Distinct classes of red/far-red photochemistry within the phytochrome superfamily

Abstract

Phytochromes are a widespread family of photosensory proteins first discovered in plants, which measure the ratio of red to far-red light to control many aspects of growth and development. Phytochromes interconvert between red-absorbing P_r and far-red-absorbing P_fr states via photoisomerization of a covalently-bound linear tetrapyrrole (bilin) chromophore located in a conserved photosensory core. From recent crystal structures of this core region, it has been inferred that the chromophore structures of P_r and P_fr are conserved in most phytochromes. Using circular dichroism spectroscopy and ab initio calculations, we establish that the P_fr states of the biliverdin-containing bacteriophytochromes DrBphP and PaBphP are structurally dissimilar from those of the phytobilin-containing cyanobacterial phytochrome Cph1. This conclusion is further supported by chromophore substitution experiments using semisynthetic bilin monoamides, which indicate that the propionate side chains perform different functional roles in the 2 classes of phytochromes. We propose that different directions of bilin D-ring rotation account for these distinct classes of red/far-red photochemistry.

Phytochromes comprise a class of red/far-red biliprotein photoreceptors that regulate photomorphogenesis, shade avoidance, and development in higher plants (1, 2). Also found in bacteria and fungi, phytochromes characteristically photoconvert between red-absorbing P_r and far-red-absorbing P_fr states (3, 4). Conversion between these 2 states involves Z/E photoisomerization of the 15/16 double bond of their linear tetrapyrrole (bilin) chromophores. Photosensory signaling by phytochromes relies on a conserved core comprising PAS (PER, ARNT, SIM), GAF (cGMP phosphodiesterase, adenylate cyclase, FhlA), and PHY (phytochrome-specific GAF-related) domains (5), with the bilin bound within a conserved pocket of the GAF domain (1, 6). The precise structure of the bilin chromophore, the nature of its covalent linkage to cysteine (Cys) side chains in the protein, and the location of those Cys residues vary among phytochromes (7), and 2 distinct subclasses can be distinguished on these grounds.

Phytobilin-containing phytochromes, which include the plant (Phys) and cyanobacterial (Cph1) phytochromes, are found exclusively in oxygenic photosynthetic organisms. The chromophore precursor for these phytochromes is either phytochromobilin or phycocyanobilin (PCB), both of which possess reduced ethylidene-containing A-rings. For these phytochromes, covalent linkage forms between a GAF-domain Cys and the α-carbon of the A-ring ethylidene (Fig. S1A). The much more widespread biliverdin-containing phytochromes (4), which include the bacteriophytochromes (BphPs) and fungal phytochromes, possess covalent linkages between a Cys residue upstream of the PAS domain and the β-carbon of the A-ring endo-vinyl group of biliverdin IXα (BV; Fig. S1B) (8). P_r is commonly the thermally stable dark state for both classes of phytochromes, with P_fr exhibiting slow thermal reversion to P_r (dark reversion). However, a small subset of biliverdin phytochromes, known as bathyphytochromes, have large amounts of P_fr at thermal equilibrium (9–11), and rare cases of atypical wavelength sensitivity have also been reported (12, 13). Such photosensory diversity suggests that rare divergent photochemical mechanisms have evolved, but most phytochromes retain spectrally characteristic P_r and P_fr states and hence are thought to share common P_r and P_fr structural features.

Microbial phytochromes have emerged as robust targets for biochemical, spectroscopic, and crystallographic analyses. These include the cyanobacterial phytochrome Cph1 from Synechocystis sp. PCC 6803, the BphPs DrBphP from Deinococcus radiodurans and RpBphP3 from Rhodopseudomonas palustris, and the bathyphytochrome PaBphP from Pseudomonas aeruginosa (11, 12, 14, 15). Structural information for photosensory cores of these phytochromes is now available (6, 8, 16–18). Cph1, RpBphP3, and DrBphP appear to share a common C5-Z,syn C10-Z,syn C15-Z,anti chromophore configuration for their P_r states (Fig. S1). The bilin chromophores of all 3 phytochromes also adopt an α-facial disposition for their D-rings (D-α_f; Fig. S1C), which contrasts with the D-β_f disposition observed for phycobiliproteins (19, 20). Only one P_fr structure has been reported to date, that of PaBphP crystallized as a thermally stable P_fr dark state (18). PaBphP's P_fr chromophore possesses a C5-Z,syn C10-Z,syn C15-E,anti configuration and adopts an α-facial disposition, suggesting that the facial disposition of BphP D-rings does not change upon P_r to P_fr interconversion.

Bilin chromophores are strongly active in circular dichroism (CD) spectroscopy; hence, CD has long been exploited to assess the structure and environment of linear tetrapyrroles in biliproteins (21). For PCB, we have shown that the CD sign of the red transition arises from the facial disposition of the D-ring (22). CD spectra of phytobilin- and BV-containing phytochromes in their P_r states share strong negative rotation for the red absorbance band (22–25). This band exhibits weakly negative rotation in the P_fr state of the BphP Agp1 assembled with a synthetic chromophore (25), consistent with structural results suggesting that the D-ring remains α-facial during P_r/P_fr interconversion of biliverdin phytochromes (8, 16, 18). By contrast, CD spectra of plant and cyanobacterial (Cph1) phytochromes show strong inversion of the red band upon P_r to P_fr photoconversion (23, 24), which raises the possibility that the P_fr states of BV-containing phytochromes differ from those of phytobilin-containing phytochromes.

Additional support for structurally-distinct subclasses of P_fr comes from mutagenesis of a conserved Tyr residue proximal to the chromophore D-ring in the P_r state (Tyr-176 in Cph1 and DrBphP). Substitution of His for this Tyr residue in both Cph1 and plant phytochromes yields intensely red-fluorescent holoproteins that are nearly photoinactive (26–28). The equivalent substitution in DrBphP and PaBphP does not block P_fr formation, despite the observation (Fig. S1) that the Tyr side chain directly contacts the BV C-ring 12-propionate side chain in the crystal structure of the PaBphP P_fr state (18, 27, 29).

The present work was undertaken to test the hypothesis that BV- and phytobilin-containing phytochromes possess distinct photochemical reaction mechanisms. Using CD spectroscopy, we establish that the P_fr state of Cph1 differs from those of the BphPs DrBphP and PaBphP. We further show that the structural requirements for P_r/P_fr interconversion in Cph1 are distinct from those of DrBphP by reconstitution experiments with synthetic chromophore 12-monoamides. Our studies provide compelling support for the conclusion that BV- and phytobilin-containing phytochromes exhibit different photochemical reaction trajectories. A mechanistic formulation of the 2 distinct red/far-red photochemical pathways is proposed.

Results

Cph1 and DrBphP Adopt Different D-Ring Facial Dispositions in the P_fr State.

Previous results have raised the possibility that phytobilin and biliverdin phytochromes have distinct P_fr states. To test this hypothesis, we examined Cph1, DrBphP, and PaBphP, for which crystal structures have been reported. Both Cph1 and DrBphP exhibit negative CD for the red absorbance band in the P_r state (Fig. 1) and positive CD for the blue absorbance band, consistent with the D-α_f disposition (22). The CD spectrum for the red-light-generated P_r/P_fr photoequilibrium of Cph1 displayed positive CD at wavelengths corresponding to the red P_fr absorbance band and more complicated behavior in the blue absorbance band (Fig. 1A). These results are in good agreement with those previously obtained for Cph1 (24) and confirm that Cph1 photochemistry proceeds with inversion of CD.

Fig. 1.

The P_fr states of Cph1 and DrBphP are distinct. (A) CD spectra of 21 μM Cph1 in the P_r state (solid line) and at P_r/P_fr photoequilibrium (dashed line). (B) CD spectra of 17 μM DrBphP in the P_r state (solid line) and at P_r/P_fr photoequilibrium (dashed line). The far-red absorbance of P_fr is indicated.

In contrast with Cph1, the CD of the P_fr red absorbance band of DrBphP remained negative (Fig. 1B). The signal strength of the P_fr state of DrBphP was notably weaker than that of P_r, indicating that the DrBphP P_fr chromophore is more planar than P_r. This observation is consistent with the weak negative CD reported for a synthetic BV chromophore bound to the BphP Agp1 in the P_fr state (25). We also observed negative CD for the thermal P_r/P_fr equilibrium of the bathyphytochrome PaBphP across the entire red absorbance band (Fig. S2). Both BphP P_fr spectra exhibited negative far-red CD.

The different signs of rotation for the P_fr states of Cph1 and BphPs demonstrate that the P_fr chromophore configurations of phytobilin and biliverdin phytochromes are distinct. We reported that red band CD of PCB chromophores reports the facial disposition of the bilin D-ring relative to the B- and C-rings (22). We extended these calculations to models for BV chromophores and to 15-E,anti configurations appropriate for P_fr (Table 1). Although the results preclude unambiguous assignment of the red band to a single calculated transition, the qualitative trend established previously is reproduced: every D-α_f configuration has negative red CD, and every D-β_f configuration has positive red CD. This finding is consistent with the D-α_f disposition observed for PaBphP (18). We thus assign P_fr in Cph1 as D-β_f; the similar inversion of CD reported for oat phytochrome (23, 24) would imply a D-β_f P_fr for plant phytochrome as well.

Table 1.

Calculated spectral parameters

Bilin	C15 configuration	D-ring facial disposition	λvert	fL	Rv
PCB	Z, anti	D-αf*	631	0.69	−332
PCB	Z, anti	D-βf*	628	0.62	+274
PCB	E, anti	D-αf	656, 642	0.14, 0.49	−112, −263
PCB	E, anti	D-βf	640	0.45	+141
BV	Z, anti	D-αf	705	0.84	−315
BV	Z, anti	D-βf	752, 643	0.24, 0.59	+85, +129
BV	E, anti	D-αf	750, 665	0.31, 0.41	−187, −200
BV	E, anti	D-βf	721	0.33	+288

*Values from ref. 22.

Apophytochromes Assemble with Chromophore Monoamides.

In the PaBphP crystal structure, Tyr-163 (Tyr-176 in DrBphP and Cph1) adopts a side-chain rotamer not observed in P_r crystal structures (Fig. S1) (6, 8, 16–18). This places the Tyr-163 side chain under the α-facial D-ring, permitting an interaction between the Tyr-163 hydroxyl and the 12-propionate side chain of the BV chromophore. Such an interaction is not plausible for Tyr-176 in the Cph1 P_fr state, because a D-β_f P_fr chromophore would sterically disfavor this Tyr rotamer. We therefore expect that the roles of the 12-propionate and this Tyr residue in P_r/P_fr interconversion will differ in these 2 classes of phytochromes. Previous studies have shown that Tyr-176 indeed performs distinct roles in the photochemistry of phytobilin and biliverdin phytochromes (8, 26–28). We tested the roles of the 12-propionate in phytochrome photochemistry by preparing 12-monoamide derivatives of BV and PCB (BV12MA and PCB12MA).

12-Monoamide chromophores were assembled with purified Cph1 or DrBphP apoprotein, dialyzed to remove free chromophore, and analyzed by spectroscopy. Both apoproteins formed covalent adducts with appropriate monoamide chromophore analogs (Fig. S3) and exhibited enhanced red absorbance relative to the free chromophores (Fig. 2). Unlike adducts with the free acids, both holoprotein spectra exhibited a pair of absorption maxima between 500 and 750 nm (Fig. 2). Both absorption bands were blue-shifted relative to the P_r peak observed with diacid chromophores (Table S1). These results indicate that bilin 12-monoamides exhibit at least 2 spectrally-distinct, thermally-stable populations on this time scale (≈0.2–0.5 s). The common “dual-P_r” behavior of Cph1 and DrBphP suggests that the 12-propionate plays similar roles in bilin binding and P_r formation in both classes of phytochromes. The reason for the presence of these 2 P_r bands is not clear, although it is possible that they represent protonated and deprotonated populations.

Fig. 2.

Apophytochromes incorporate chromophore 12-monoamides. (A) PCB12MA (solid line) was assembled with apoCph1 to give the PCB12MA adduct Cph1-PCB12MA, which has a dual-P_r spectrum (dashed line; peaks indicated). (B) BV12MA (solid line) was assembled with apoDrBphP to give the BV12MA adduct DrBphP-BV12MA, which has a similar dual-P_r spectrum (dashed line; peaks indicated). Spectra are normalized to the blue/UV (Soret) band.

Cph1 Requires the 12-Propionate for P_fr Formation, Whereas DrBphP Does Not.

To examine the role of the 12-propionate in Cph1 photoconversion, we illuminated the PCB12MA adduct with red light matching the longer dual-P_r peak and collected spectra as a function of illumination time (Fig. 3A). We observed slow loss of red absorbance without appearance of obvious photoproducts. Loss of both dual-P_r peaks upon irradiation could arise from an equilibrium between the 2 P_r populations or spectral overlap of the shorter red absorbance peak with the illuminating light. Control samples assembled with authentic PCB exhibited characteristic peak/trough pairs in the blue and red/far-red regions of the difference spectra (Fig. S3C), but samples assembled with PCB12MA displayed only very small positive peaks in the difference spectra (Fig. 3B). We cannot rule out formation of a photoproduct with a peak wavelength of ≈600 nm, but such a product would not correspond to P_fr (Table S1). We therefore conclude that the 12-propionate is essential for formation of stable P_fr by Cph1.

Fig. 3.

Distinct roles for the 12-propionate in Cph1 and DrBphP photochemistry. (A) Cph1-PCB12MA was illuminated with 650 ± 20 nm light (indicated), and spectra were taken as a function of time. (B) The photochemical difference spectrum is plotted for the data in A. (C) DrBphP-BV12MA was illuminated with 670 ± 20 nm light (indicated) as in A. Arrows indicate peak/trough regions in the difference spectrum. (D) Dark reversion of the dual-P_fr photoequilibrium was examined. Arrows are as in C. (E) Difference spectra are shown for conversion of DrBphP-BV12MA from P_r to P_fr with 670 ± 20 nm light (blue), for subsequent conversion of the same sample with 600 ± 5 nm light (green), for conversion of the 670-nm photoequilibrium mixture with 750 ± 20 nm (red), and for dark reversion of the 600-nm photoequilibrium mixture to the P_fr state (purple). (F) Normalized absorbance at 750 nm is shown for conversion of the dual-P_r/dual-P_fr photoequilibrium mixture of DrBphP-BV12MA to dual-P_r either via dark reversion (blue) or in the presence of 750 ± 20 nm light (red). Illum, illumination.

P_fr formation was readily detectable upon illumination of the BV12MA adduct of DrBphP (Fig. 3). The resulting P_fr exhibited a slight blue-shift from 750 to 738 nm by comparison with wild-type DrBphP. An additional photoproduct with a peak wavelength falling in between the 2 P_r peaks was also formed for the BV12MA adduct of DrBphP (Table S1). Dual-P_r thus gives rise to dual-P_fr. Moreover, irradiation of either P_r peak depleted both P_r peaks to yield both P_fr peaks (Fig. 3C and Fig. S3E), indicating that the dual-P_r states are in equilibrium. These results demonstrate that the 12-propionate is not necessary for P_fr formation by DrBphP.

Spontaneous thermal reversion of these dual-P_fr photoproducts to dual-P_r could be observed over a time scale of several hours (Fig. 3D), which indicates that dark reversion, 1 of 2 routes for conversion of P_fr to P_r in DrBphP, can still occur with the BV12MA adduct. Illumination of the dual-P_fr photoequilibrium mixture with 750-nm light (Fig. S3F) resulted in formation of both dual-P_r species, suggesting that the dual-P_fr states are also in equilibrium. The difference spectra for dual-P_fr formation and dual-P_r formation show similar features (Fig. 3E): a P_r/P_fr doublet in the blue and a pair of P_r/P_fr doublets in the red/far-red, with very similar peak wavelengths (Table S1). Dual-P_r formation by 750-nm illumination was faster than dark reversion (Fig. 3F), indicating that the BV12MA adduct is competent for photochemical conversion of P_fr to P_r. DrBphP therefore does not require the 12-propionate side chain of BV for interconversion of P_r and P_fr states, whereas Cph1 does.

Discussion

We present evidence that the red/far-red photocycles of the phytochrome superfamily can be divided into at least 2 classes that correlate to the BV- and phytobilin-containing phytochrome subfamilies. Both classes exhibit red/far-red photoconversion, and their P_r states are structurally similar as assayed by both CD spectroscopy and crystallography (8, 17). The roles of the bilin 12-propionate side chain during assembly of the P_r state seem similar in these 2 classes as well (Fig. 2). However, P_fr formation in phytobilin phytochromes proceeds with inversion of CD and requires the free 12-propionate side chain, whereas P_fr formation by biliverdin phytochromes does not lead to CD sign inversion and does not require the 12-propionate. These 2 classes also possess distinct requirements for a conserved Tyr residue in stable P_fr formation (27, 29). We therefore conclude there are structurally-distinct variants of red/far-red photochemistry within the phytochrome superfamily.

CD spectroscopy of photoactive biliproteins is most easily interpreted in terms of the facial disposition of the D-ring (22): negative CD for the long-wavelength transition is associated with the D-α_f disposition, whereas positive CD corresponds to D-β_f. This interpretation is consistent not only with all available phytochrome crystal structures (6, 8, 16–18), but also with structural data for α-phycoerythrocyanin (α-PEC), a phycobiliprotein that exhibits inversion of CD upon Z/E photoisomerization (20, 30, 31). Although confirmation will require crystallographic information on the Cph1 P_fr state, we assign the P_fr states of phytobilin phytochromes as D-β_f, distinct from the D-α_f P_fr predicted for DrBphP and observed for PaBphP.

We propose that different directions of rotation in BphPs and Cph1 account for the formation of their distinct P_fr states. As an initial test of this hypothesis, we modeled the motions involved in such rotations by manually displacing a model compound for the C15-Z,anti chromophore of the DrBphP P_r state in either direction, followed by semiempirical geometry optimization (Movies S1 and S2). If D-ring rotation were counterclockwise about the 15/16 bond (as viewed from the C16 side: Fig. 4), the resulting C15-E,anti geometry would be quite twisted. A significant steric clash would arise between the bilin methyl groups at C13¹ and C17¹, repelling the D-ring down to the β-face of the bilin ring system and resulting in a D-β_f P_fr state (Movie S1). By contrast, an initial clockwise rotation would move the D-ring nitrogen further toward the B/C plane (Fig. 4). This would move the D-ring NH proton into a steric clash with the C13¹ methyl group. Energy to overcome this barrier could be gained by relaxing the strained C15/C16 dihedral angle observed in BphP P_r structures but not in Cph1 (6, 8, 16, 17). Once this barrier is overcome, the D-ring is free to continue rotating and does not encounter a similar steric clash between methyl groups (Movie S2). The resulting geometry retains a D-α_f disposition that closely resembles the observed D-α_f P_fr of PaBphP. Although these simulations should not be considered accurate representations of the reactions occurring in phytochrome, they illustrate that different directions of rotation can account for the distinct P_fr states of Cph1 and DrBphP.

Fig. 4.

Different directions of D-ring rotation in phytochrome photochemistry. (Upper) The ring skeleton for the P_r chromophore is shown. Rotation of the D-ring to a formally E,anti configuration will lead to a strained 15/16 bond. (Lower Left) Counterclockwise rotation of the α-facial D-ring about the 15/16 bond (blue) will result in a steric clash between the indicated methyl groups at C13¹ and C17¹ as the 15/16 dihedral relaxes. This steric repulsion causes the D-ring to slump to the bilin β-face and results in a D-β_f C15-E,anti configuration (Movie S1). (Lower Right) Clockwise rotation of the D-ring (red) does not produce this clash upon 15/16 dihedral relaxation and permits further rotation to a D-α_f C15-E,anti configuration (Movie S2).

The structural differences between these P_fr states are also consistent with the distinct phenotypes of GAF-domain tyrosine mutants in the 2 classes of phytochromes. The interaction between GAF-domain Tyr-163 and the C12-propionate in the PaBphP P_fr state (18) would be sterically disfavored in the D-β_f P_fr state of Cph1. The role of Tyr-176 in Cph1 would thus have to be different from that found in BphPs, and indeed Y₁₇₆H and equivalent mutations have profoundly different effects in the 2 classes of phytochromes (26–29). In Cph1 (and plant phytochromes), the Y₁₇₆H mutation results in loss of photochemistry and supports intense red fluorescence (26, 27). This phenotype could be explained were the mutant His residue to adopt the side-chain rotamer observed in PaBphP's P_fr dark state, disfavoring movements required for formation of the normal D-β_f P_fr state of Cph1. Residual P_fr formation observed for Y₁₇₆H Cph1 (26, 27) could arise either from formation of aberrant D-α_f P_fr or from a population of holoprotein with His-176 in the rotamer observed in wild-type Cph1. In biliverdin-containing phytochromes, equivalent mutations neither ablate P_r/P_fr interconversion nor enhance fluorescence (27, 29). Assembly of DrBphP with BV12MA also does not ablate photochemistry or dark reversion. Perhaps neither substitution of this Tyr with His nor introduction of the amide moiety on the C-ring ablates the interaction between Tyr-163 and the C12-propionate seen in the PaBphP structure.

By contrast with BphPs, Cph1 requires both Tyr-176 and the 12-propionate for efficient photoconversion of P_r to P_fr. Indeed, no other residue can compensate for Tyr at this position in Cph1 (27), and PCB12MA does not support P_fr formation (Fig. 3). That said, the 12-propionate side chain appears to regulate formation of the protonated, extended C15-Z,anti configuration in similar ways in both classes, as revealed by the dual-P_r spectra of both 12MA adducts. The native propionate side chain may thus modulate the pK_a of the bilin ring system.

Structurally-distinct red/far-red photocycles within the phytochrome superfamily have implications for engineering of plant phytochromes for agricultural purposes. In particular, the chromophore–protein interactions of plant phytochrome P_fr are likely to be quite different from those of PaBphP. However, it is conceivable that such differences could be offset by changes in the structure of the protein, such that the conformational changes in the protein that lead to changes in the signaling state are still conserved. Such offsetting changes may well occur in the “tongue” linking the PHY domain to the chromophore (17, 18), permitting distinct P_fr states to proceed through a single, conserved signal transmission pathway to the various phytochrome output domains.

Materials and Methods

Bilin Pigments.

PCB and BV were prepared for in vitro assembly reactions as described (32–34). BV12MA was prepared by acid scrambling of appropriate precursors and was used to prepare PCB12MA via enzymatic conversion.

Holophytochrome Expression and Purification.

CD experiments used full-length phytochromes including histidine kinase domains. Strep-tagged PaBphP was purified as described (27). Full-length Cph1 was coexpressed with PCB biosynthetic machinery in Escherichia coli (35) from plasmid pBAD-Cph1FL-CBD, which contains the pBAD promoter and Cph1 reading frame described (14, 27, 35) fused to the intein/chitin-binding-domain region of pTYB2 (NEB). Cph1 holoprotein was purified as a chitin-binding fusion in accordance with the manufacturer's directions, followed by overnight dialysis into TKKG buffer [25 mM TES·KOH (pH 7.5), 25 mM KCl, 10% glycerol]. A full-length DrBphP construct (gift of Richard Vierstra, University of Wisconsin, Madison) was subcloned into pTYB2 (NEB), and the resulting DrBphPFL-CBD fusion was subcloned into plasmid pBAD-Cph1FL-CBD to yield pBAD-DrBphPFL-CBD. Expression and purification of DrBphP proceeded as for Cph1 using the ho1-expressing plasmid pAT-BV, derived by excising PcyA from pAT103 (36), to drive BV production.

Spectroscopy and Photoconversion.

Absorption spectra were recorded at 480 nm/min at 25 °C in a Cary 50 UV/Vis spectrophotometer equipped with a temperature-controlled cuvette holder. A 75-W Xe source (PTI) passed through a water filter and an appropriate bandpass interference filter was used to irradiate sample solutions in the Cary 50 via a liquid light guide. Bandpass filters used for triggering photochemistry were 650-nm center/40-nm width, 700 nm/40 nm, 750 nm/40 nm, 670 nm/40 nm, and 600 nm/10 nm. This apparatus permitted efficient and saturating photoconversion (Fig. S2). CD spectra were acquired on a Jasco J-720 at room temperature and are presented as the smoothed average of 2–3 scans with buffer subtraction.

In Vitro Assembly Reactions.

Truncated Cph1Δ (amino acids 1–514) and full-length DrBphP were expressed and purified as apoproteins by omitting plasmids encoding chromophore biosynthetic machinery. Apoprotein concentrations were estimated by using a modified Bradford assay (Bio-Rad). Chromophore (4–5 nmol, in DMSO) and apoprotein (16–20 nmol) were mixed in 1 mL of TKKG buffer with 1 mM Tris(chloroethyl)phosphine. Final DMSO levels were <2% vol/vol. After 2 h, reactions were dialyzed overnight in TKKG buffer to remove free chromophore. Presence of covalently attached chromophore after dialysis and competence of apoproteins for forming active holophytochrome were confirmed in control experiments (Fig. S3).

Ab Initio Calculations.

Model compounds lacked propionate side chains and thioethers as described (22). Gas-phase B3LYP/6–31+G* geometries were used for excited-state BLYP/6–311+G* calculations (22). BV in the C15-Z,anti D-α_f configuration was adjusted manually to match the DrBphP crystal structure. Oscillator strength f_L is reported in the length representation; rotational strength R^v is reported in the velocity representation as (esu² cm²) × 10⁴⁰. Movement of the model compound to different C15-E,anti geometries was examined by manually rotating the 15/16 dihedral angle of the BV P_r geometry from +35.8° to −100° or +103° (formally E). The resulting structures were then optimized at the AM1 level in GAMESS (37). Movies were made in MacMolPlt (38).