Green Cyanobacteriochrome AnPixJ

Article pubs.acs.org/JPCB

Color Tuning in Red/Green Cyanobacteriochrome AnPixJ: Photoisomerization at C15 Causes an Excited-State Destabilization Chen Song,†,‡ Rei Narikawa,§,∥,⊥ Masahiko Ikeuchi,§,# Wolfgang Gar̈ tner,∇ and Jörg Matysik*,†,‡ †

Leids Instituut voor Chemisch Onderzoek, Universiteit Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands Institut für Analytische Chemie, Universität Leipzig, Johannisallee 29, D-04103 Leipzig, Germany § Department of Biological Science, Faculty of Science, Shizuoka University, Ohya, Suruga-ku, Shizuoka 422-8529, Japan ∥ Graduate School of Art and Sciences, University of Tokyo, Komaba, Meguro, Tokyo 153-8902, Japan ⊥ Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan # Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan ∇ Max-Planck-Institut für Chemische Energiekonversion, Stiftstraße 34−36, D-45470 Mülheim an der Ruhr, Germany ‡

S Supporting Information *

ABSTRACT: Cyanobacteriochromes (CBCRs) are cyanobacterial phytochrome-like photoreceptors that carry a single or several GAF (cGMP phosphodiesterase/adenylyl cyclase/ FhlA) domains in a repetitive manner. Unlike phytochromes that photoswitch between red-absorbing 15Z Pr and far-redabsorbing 15E Pfr states, CBCRs exhibit a much wider spectral activity. One of the best-characterized CBCRs, the phototaxis regulator PixJ of Anabaena sp. PCC 7120, AnPixJ can adopt two thermally stable photoreversible states, a red-absorbing dark state (Pr) and a green-absorbing photoproduct (Pg). Cross-polarization magic-angle spinning (CP/MAS) NMR spectroscopy on AnPixJ assembled in vitro with uniformly 13C- and 15 N-labeled phycocyanobilin (PCB) chromophore identifies changes of the electronic structure of the chromophore between the two states. Results are compared with the data from red- and far-red-absorbing forms of the complete sensory module of cyanobacterial phytochrome Cph1 aiming at a conceptual understanding of the distinct photoproduct (Pg vs Pfr) absorbances upon Pr photoconversion. The PCB chromophore in the Pr state of both photosensors exhibits very similar spectral features. The photoconversion of Cph1 and the red/green switching AnPixJ C15-Z/E photoisomerization result in a very similar chemical-shift difference (Δδ) pattern having, however, opposite sign. The persistence of this pattern confirms the identity of the photochemical isomerization process, while the difference in its sign demonstrates that the same electronic factors drive into opposite direction. It is proposed that the LUMO energy of the 15E photoproduct is stabilized in Cph1 but destabilized in AnPixJ leading to opposite color shifts upon phototransformation.

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INTRODUCTION

thioether linkage between the chromophore A-ring ethylidene side-chain (C31) and a conserved Cys residue.9−11 The primary photochemistry of the chromophore in phytochromes triggers a photoconversion typically between the red-absorbing Pr and far-red-absorbing Pfr states with a Z-to-E isomerization about C15C16 double bond.12,13 The absorbance of the photoproducts around 710−730 nm (for PCB and PΦB) or 750 nm for BV chromophores is found virtually unmodified for phytochromes.11 Remarkably, certain eukaryotic algal phytochromes have recently been demonstrated to exhibit an

Phytochromes from higher plants were the first protagonists of bilin-binding photosensory proteins1 that regulate multiple aspects of development via their red/far-red photosensitivity. Numerous phytochromes have been identified later in a wide range of organisms, including cyanobacteria,2,3 algae,4 fungi,5 and nonphotosynthetic bacteria.6 The sensory module of canonical phytochromes, irrespective of their use of PCB, phytochromobilin (PΦB) or biliverdin IXα (BV) as chromophore, comprises three domains, in which a central GAF domain is situated between an N-terminal Period/ARNT/ Single-minded (PAS) domain and a C-terminal phytochromespecific (PHY) domain.7−10 Plant phytochromes and cyanobacterial phytochromes like Cph1 incorporate either PΦB or PCB within their GAF domains (Figure 1a) via a covalent © 2015 American Chemical Society

Received: May 15, 2015 Revised: June 26, 2015 Published: June 26, 2015 9688

DOI: 10.1021/acs.jpcb.5b04655 J. Phys. Chem. B 2015, 119, 9688−9695

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The Journal of Physical Chemistry B

folds, very superimposable with the GAF domain of Cph1 15Z Pr structure (PDB code 2VEA, ref 8).23 On the other hand, major structural differences between the red-/green-absorbing CBCR AnPixJg2 and the Cph1 GAF domain include the following: (i) The binding pocket in 2VEA is completely sealed by the tongue region extending “backwards” from the PHY domain,9 whereas the isolated GAF domains in CBCRs cause partial solvent access to the pyrrole rings A and B.23 (ii) In 2VEA, the pyrrole water (pw, Figure 1a) at a central position in the center of nitrogens of rings A−C is closely associated with H260 and D207 (homologous to H322 and D291 in AnPixJg2) as well as the nitrogen atoms of rings A−C, but it is clearly absent in 3W2Z (Figure 1b). In phytochromes, this water contributes to maintain the bilin conformation and its protonation state.8−10 (iii) The conserved D291 in AnPixJg2 is oriented differently relative to D207 in Cph1 leading to distinguished bilin interactions: in 2VEA, the acidic side-chain of aspartate in the invariant DIP-motif points away from the bilin to interact with an arginine in the tongue, whereas the corresponding residue D291 in 3W2Z forms hydrogen bonds to the nitrogen atoms of rings A−C via its side-chain (Figure 1b), thus possibly acting as a counterion for the positively charged tetrapyrrole system. (iv) Red/green-type CBCRs as AnPixJ are unique in carrying a Trp located between rings A and D (W289 in AnPixJ, Figure 1b) which probably has a direct impact on the red absorption in Pr and in the formation of the blue-shifted Pg photoproduct.23 Moreover, a pair of Phe residues (the β2 Phe and helix Phe) conserved in some red/ green CBCRs (F268 and F329 in AnPixJg2, Figure 1b) was proposed to play a role in tuning photoproduct absorption,24,37 as it may stabilize a strongly twisted D-ring conformation, such that the ring is effectively out of conjugation. Also, a Phe residue on β1 was identified to be responsible for photoproduct tuning in NpR3784 which has a similar photocycle to that of canonical red/green CBCRs like AnPixJ.25 In the present work, uniformly 13C- and 15N-labeled PCB (u[13C,15N]-PCB) chromophore is in vitro reconstituted into red/ green CBCR AnPixJg2, enabling a comprehensive 13C MAS NMR analysis of a CBCR chromophore in both Pr and Pg. Here we compare these data with the 13C shifts of the Pr- and Pfr-state chromophore in the N-terminal sensory module of Cph1 (Cph1Δ2). The analogous 15Z-to-15E photoconversion of both proteins causes virtually opposite signal shifts (ΔδC pattern) of the chromophore in a region including entire ring C, C15-methine bridge, as well as C16 and C17 of ring D. The pattern allows a new, very detailed insight into the orbital architecture of the chromophore in both sensors and thus a detailed view on the color tuning mechanism of bilins bound to proteins. We propose destabilization of the LUMO level as the origin of the hypsochromic absorption shift of the greenabsorbing photoproduct in AnPixJg2.

Figure 1. Close-up views of 15Z PCB in the GAF pockets of Cph1 (a) and AnPixJ (b) crystal structures. The central α-helices and β-sheets of AnPixJ were labeled (α3′ helix omitted for clarity). Both PCB chromophores adopt the 15Za geometry covalently bound to the Sγ of a Cys residue (C259/C321 in Cph1/AnPixJ) via its ethylidene group of ring A (C31). Two conserved Phe residues, F268 and F329, proposed to be important for the green-absorbing photoproduct in red/green CBCRs,24 are highlighted (b). Pyrrole rings A−D are labeled for reference. Dashed lines indicate hydrogen bonds around the chromophore and its nearby groups. Water molecules in the tetrapyrrole cavities are marked as red spheres. Pyrrole water (pw) closely associated with the rings A−C in Cph1 (a) is absent in AnPixJg2 (b).

astonishing variety of photoproduct absorbances that cover the entire visible spectrum.14 CBCRs, a recently discovered family of photoreceptors in cyanobacteria, possess a simpler domain architecture relative to phytochromes but exhibit diverse photocycles spanning the visible to the near-ultraviolet spectral range.15−18 CBCRs can be classified into at least four subfamilies according to their characteristic differences in primary sequences and photocycles,19 i.e., two of them feature opposite photocycles: red/ green20−26 and green/red27,28 as well as the so-called DXCF29−32 and insert-Cys18,33−36 subfamilies exhibiting twoCys photocycles. Also, there is a large group of simply sequentially identified, but functionally still undefined, members.16,21 Despite their remarkably variable photocycles, CBCR sensors require only a bilin-binding GAF domain to achieve fully reversible photochemistry,15,16 yet several CBCRs contain multiple repeats of GAF domains, sometimes more than one being capable of binding PCB, thus exhibiting complex light sensing ability.18,21,22 Like phytochromes, CBCRs use bilins as chromophores. The identity of the chromophore and the autocatalytic binding, its attachment site, and the similarity of the primary photochemistry as well as a high level of conservation in key structural residues between CBCRs and phytochromes have been confirmed by a number of recent high-resolution crystal structures of CBCRs: a red/green AnPixJg2 (second GAF domain in AnPixJ) in the 15Z Pr state (PDB code 3W2Z) and a DXCF-type blue/green TePixJg (PixJ of Thermosynechococcus elongatus BP-1) in the 15E Pg state (PDB code 3VV4).23 Moreover, two crystal structures of TePixJg in its 15Z blue-absorbing (Pb) state (PDB codes 4FOF and 4GLQ) confirmed the presence of second Cys-based thioether linkage to the bilin at C10 besides the conventional Cys linkage involving C31.35 Three-dimensional crystal structures revealed a high similarity to the “canonical” GAF

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EXPERIMENTAL METHODS Sample Preparation. Cph1Δ2. The u-[13C,15N]-PCBCph1Δ2 in both Pr and Pfr states at 100% occupancy were obtained as described.12 No additional illumination was applied to the samples before and during the data collection. AnPixJg2. E. coli C41 cells generating apo-AnPixJg2 were disrupted by a French press as previously described.20 After centrifugation, the supernatant was separated from the pellets and was mixed stepwise with small amount of u-[13C,15N]-PCB aliquots, until no further increase of the Pr peak was detected. Subsequently, the holo-AnPixJg2 was purified by nickel affinity 9689


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C(O)O− signal at 176.04 ppm on the TMS scale. Data were processed with Topspin 3.1 (Bruker).

chromatography as previously described.20 Prior to measurement of the Pr form, the purified protein was loaded into a 1 mm bore glass capillary syringe at 20 °C and illuminated with 525 nm light for 2 min from an array of appropriate LEDs to ensure maximal Pr formation.20 We note that the remaining fraction of Pg was negligible for NMR measurements. Pg data were acquired after illumination at 639 nm under otherwise identical experimental conditions. MAS NMR Data Collection. All 13C CP/MAS experiments were performed on a Bruker AV-750 WB spectrometer (Karlsruhe, Germany) equipped with a 4 mm triple resonance MAS probe (Bruker). Samples of 15 mg of Cph1Δ2 and 1.3 mg AnPixJg2 were used in this study. Each sample was loaded into a 4 mm zirconia MAS rotor and cooled to −40 °C in the magnet. The MAS rate of all experiments was maintained at 13 kHz ± 5 Hz. Typical 1H π/2 and 13C π pulses were 3.0 and 5.2 μs, respectively. 13C transverse magnetization created by the ramped CP was transformed from 1H with a contact time of 2.048 μs for all spectra (Figures 2 and 3). For 1H decoupling,

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RESULTS AND DISCUSSION AnPixJg2 and Cph1Δ2 in Their Pr Dark States. The Pr state of AnPixJg2 is similar in secondary and tertiary structure to the GAF domain of Cph1 with a root-mean-square deviation of 1.5 Å.23 This is in particular so with respect to the geometry of the chromophore and also the conservation of instrumental amino acids in direct vicinity of the chromophore serving to maintain its conformation and hydrogen-bonding interactions (Figures 1 and 2, inset). In both proteins, electrostatics and hydrogen-bonding interactions fix pyrrole rings A−C by a central aspartate residue (either through the carboxylate sidechain in AnPixJ or indirectly via an intermittent water molecule (“pw”) in Cph1, Figure 1), and the propionate side-chains at rings B and C in dissociated state are similarly stabilized by amino acid side-chains. Not surprisingly, similar 13C Pr spectra are obtained for the chromophores of both photosensors (Figure 2; for tentative assignment of AnPixJg2, see Supporting Information). In particular, the positions of most of 13C signals remain identical; e.g., all peripheral methyl/methylene sites ranging from 0 to 50 ppm are barely affected by the binding pocket (|ΔδC| ≤ 1.3 ppm) suggesting no significant difference in the chromophore−protein interaction in both Pr forms (Supporting Information Table S1 and Figure 4c). Moreover, the persistence of C6, C9, and C11 peaks for both samples (Figure 2) implies that in Cph1Δ2 and AnPixJg2 the chromophore remains cationic with all four nitrogens being protonated. The C5 signals differ by only 1.7 ppm, corresponding nicely to a similar tilt between rings A and B in 3W2Z (11.4°) and 2VEA (9.8°). However, notable differences are found for the line width of some signals; e.g., the C5 peak of Cph1Δ2 is much narrower (ν1/2 = 258 Hz) than that from AnPixJg2 (605 Hz), suggesting a loss of local order of this methine bridge in the latter case. Contrary to C5, the line width of C4 in Cph1Δ2 is broader than that from AnPixJg2, and a similar broadening is also seen for other A-ring carbons like C3 and C31 implying the disturbance of crystal packing in Cph1, consistent with the observation that C31 atom coexists in Cph1-Y263F mutant as diastereomers.38 The most prominent ΔδC is noted for the C15 signals (3.8 ppm) presumably caused by the much larger distortion angle of ring D (relative to the coplanar B/C plane) in 3W2Z (60.8°) compared to that in 2VEA (26.3°). Further, a relatively large ΔδC of 3.2 ppm is found for C31 that can be explained as a consequence of opposite stereochemistry at this position in both cases (Figure 2, inset). It should be noted that in AnPixJg2 the signal intensities of the methylenes occurring between 20 and 50 ppm show an overall increase over those of Cph1Δ2. A similar spectral change is also found in the region below 20 ppm containing the D-ring ethyl and four bilin methyl groups. Increased peak intensities lead to relative narrowing of the lines suggesting a more efficient CP by reduction of dynamics of the chromophore in the binding pocket of AnPixJg2 relative to Cph1Δ2 (see the associated difference spectrum in Figure 2, AnPixJg2 peaks dominating the region below 50 ppm). Pr → Pg Photoconversion in AnPixJg2. The lightinduced ΔδC values of AnPixJg2 PCB between Pr and Pg (Figures 3a and 4b) denote substantial changes of the electronic structure of the chromophore as well as its interaction with the binding pocket upon photoisomerization. Two key aspects of the alternating pattern are as follows: (1)

Figure 2. Comparison of 15Z PCB Pr states in AnPixJg2 and Cph1Δ2. 13 C MAS NMR spectra of u-[13C,15N]-PCB-AnPixJg2 and u-[13C,15N]PCB-Cph1Δ2 are shown in red and purple, respectively. δC values are indicated by vertical lines (for detailed AnPixJg2 assignments, see Supporting Information). The assignments of C14 and C16 in AnPixJg2 (underlined) may be interchanged. Normalized difference spectrum (bottom) was calculated as Cph1Δ2 − AnPixJg2. Peaks arising from the natural abundance glycerol carbons are denoted by asterisks. Pair-fitted 15Z PCB structures of both proteins with selected atoms labeled by numbers are displayed as inset.

two-pulse phase modulation scheme with a pulse duration of 5.5−7 μs and a 1H rf field strength of ∼78 kHz were employed. 13 C spectra were recorded with 8k and 32k scans for Cph1Δ2 and AnPixJg2, respectively. A recycle delay of 1.5 s was used in all cases. A Lorentzian apodization function with line broadening factors of 10 Hz was applied to the data processing. 13 C chemical shifts were externally referenced to α-glycine 9690


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Figure 3. Comparison of the 15Z Pr and 15E Pg states of AnPixJg2 and the two 15E PCB photoproducts of AnPixJg2 (Pg) and Cph1Δ2 (Pfr). (a) 13 C MAS NMR spectra of u-[13C,15N]-PCB-AnPixJg2 in its 15Z dark state (red) and 15E photoproduct (blue) with δC indicated by vertical lines. The assignments of C14 and C16 for 15Z PCB may be interchanged, also, for C14, C16, and C18 for 15E PCB. (b) 13C MAS NMR spectra of two 15E photoproducts of AnPixJg2 (blue) and Cph1Δ2 (brown). Normalized difference spectrum (bottom) was calculated as 15E − 15Z (a) and Cph1Δ2 15E − AnPixJg2 15E (b).

evidenced by the significant δC change at C9 position (5.2 ppm). As shown with Raman spectroscopy, for in vivo generated AnPixJg2, all four pyrrole nitrogens are protonated in both states40 as seen in phytochromes.41−44 Intriguingly, in vitro generated sample exhibits the almost identical dark state and photoproduct absorptions as the native protein (Supporting Information Figure S1), although for such preparation the photoconversion generates a deprotonated 15E photoproduct. In any case, it is evident that the effect of the de/protonation remains very local and that the conjugation network is robust against this change (Figure 4b) implying that the observed changes on rings C and D can be interpreted independently of the protonation state. (2) For the change in chromophore− protein interaction, W289 of AnPixJg2 connects in the Pr state both terminal pyrrole rings by forming π-stacking interactions with ring D via its indole ring and by hydrogen bonding to the A-ring carbonyl (Figure 1b). Upon photoconversion to Pg, the interaction of ring A seems to be maintained, as evidenced by the subtle ΔδC of A-ring carbons (Figure 4b). On the other hand, rotation of ring D would destabilize or disrupt the strong π-stacking interactions and thus result in a pronounced positional change of this indole. Indeed, these interactions have been proposed to be critical for the color tuning in AnPixJg2.40 Furthermore, to generate Pg, ring D must be released from its Pr association with Y352 (C19O···Y352Oη, ∼2.6 Å, Figure 1b). As implied by the two Pg structures of TePixJ, 3VV423 and 2M7V,45 the ring D of 15E AnPixJg2 chromophore is likely to be stabilized by the interaction with the side-chain of D291 via its nitrogen, whereas no hydrogenbonding partner is apparent for its carbonyl. Weakening of the hydrogen bond at this position is evidenced by the striking upfield shift of C19 (ΔδC = 5.1 ppm). The decreased

For C15-Z/E photoisomerization, the same primary photochemistry in both canonical phytochromes and CBCRs has recently been confirmed to involve a rotation of ring D around the C15C16 double bond.23 Our current data on the Pr → Pg photoconversion in AnPixJg2 are fully consistent with such a statement, suggesting that the absorption properties of the photoproduct are not controlled simply by the initial photoisomerization but regulated by subsequent changes in the chromophore−protein interactions. As seen in Figure 3a for the two states of AnPixJg2, C5 and C10 signals of Pg occur at positions almost identical to their Pr counterparts. However, a large ΔδC of 2.3 ppm was found to be associated with the C15methine bridge, fully in line with the fact that the Z/E isomerization occurs at this position. This is further supported by the striking ΔδC of C14 and most of D-ring pyrrolic carbons (Supporting Information Table S1). The current data do not allow an unequivocal one-to-one assignment for C14, C16, and C18 signals of the Pg state at 139.7, 137.0, and 141.3 ppm. However, in any case, the Pr signals of C14 and C16 (δC = 147.1/145.4 ppm) undergo dramatic upfield shifts upon Pg formation, and on the contrary, the C18 Pr-signal is displaced downfield by at least 5.0 ppm (see Supporting Information). Another prominent change in the spectrum is found in the lowfield region, referring to the D-ring carbonyl (C19) that shifts upfield from 174.0 as Pr to 168.9 ppm as Pg (see below). On the other hand, the photoconversion has little effect on all Aring carbons (Supporting Information Table S1). For this reason, an A-ring rotation about C5 can be ruled out although one of its adjoining carbons, C6, exhibits a 5.0-ppm downfield shift that can be ascribed to the Pg deprotonation of the B-ring pyrrole nitrogen.39 The state-induced protonation change of the AnPixJg2 chromophore when generated in vitro is also 9691


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facilitate such chromophore movement during photoconversion, this Tyr would have to adopt a new side-chain conformation and thereby modify its Pr interaction with 12carboxylate. Surely, the discussed conformational changes of both aromatic side-chains (W289 and Y302) found in close proximity to the π-electrons of the chromophore cause an electronic rearrangement in the conjugated double bond system of the bilin. The photoconversion in canonical cyanobacterial Cph1Δ2 is intimately linked to a mesoscopic increase in rigidity of chromophore and its direct protein environment, evident from the line-width reduction in 13C signals of the chromophore and in its interfacial 1H contacts.46 This appears not to be the case for the formation of 15E photoproduct in AnPixJg2 which causes broadening of most 13C signals from pyrrolic carbons, indicating increased mobility relative to the more rigid 15Z Pr. Heterogeneous mixtures of 15E species have shown to be widespread in both phytochrome47,48 and in the red/green CBCR subfamily.37,49,50 Comparison of the PCB-binding pockets of AnPixJg2 and Cph1Δ2 (Figure 1) identified a number of differences in critical chromophore−protein contacts, e.g., the absence of pyrrole water in AnPixJg2 and different roles of the conserved aspartate (D291/207 in AnPixJg2/Cph1), causing differences in the Coulomb architecture of the chromophore-binding pocket which might prevent the occurrence of a charge-driven mesoscopic phase transition as observed in Cph1Δ2.46 15Z-to-15E Isomerization of the Chromophore in the Two Photosensors. Despite the 13C spectral similarity for the PCB isomers of AnPixJg2 and Cph1Δ2 in their 15Z Pr states (Figures 2 and 4c), both proteins in their respective 15E states demonstrate significantly distinguished ΔδC for most of the pyrrolic carbons (Figures 3b and 4d). In particular, C13−C17 at and around the methine bridge between rings C and D show a |ΔδC| ≥ 7.7 ppm (Supporting Information Table S1). Such ΔδC cannot be explained only by the configurational changes of the chromophore occurring upon photoproduct formation, but must also involve large-scale rearrangement of the critical interactions with its binding pocket and therefore with the protein counterions. Intriguingly, the prevalence of blue spheres for all C-ring pyrrolic carbons (Figure 4d) indicates the electron density at this ring in AnPixJg2 15E PCB is increased relative to that of Cph1Δ2. It is also evidence that the origin of the changes in conjugated π-system of the two 15E PCB photoproducts locates here. Moreover, as shown in Figure 4a, the photoconversion of Cph1Δ2 from Pr to Pfr affects mostly the rings C and D (indicated by the green-shaded region), whereas for the Pg formation in AnPixJg2, the analogous 15Z-to-15E isomerization causes an exactly opposite ΔδC pattern in this region, including the entire ring C, C15-methine bridge, as well as C16 and C17 of ring D (Figure 4b). The persistence of the pattern points to the conservation of the same photochemical mechanism, occurring, however, with an inverted electronic effect. Since the photoisomerization of free bilins in organic solution shows an entirely different ΔδC pattern from that observed for both proteins,39 we take this continuity as convincing evidence for a double-bond photoisomerization by the same mechanism. In the case of Cph1Δ2 (Figure 4a), red spheres are dominant in this green-shaded region, indicative of a decrease of local electron density for its 15E PCB as Pfr, whereas formation of the green-absorbing 15E species of AnPixJg2 (Figure 4b) leads to an increase of local electron density in the same region.

Figure 4. Schematic of ΔδC of PCB accompanying 15Z-to-15E isomerization in AnPixJg2 and in Cph1Δ2 as well as differences between their 15Z dark states and 15E photoproducts. ΔδC values taken from Supporting Information Table S1 are illustrated in panels (a−d) for changes from 15Z PCB to 15E PCB in Cph1Δ2 (15E − 15Z, a), from 15Z PCB to 15E PCB in AnPixJg2 (15E − 15Z, b), and for differences between two 15Z Pr states (AnPixJg2 Pr − Cph1Δ2 Pr, c) and two 15E photoproducts (AnPixJg2 Pg − Cph1Δ2 Pfr, d), respectively. Carbons and nitrogens are colored gray and blue, respectively. PCB atoms in the shaded regions (a and b) exhibit opposite ΔδC patterns upon 15E photoproduct formations in both proteins. The red and blue spheres represent down- and upfield shifts, respectively.

polarization at the terminal group of the conjugated π-system might impair the full D-ring electronic conjugation with the rest of the chromophore; however, this alone would hardly account for the large hypsochromic shift of the 15E photoproduct absorbance in AnPixJg2.21 The C-ring propionate carboxylate (C123) undergoes a 4.1ppm upfield shift during Pg formation (Supporting Information Table S1). A plausible explanation could be the rearrangement of its hydrogen-bonding network caused by a rotation shift of the chromophore within the binding pocket subsequent to its initial D-ring flip. In AnPixJg2 Pr, 12-carboxylate forms a bidentate salt bridge with the conserved R301 (Figure 1b). Photoconversion to Pg might be associated with a partner swap of R301 from 12-carboxylate as Pr to 8-carboxylate as Pg due to the chromophore rotation as implied by a comparison between the two structures of TePixJ, Pr 4GLQ35 and Pg 3VV4.23 The positional shift would also cause a steric clash between the 12carboxylate and the phenyl ring of Y302 (Figure 1b). To 9692


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The Journal of Physical Chemistry B To explain the hypsochromic shift of the first absorption transition of AnPixJg2 photoproduct in a simple model, we assume that a simple two-dimensional Hückel approach can be successfully applied to the extended conjugated π-system of open-chain tetrapyrroles considering “regular” π−π* transitions. The results are analogous to the four-orbital model by Gouterman explaining the Q- and B-bands of macrocyclic tetrapyrroles as first and second electronic transitions, respectively, and their almost complete absence of vibronic fine structure.51,52 In fact, the high-energy shoulder of the first electronic transition is also observed in pure Pfr of Cph153 and Pg (Supporting Information Figure S1) and might be a common feature of bilin proteins independent of their actual conformational and protonation state. The simple orbital scheme shown in Figure 5 explains the relative energy levels

orbital energies. This point-charge control appears to affect the LUMOA localized in particular on rings C and D. Strong effects of counterions on the energy of a transition are well-known, e.g., in retinal proteins.54 However, such a direct correlation between NMR Δδ patterns and the electronic transition energies has not yet been reported. Comparison to NpR6102g4. NpR6102g4 is a photochromic red/green-active CBCR, closely related to AnPixJg2. Very recently, solution-state NMR studies on NpR6102g4 provided the complete 13 C assignments of the PCB chromophore for both Pr and Pg states37 which are compared to those of AnPixJg2 and Cph1Δ2 presented here (Supporting Information Figure S2 and Table S2). Again, the δC of NpR6102g4 15Z Pr is similar to that of Cph1Δ2 (Supporting Information Figure S2e) although some differences at ring A and the C-ring propionate reflect the changed chromophore− protein interactions. The light-induced changes upon Pg formation in NpR6102g4 are less pronounced (Supporting Information Figure S2c) compared to AnPixJg2 (Supporting Information Figure S2b); however, a dramatic change is observed at the A-ring covalent linkage to the protein (Supporting Information Figure S2c). Comparison of the two green-absorbing photoproducts with the far-red one (Supporting Information Figure S2g,h) reveals the same ΔδC pattern at rings C and D (shaded in green) but in a less pronounced manner in the case of NpR6102g4 (Supporting Information Figure S2h). It appears that also in NpR6102g4 the same change of orbital architecture occurs, although probably to a lesser extent. There are, however, additional effects in NpR6102g4, in particular on the ethylidene carbons of ring A as well as on the three methine bridges indicating a stronger mechanical distortion upon phototransformation. The two Pg photoproducts are compared in Supporting Information Figure S2i which reveals an additional electronic effect in NpR6102g4: red spheres at most of the pyrrolic carbons of rings B and C (representing higher ppm values in NpR6102g4) and blue spheres at the two terminal rings, suggesting that in NpR6102g4 the charge is pushed out of the central part toward the terminal parts of the chromophore by an additional electrostatic effect specific for the more complex changes occurring in NpR6102g4.

Figure 5. Simplified four-orbital model for color transition in AnPixJg2 and Cph1Δ2 in their photoproduct states. Two near-by electronic ground states (HOMOA and HOMOB) and two more distant electronically excited states (LUMOA and LUMOB) allow for first (B) and second (Q) electronic transition. Both the B- and Q-bands are split into two transitions and polarized within the plane of the chromophore (x, y) as high-frequency Bx/y and low-frequency Qx/y. The LUMOA’s of two photoproducts in AnPixJg2 and Cph1Δ2 are labeled as LUMOA (Pg) and LUMOA (Pfr), respectively. The LUMOA is destabilized in AnPixJg2 and stabilized in Cph1Δ2. The B-band transitions are not affected by the change of the LUMOA energy.

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of the electronic transitions of these compounds between two highest occupied molecular orbitals (HOMOA and HOMOB) and two lowest unoccupied molecular orbitals (LUMOA and LUMOB). Due to an extended multipole structure, the excitedstate orbitals are more strongly split than those of the ground state; essentially two slightly split absorption bands occur: a first transition from HOMOA and HOMOB to LUMOA and second from HOMOA and HOMOB to LUMOB. Both transitions are shown to be composed of two components caused by transitions of different polarization (x and y). Applying this concept to the 15Z PCB Pr states of Cph1Δ2 and AnPixJg2, we conclude that mainly the energy of LUMOA is shifted in both 15E PCB photoproducts: stabilized in Pfr [LUMOA (Pfr)] but destabilized in Pg [LUMOA (Pg)]. The maintenance of the same photochemical process (i.e., the C15C16 double-bond isomerization) leads to conservation of the Δδ pattern. The opposite sign of the pattern, however, is caused by the difference in LUMOA stabilization. This simple concept implies that differences in absorption properties of the photoproducts of Pfr and Pg are difficult to rationalize by a possible change in the effective conjugation length, but more probably are caused by matrix effects as for example by counterions of the protein environment controlling

CONCLUSIONS In this study, by using 13C MAS NMR spectroscopy, we demonstrate that the red/green CBCR AnPixJg2 has a 15Z PCB Pr state very similar to that of Cph1Δ2. Also, the bilin photoconversion occurs under maintenance of the ΔδC pattern as in Cph1Δ2, implying the same site (C15-methine bridge) and mechanism of double-bond isomerization. The sign of the pattern, however, is inverted. While the electron density at rings C and D is increased in AnPixJg2 as Pg, it is decreased in Cph1Δ2 as Pfr. This difference in charge architecture points to a destabilization of a LUMO of AnPixJg2 in the Pg form resulting in the observed blue shift of its photoproduct. It appears that the comparison of AnPixJ and Cph1 is a fortunate case for the dissection of the origin of the electronic spectra and their changes of bilin proteins since the “orbital lift” pattern is not superimposed by further effects.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed NMR assignment of AnPixJg2 in both states, and additional spectroscopic data. The Supporting Information is 9693


Article


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available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b04655.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+49) 341 9736112. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We thank Prof. J. Hughes (Gießen) for providing labeled chromophores and his helpful comments, and Profs. P. Hildebrandt (Berlin) and D. Sebastiani (Halle an der Saale) for stimulating discussions. We are grateful to F. Lefeber and K. B. Sai Sankar Gupta (Leiden) for instrumental assistance. This work was funded by the DFG (Hu702/8), the NWO (DN 89190 and ALW 822.02.007), the Max-Planck-Society, and the JST.

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Green Cyanobacteriochrome AnPixJ

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