket. The water network plays an important role in determining the spectral responses and (de)protonation events of the BV. 2 Results To study the role of residues H260 and Y263 in the structure and photoresponses, we generated variants of the CBD fragment of DrBphP by site-directed mutagenesis. For H260, we chose alanine mutation (H260A), enabling a small nonpolar substitution that is in close proximity to the four pyrrole nitrogens of the BV and the highly coordinated pyrrole water. As for Y263, we removed its hydroxyl group with Y263F mutation [21]. As a result, we ended up having a set of four CBD variants for our studies: wild-type CBD (CBD-WT), CBD-Y263F, CBD-H260A, and CBD-H260A/ Y263F. 2.1 H260 imidazole andY263 hydroxyl affect thewater content ofthepocket To assess the structural features in the chromophore vicinity caused by the mutations, we solved the crystal structures of CBD-H260A and CBD-H260A/Y273F. For crystallizing the variants, the same conditions were used as previously reported for CBD-WT and CBD-Y263F [18, 21]. The use of identical crystallization conditions enabled a direct comparison of all four variant structures. The H260A variants appeared to have reduced tendency to crystallize, which may indicate that they are structurally less stable than the CBD-WT and CBD-Y263F. The CBD-H260A and CBD- H260A/Y263F variants diffracted up to 1.88Å and 1.48Å, respectively (Table3), and the overall structures were highly similar to the CBD-WT and CBD-Y263F. The rmsd values relative to CBD-WT were 0.13Å for CBD-Y263F (8/317 residues rejected), 0.15Å for CBD-H260A (14/314), and 0.18Å for CBD-H260A/Y263F (15/315). The BV orientation was highly similar among the variants, and no differences were detected within the limits of the resolution (Supplementary Fig.1). Most significant differences between the structures resided in the vicinity of the mutated residues at the chromophore-binding pocket. Direct comparison of all four crystal structures (CBDWT, CBD-Y263F, CBD-H260A, and CBD-H260A/Y263F) is shown in Fig.2. All CBD variants contain a pyrrole water (W1), and a weaker density indicating a second water between the pyrrole water and the phenyl group of residue 263 (W2). However, in CBD-Y263F, this water density is weak, concurrent with the closer position of F263 to the pyrrole water. The chromophore-binding pockets of these CBD variants, including the coordinated waters, are very similar to the CBD-PHY fragment, except for the PHY tongue extension that binds to the pocket vicinity (see Supplementary Fig.1C). In the CBD-H260A and CBD-H260A/Y263F, the removal of the histidine side chain however allowed 2–3 additional water molecules (W3 and W4) to occupy the positions of imidazole nitrogens (Fig.2C, D). In addition, the electron density supports a fifth water molecule near W4 (W5), but its proximity to W4 (2.1Å) and weak density indicate that this may be either an alternative position for W4 or a transient water. Waters W4 and W5 were therefore modeled with partial occupancy. The lack of the histidine side chain and the involvement of additional waters also changed the hydrogen bonding network in the H260A variants (Fig.2E, F). In CBD-WT and CBD-Y263F, the imidazole hydrogens of H260 interact with the pyrrole water and C-ring propionate. In H260A variants, these imidazole interactions are replaced by hydrogen bonds Photochemical & Photobiological Sciences 1 3 between W3 and A260 main chain hydroxyl, and W4 and C-ring propionate. Overall, the amount of hydrogen bonds in H260A variants has increased considerably. 2.2 Water coordination inthebinding pocket directs theentrance toreaction pathway The two mutations, H260A and Y263F, had striking effects on the water network in the chromophore-binding pocket (Fig.2). To study the effects of these mutations on the initial photoreactions (Fig.1B), time-resolved absorption and fluorescence measurements were conducted. We concentrated on the wild-type CBD and its single mutations (H260A and Y263F) to pinpoint the separate effect of these residues on the photocycle. The measurements were conducted at pH 8.0, like in other similar experiments [13, 14, 54]. The timeresolved absorption and fluorescence spectroscopy studies of CBD-PHY under similar conditions are published elsewhere [14, 21]. We used time-resolved absorption spectroscopy (pumpprobe) from a femtosecond to nanosecond time scale to monitor the relaxation processes of the photoexcited samples. Pump-probe measurements for all samples were done by exciting the BV at 700nm and monitoring in the spectral range of 530–770nm. For the spectra, see Supplementary Fig.2. At a fast time scale, one can recognize ground state bleaching of the Q band together with stimulated emission. At a long time scale, an induced absorption at 730nm appears, which can be assigned to Lumi-R state formation [13]. In Fig.3A, we show trace curves at 730nm with their fits for CBD-WT, CBD-H260A and CBD-Y263F. Typically, multi-exponential data fitting is used to analyze the time-resolved absorption traces, and this approach was required for all CBD variants in this study. The parameters are summarized in Table1. Until 6ns, the resulting transient absorption traces of CBD-WT show features that have been previously reported [14]. Only CBD-WT shows induced absorption at 730nm typical of Lumi-R, whereas CBD-Y263F and CBD-H260A do not display this absorbance (Fig.3A). This is consistent with our steady-state measurements at pH 8.0 (Supplementary Fig.3A–C), and our published results with the Y263F variant [21], where only minor absorption at around 750nm appeared in CBD-Y263F and CBD-H260A after illumination with red light. Despite this small absorption signal, urea denaturation experiments indicate, however, that BV isomerization occurs in illuminated CBD-H260A to a similar extent as in wild-type CBD and CBD-PHY fragments (Supplementary Fig.3E–I). WT Y263F H260A Y263 H260 A260 D207 H290 H290 H290 H290 F263 D207 D207 D207 H260 A260 Y263 F263 W3 W4 W5 W1 W2 W3 W4 W5 W1 W2 W1 A H260A/Y263F W1 W2 W1 W2 WT +Y263F H260A+ H260A/Y263F H290 D207 H260 H290 D207 A260 W3 W4 W5 W1 W2 B CD E F Fig. 2 Crystal structures and comparison of CBD variants. A–D Water omit maps at biliverdin surroundings of the four CBD variants: A CBD-WT [18], B CBD-Y263F [21], C CBD-H260A, and D CBD-H260A/Y263F. All variants contain a pyrrole water (W1) and usually a water between it and the residue 263 (W2). The variants containing the H260A mutation have extra space created by the missing imidazole side chain, which is occupied by additional water molecules (W3–W5). The Fo-Fc omit map (green mesh) is shown at 4.0 rmsd. The maps were calculated by repeating the final step of each refinement without waters W1–W5. See [18] and [21] for refinement details of CBD-WT and CBD-Y263F, respectively. E–F Interactions of the waters W1–W5 in the biliverdin-binding pocket. These selected interactions are shown as black dashed lines. CBD-WT and CBD-Y263F are overlaid in panel E, whereas CBD-H260A and CBD- H260A/Y263F are overlaid in panel F Photochemical & Photobiological Sciences 1 3 Isomerization of illuminated CBD-Y263F was only 23% of that observed for CBD-WT (Supplementary Fig.3H). The time-resolved fluorescence measurements of CBDWT, CBD-H260A, and CBD-Y263F (Fig.3B) demonstrate that the fluorescence lifetimes correlate well with the second time constants (τ2) of pump-probe measurements (Table1). The excitation decay properties of the BV in the binding pocket can be studied using 660nm excitation and 720nm monitoring wavelengths. Similar to previous time-resolved studies of phytochrome systems [35], the fluorescence decays were fitted with multi-exponential functions to obtain the excited-state lifetimes of the CBD variants. We found two categories: first category contained CBD-WT and CBD- H260A, whose decay profile required two exponential components. In the second category, a mono-exponential fit was sufficient to describe the fluorescence decay of CBD-Y263F. The average decay times were 430ps (CBD-WT), 670ps (CBD-Y263F), and 420ps (CBD-H260A). 2.3 UV–Vis absorption ofpH titrations inthePr state show two distinct BV protonation states The time-resolved measurements indicated that the water content in CBD variants affect the fluorescence properties and the production of a Lumi-R-like state (Fig.3). Previous studies have shown that H260 and Y263 play a role in the BV protonation [50, 52]. For example, Y263F mutation in DrBphP CBD-PHY fragment increases the occurrence of a pH-dependent BV population that absorbs at 650nm when deprotonated [52]. To study whether these results apply for the CBD fragment, we applied the same four variants for pH-dependent absorption spectroscopy (Fig.4). Similar to pH titrations of CBD-PHY fragment [52], an increase in pH led to a decreased absorption of the Q band centered at 700nm, which has been attributed to deprotonation of the biliverdin chromophore [50, 55, 56]. The absorption at 700nm as a function of pH is shown for each variant fit to a single pKa model in Fig.4E. For the CBD-WT and CBD-Y263F, the midpoint pH of the titration is greater than 10 (Fig.4E), similar to the CBD-PHY counterparts [52]. Due to the protein instability at high pH values, the endpoint of the titration could not be measured and therefore accurate pKa values determined. For CBD-H260A and CBD-H260A/ Y263F, the lack of H260 imidazole decreases the midpoint of the pH titration by over two pH units. As a result, the approximate pKa value was 7.6 for both CBD-H260A and CBD-H260A/Y263F, similar to the pKa values of near 8 determined in their CBD-PHY counterparts [52]. Due to the above effects, the BV deprotonation starts to become significant at and above pH 9 in WT and Y263F. The absence BA ]DOm[037A∆ Time [ps] 0 -4 -8 0510 100 1000 ]stnuoc[ ytisnetnI Time [ns] 10 5 0 ×103 012 WT H260A Y263F IRF WT H260A Y263F Fig. 3 Transient absorption and emission decays of three CBD variants. A Transient absorption traces of CBD-WT, CBD-H260A and CBD-Y263F at pH 8.0. Samples were excited at 700nm and absorption was monitored at 730nm. Solid lines show the multi-exponential fit of the data. B Emission decays of CBD-WT, CBD-H260A and CBD-Y263F at pH 8.0. Samples were excited at 660nm and emission was monitored at 720nm. IRF is the instrument response function Table 1 Time-resolved spectroscopic characteristics of the CBD-WT, CBD-Y263F, and CBD-H260A at pH 8.0. TCSPC stands for Time-Correlated Single-Photon Counting *The average decay values of the two exponential decay components are presented for CBD-WT and CBD- H260A. In the case of CBD-Y263F, one component was applied Variant TCSPCτave (ps) pump-probe τ1 (ps) pump-probe τ2 (ps) pumpprobe τ3 (ns) CBD-WT 430 ± 30* 10 ± 5 380 ± 30 > 6 CBD-Y263F 670 ± 30* 27 ± 10 680 ± 30 – CBD-H260A 420 ± 30* 16 ± 8 370 ± 30 – Photochemical & Photobiological Sciences 1 3 of the PHY tongue, an extension of the PHY domain that shields the chromophore vicinity of CBD, does not notably affect the pKa of the BV [52]. Previous studies with CBD-PHY fragment indicated a pH-induced deprotonation of the BV and the Y263 hydroxyl [52]. In CBD-H260A, the residuals from the fit to a single pKa indicate some deviation from two-state behavior (see Supplementary Fig.4), similar to CBD-PHY H260A variant. However, this deviation is also observed for CBD-H260A/ Y263F, which does not have the hydroxyl group at position 263, thus providing no indication of Y263 deprotonation in CBD-H260A. Using singular value decomposition (SVD), two deprotonation events are observed for CBD-WT while only one is indicated for CBD-Y263F (Supplementary Fig.5), which suggests Y263 deprotonation in CBD-WT. SVD also gives some indication for Y263 deprotonation in CBD-H260A, but not as clear as observed in CBD-PHY. 2.4 Fluorescence properties ofthepH‑dependent populations The existence of the pH-dependent populations in the CBD variants (Fig.4) led us to assume that these populations were also visible in the fluorescence spectra. We therefore continued to measure the absorption, fluorescence emission, and fluorescence excitation spectra at various pH values. We chose three specific pH values to cover different degrees of protonation of the H260 variants: greater than 99% protonated at pH 5.5, ~ 30% protonated at pH 8.0, and ~ 3% protonated at pH 9.0 (Fig.4E). With CBD-WT and CBD-Y263F this trend is not as prominent, as BV remains at least 99.9% protonated at pH values 5.5 and 8.0, and approximately 98% protonated at pH 9.0. The resulting spectra at three selected pH values are shown in Fig.5, and all parameters are summarized in Table2. The shapes of the CBD-WT emission spectra (Fig.5E) were similar at all pH values, consistent with the absorption measurements (Fig.5A). However, a small blue-shift of 4nm could be observed when increasing the pH, concomitant with the decrease of the fluorescence QY from 3.0% (pH 5.5) to 1.3% (pH 9.0). Compared to CBD-WT, the fluorescence emission of CBD-Y263F was less sensitive to changes in pH (Fig.5F). The shape and the QY of the spectra remained almost unchanged between pH 5.5 and 8.0, and only a small additional emission peak appeared around 650nm at pH 9.0. In CBD-H260A and CBD-H260/Y263F, the shape and intensity of the emission spectra however did not remain as constant at different pH values (Fig.5G, H). Both variants had two emission maxima, at 650nm and 722nm. The 722nm emission peak decreased abruptly with increasing pH. This decrease was accompanied by an increase of the 650nm emission peak, which levels off at pH 8.0. These pH-induced changes in spectra are even more significant for the double variant, which shows a clear 651nm peak already at the lowest pH value (pH 5.5) and a near disappearance of the 722nm peak at the highest pH value (pH 9.0). As the fluorescence spectra of CBD-H260A and CBD-H260A/Y263F did not show PPIXa-specific emission peaks at 622nm and 688nm [44], we conclude that the entire emission profile originates from the BV molecules covalently bound to protein. To demonstrate the fluorescence properties at 650nm and 750nm further, we measured the excitation spectra of the CBD variants detected at these two different wavelengths 0 1 2 3 0 1 2 Wavelength nm[ ] ].u.a[AdezilamroN bsorbance H260A Y263F/ H260A Y263F 5.0 6.9– 7.0 7.5– 7.6 8.0– 8.1 8.9– 9.0 9.5– 9.6 10.4– 10.4 – 1.21 pH 300 500 700 300 500 700 WT AB CD pH 0 ].u.a[ 007A 5678910 11 0.5 1.0 1.5 2.0 2.5 WT H260A H260A/Y263F Y263F E Fig. 4 Absorption spectra of CBD variants in the Pr state at different pH values. A CBD-WT, B CBD-Y263F, C CBD-H260A, and D CBD-H260A/Y263F. All spectra are color-coded according to the measured pH. E The absorbance value at 700nm plotted as a function of pH for CBD variants. The solid lines represent the fit to a deprotonation model with one pKa [52]. For WT and Y263F, the endpoint absorbance values at high pH were fixed to those obtained for H260A and H260A/Y263F, respectively, resulting in fitted pKa values of 10.7 (WT) and 10.6 (Y263F). The fitted pKa value for both H260A and H260A/Y263F is 7.6. The pH values selected for the following experiments (5.5, 8.0, and 9.0) are indicated as vertical dashed lines Photochemical & Photobiological Sciences 1 3 Wavelength nm[ ] ].u.a[ ytisnetnI ×106 0 2 4 0 1 E WT H H260A Y263F/ G H260A F Y263F 650700 750 800650700 750800 pH 5.5 8.0 9.0 0 1 2 3 IWT JY263F 0 1 2 KH260A LH260A Y263F/ ].u.a[ ytisnetnI dezilamroN Wavelength nm[ ] 400 500 600 700400 500 600 700 0 1 2 3 0 1 2 ].u.a[ ytisnetnI dezilamroN M H260A WT OH260A/Y263F P Y263F N Wavelength nm[] 400500 600700400500 600700 pH 5.5 8.0 9.0 0 1 2 3 0 1 2 dezilamroN ].u.a[Absorbance A H260A WT CH260A/Y263F D Y263F B Wavelength nm[] 400 500 600 700400 500 600 700 pH 5.5 8.0 9.0 pH 5.5 8.0 9.0 Fig. 5 Steady-state absorption, emission, and excitation spectra of the CBD variants at three different pH values: 5.5 (red), 8.0 (green) and 9.0 (blue). A–D Absorption spectra of WT (A), Y263F (B), H260A (C), and H260A/Y263F (D). The spectra were normalized at the Soret band (around 400nm). E–H Emission spectra of WT (E), Y263F (F), H260A (G), and H260A/Y263F (H) excited at 550nm. The fluorescence intensities were corrected for the number of absorbed photons. I–L Excitation spectra of WT (I), Y263F (J), H260A (K), and H260A/Y263F (L) at 750nm emission. M–P Excitation spectra of WT (M), Y263F (N), H260A (O), and H260A/ Y263F (P) at 650nm emission. All excitation spectra are normalized at the Soret band (around 400nm) and therefore the intensities are not directly comparable between the variants Table 2 Steady-state spectroscopic characteristics of the CBD variants at three different pH values a Absorbance and emission maxima and fluorescence quantum yield as reported in [33] b The fluorescence quantum yields were determined at 550nm Variant pH Abs. max (nm) Q/Soret ratio Exc. max (nm) at 650nm emis. Exc. max (nm) at 750nm emis. Q/Soret ratio at 750nm emis. Em. max (nm) ΦF (%)b WT 5.5 700 2.8 – 700 2.0 723 4.2 8.0 696 2.7 – 696 2.3 719 2.6 9.0 696 2.4 – 696 2.8 719 1.7 Y263F 5.5 701 2.5 – 700 1.6 723 4.5 8.0 701a2.5 – 700 1.6 723a4.2a 9.0 701 2.4 600 700 1.7 653 and 723 3.3 H260A 5.5 700 1.9 600 700 2.1 722 4.5 8.0 697 1.0 600 700 2.1 653 and 722 1.1 9.0 625 and 688 0.4 600 622 and 698 1.3 653 and 722 1.0 H260A/Y263F 5.5 698 2.1 600 700 1.8 651 and 722 4.1 8.0 616 and 698 1.1 600 700 1.6 651 and 722 1.6 9.0 606 and 696 0.4 600 612 and 700 0.9 651 1.4 Photochemical & Photobiological Sciences 1 3 (Fig.5I–P). The excitation spectra at 750nm emission (Fig.5I–L) showed that all variants have a component that absorbs at the Q band. The excitation spectra at 650nm emission for CBD-WT and CBD-Y263F (Fig.5M, N) had very similar shapes at almost every pH value, but the excitation spectrum for CBD-Y263F at pH 9.0 had a small broad peak at around 600nm. This peak indicates a second fluorescent component with low 600nm absorption. The excitation spectra of CBD-H260A and CBD-H260/Y263F confirm the existence of at least two fluorescing components. Their excitation spectra at 750nm emission (Fig.5K, L) show that the second component is present mainly at the highest pH values, and the excitation spectra at 650nm emission (Fig.5O, P) show that the second component absorbs widely at around 600nm. To aid comparison, the excitation and absorption spectra (Fig.5A–D) are normalized to the maximum of the Soret band at approximately 400nm. The overall similarity between the excitation spectra at 750nm emission (Fig.5I–L) and absorption spectra (Fig.5A–D) for all variants, suggests that this part of the emission is due to normally oriented BV [22]. However, a closer inspection indicates that the ratios between the Q and Soret band intensities are different in absorption spectra and excitation spectra (Fig.5 and Table2). This is most clearly observed in CBD- Y263F and CBD-WT. The Q/Soret ratio for CBD-Y263F was lower in the excitation spectra than in the absorption spectra at all pH values. For CBD-WT, however, the ratio in the excitation spectra changes more significantly as a function of pH, being lower than in the absorption at pH 5.5 but higher at pH 9.0. This suggests that the excited-state energy surfaces, responsible for Q and Soret absorption, are similarly affected at pH 5.5 but differ as the pH increases. Since the pH titration in Fig.4 indicates no significant deprotonation of BV, a logical candidate for these effects is deprotonation of the Y263 hydroxyl. The H260A and H260A/Y263F variants can also be used to distinguish the effects of pH with and without the Y263 hydroxyl, although the interpretation is complicated by significant deprotonation of the BV. The Q/Soret ratio for CBD- H260/Y263F was lower in excitation spectra than absorption spectra at pH 5.5 and higher at pH 8.0 and 9.0, whereas the ratio for CBD-H260A in excitation spectra was higher than absorption spectra at all pH values. Given that the pKa values of these variants are similar, it suggests that Y263 hydroxyl is deprotonating. Y263 was also observed to deprotonate around pH 8.0 in pH titration experiments of CBD-PHY- WT and CBD-PHY-H260A [52]. In this study, SVD analysis indicates Y263 hydroxyl deprotonation for CBD-WT. However, the evidence is not as clear for deprotonation of this tyrosine in CBD-H260A (Supplementary Fig.5). Possibly the non-two-state behavior observed in both CBD-H260A and CBD-H260A/Y263F (Supplementary Fig.4) makes the detection of Y263 deprotonation difficult, as several pH- dependent processes seem to occur concurrently between pH 6 and 9. 3 Discussion The available space, amount of water molecules, and the flexibility of the chromophore-binding pocket have been proposed as the main factors to explain the spectral behavior of phytochromes [31, 54, 57]. In this study, we have studied H260 and Y263 in the function of the CBD fragment of D. radiodurans phytochrome. Namely, we showed that these residues have an important role in the Pr state and water content of the chromophore-binding pocket. Through these waters, the residues influence the photoreaction pathway taken, by selectively favoring either Lumi-R state, fluorescence, or non-radiative relaxation pathway. We also showed that these residues protect BV against changes in solvent pH by excluding water molecules from the BV-binding pocket. The main conclusions are summarized in Fig.6. The initial steps in the photocycle of phytochromes, i.e., from the Pr state to the Lumi-R state, have been extensively studied by time-resolved absorption spectroscopy [13–15, 24, 54, 58–60]. The kinetics of the various BV-binding phytochrome photoreactions do not differ significantly. Mutational studies have identified highly conserved key amino acids, like Y263, D207, H260, and H290, in the immediate vicinity of the D-ring, which directly affect the excited-state Water content Reaction pathway His260 Protonation state Tyr263 A Lumi-R Fluorescence Relaxation Tyr263 hydroxyl His260 imidazole Water content Pocket rigidity B Protonation (pKa) -- Fig. 6 Summary of the results obtained in the study. A Simplified scheme of the events related to the coordinated water content in the chromophore-binding pocket. H260 and Y263 affect the water network, which then affects the chosen reaction pathway (Lumi-R, fluorescence, or non-radiative relaxation) and the protonation state of the biliverdin. Only relations through the coordinated waters within the pocket are shown. B The effects of H260, Y263, and pocket parameters on the photoreaction pathway taken and on the pKa Photochemical & Photobiological Sciences 1 3 refinement of macromolecular crystal structures. Acta Crystallographica Section D Biological Crystallography, 67, 355–367. https:// doi. org/ 10. 1107/ S0907 44491 10013 14 72. Sirbu, D., Turta, C., Benniston, A. C., Abou-Chahine, F., Lemmetyinen, H., Tkachenko, N. V., etal. (2014). Synthesis and properties of a mesotris–ferrocene appended zinc(ii) porphyrin and a critical evaluation of its dye sensitised solar cell (DSSC) performance. RSC Advances, 4, 22733–22742. https:// doi. org/ 10. 1039/ C4RA0 3105A Authors and Affiliations HeliLehtivuori1,2 · JessicaRumfeldt3 · SatuMustalahti4 · SamiKurkinen3,5 · HeikkiTakala3,6 1 Nanoscience Center, Department ofPhysics, University ofJyvaskyla, 40014Jyvaskyla, Finland 2 Research andInnovation Services, Tampere University, 33014Tampere, Finland 3 Nanoscience Center, Department ofBiological andEnvironmental Sciences, University ofJyvaskyla, 40014Jyvaskyla, Finland 4 Nanoscience Center, Department ofChemistry, University ofJyvaskyla, 40014Jyvaskyla, Finland 5 Institute ofBiomedicine, Integrative Physiology andPharmacy, University ofTurku, 20014Turku, Finland 6 Department ofAnatomy, Faculty ofMedicine, University ofHelsinki, Box63, 00014Helsinki, Finland