Phytochromobilin C15-Z,syn → C15-E,anti isomerization: concerted or stepwise?

The C15-Z,syn → C15-E,anti isomerization of phytochromobilin that underlies the photoactivation of phytochrome, the plant photoreceptor responsive to red and far-red light, is investigated by means of quantum chemical methods.

By calculating ground and excited-state potential energy surfaces for a phytochromobilin model comprising the full tetrapyrrolic skeleton, and taking into consideration rotations around the C14−C15 and C15C16 bonds constituting the methine bridge between pyrrole rings C and D, it is found that a stepwise ZE, synanti mechanism is energetically preferable over a concerted Z,synE,anti mechanism.

In particular, on the basis of the calculated potential energy surfaces, it is proposed that the primary photochemical reaction involves a ZE isomerization only, and that the subsequent synanti isomerization proceeds thermally.


Phytochromes make up a class of regulatory biliprotein photoreceptors in plants that monitor the quality and quantity of light in the environment and govern the adaptation of growth and developmental patterns in response to the prevailing light conditions by means of so-called photomorphogenic processes.1–3

The chromophore of phytochrome, phytochromobilin (PΦB, Fig. 1), is an open-chain tetrapyrrole which is covalently bound to a cysteine residue of the apoprotein through a thioether linkage.4

Phytochromes exist in two thermostable forms: the physiologically inactive red-light absorbing form (Pr, λmax = 660 nm) and the physiologically active far-red-light absorbing form (Pfr, λmax = 730 nm).5

These two are photointerconvertible, and Pr/Pfr thus constitutes a photochromic system.

Illumination with 660-nm light shifts the in vitro equilibrium between the two towards approximately 85% Pfr and triggers a physiological response in vivo, whereas illumination with 730-nm light yields about 99% Pr and cancels the physiological effect5.The kinetics of the Pr → Pfr phototransformation has been extensively studied by a variety of time-resolved spectroscopic techniques,6–14 and a number of metastable intermediates states with lifetimes in the micro to millisecond regime and distinctive absorption characteristics (λmax) have thereby been identified.

It has also been established that only the first step of the reaction, formation of the lumi-R intermediate, requires light; the subsequent steps leading to Pfr take place in the dark (cfref. 14.).

As for conformational changes of the protein and the chromophore during the phototransformation, a detailed understanding is still missing (e.g., X-ray crystallographic structures are not available).

In 1983, Rüdiger and co-workers recorded 1H NMR spectra of chromopeptide fragments of phytochrome showing that while the chromophore in Pr has a Z configuration around the C15C16 bond (C15-Z), the chromophore adopts a C15-E configuration in Pfr.15

Accordingly, they proposed that the primary photochemical reaction underlying the activation of phytochrome involves a ZE isomerization of the chromophore around its C15C16 bond.

In later studies resonance Raman (RR) techniques, which offer a means to investigate the in situ structure of chromophores in proteins, have been used to probe the conformation of PΦB at different stages of the photocycle.16–20

The consensus of these studies is that the configuration of PΦB indeed is C15-Z in Pr and C15-E in Pfr.

Furthermore, the C15-Z → C15-E photoisomerization appears to take place prior to the formation of the first intermediate (lumi-R),17,20 which is in accordance with the observation that the lumi-R → Pfr conversion does not require light (cfref. 14.).

Apart from implicating different configurations around the C15C16 bond, RR vibrational spectra also argue for different configurations with respect to the C14−C15 bond in Pr and Pfr (C15-syn and C15-anti, respectively).16,19

This suggests that the geometry of the methine bridge between pyrrole rings C and D changes from C15-Z,syn to C15-E,anti during the course of the reaction (Fig. 1), i.e., the conversion of Pr into Pfr involves a ‘two-fold’ isomerization of PΦB.

The isomerization about both a double bond and an adjacent single bond is commonly referred to as a ‘hula-twist’ isomerization.21

While the ZE isomerization in all likelihood proceeds photochemically prior to the formation of lumi-R, the nature and starting of the synanti isomerization remain elusive issues.

It has been argued that the primary photochemical reaction corresponds to the two isomerizations occurring simultaneously in a concerted fashion, as this mode may require less space in the protein pocket harbouring rings C and D.5,22

On the other hand, RR vibrational spectra of Pr, lumi-R, and Pfr suggest that the synanti isomerization is not initiated until after the formation of lumi-R, which implies that synanti proceeds thermally.19

The possibility that the protonation state of PΦB changes during the Pr → Pfr transformation is another issue that has attracted considerable interest.

As for Pr, spectroscopic data from RR17,18,20 and Fourier transform infrared (FTIR)23,24 studies unambiguously show that the chromophore is protonated (Fig. 1).

In Pfr, on the other hand, the RR spectra due to Mizutani et al. favour a deprotonated chromophore,17 while subsequent and arguably more reliable RR spectra recorded by Hildebrandt and co-workers using near-infrared Fourier transform techniques, which reduce the interference by inherent fluorescence on RR signals, suggest that the chromophore remains protonated throughout the Pr → Pfr transformation.18,20

FTIR results are also indicative of the chromophore being protonated not only in Pr and Pfr, but also in all intermediates connecting these.23,24

In light of the above discussion, the main objective of the present work is to investigate the C15-Z,syn → C15-E,anti isomerization of PΦB using quantum chemical methods.

By calculating ground and excited-state potential energy surfaces (PES) taking into account both concerted (Z,synE,anti) and stepwise (ZE followed by synanti) reaction pathways, we will in particular herein be able to present valuable new information with regard to the nature and starting of the synanti isomerization (photochemical isomerization simultaneously with ZE or thermal isomerization after ZE).

From relatively recent quantum chemical studies of the chromophore photoisomerization underlying the biological activity of the rhodopsin family of proteins, it has been suggested that only two electronic states of the chromophore (a retinal molecule) are of importance for this reaction; the ground state (S0) and the strongly absorbing first singlet excited state (S1).25–27

This two-state model, which challenges a proposed three-state model involving the optically forbidden second singlet excited state (S2) as well,28–29 has later also been implicated in other photoisomerizations of conjugated protein-bound chromophores, including, e.g., that of p-coumaric acid responsible for the activation of the bacterial photoreceptor photoactive yellow protein.30

It is, consequently, of interest to explore whether a two-state model is applicable to PΦB isomerization as well.

It should here be pointed out that the above studies25–27,30 were carried out using multiconfigurational complete active space self-consistent field (CASSCF) methods, which constitute the most appropriate theoretical framework for the modeling of photochemical reactions.31

However, such methods are inevitably marred by substantial computational demands, rendering them inapplicable to a molecular system as large as PΦB.

Actually, even subjecting to CASSCF calculations a simplified PΦB model comprising only rings C and D is a prohibitively difficult task if many points on a PES are of interest (an active space consisting of 12 electrons and 12 orbitals is a recent estimate32 of the practical limit for asymmetric molecules).

The density functional theory (DFT)-based approach undertaken in this work, on the other hand, enables the necessary calculations, but provides a less accurate description in regions where two potential energy surfaces come close in energy.

None of the conclusions emerging from the present work is however affected by this shortcoming.

Finally, even though quantum chemical investigations of direct relevance for phytochrome are scarce, it is worth mentioning that calculated RR spectra of hexamethylpyrromethene (a dipyrrole) in fair agreement with experiments have been reported,33,34 and that semiempirical calculations have yielded rather accurate electronic absorption spectra of PΦB35.

Theoretical methods

All calculations were carried out with the Gaussian 03 suite of programs.36

Ground-state geometries were optimized using the B3LYP hybrid density functional37–39 in conjunction with the 6-31G(d) basis set, excited-state geometries were optimized using the ab initio configuration interaction singles (CIS) method within the frozen-core approximation and employing the same basis set.

Vertical and adiabatic excitation energies were calculated on the basis of B3LYP ground-state and CIS excited-state geometries, respectively, using the Gaussian 03 implementation40 of time-dependent density functional theory (TD-DFT).41

The TD-DFT single point calculations were carried out with the B3LYP functional, and made use of the 6-31G(d) basis set unless otherwise explicitly noted.

Due to the fact that PΦB contains 80 atoms and PES comprising in total >200 structures had to be computed, simplified PΦB models were employed. The principal model use, hereafter referred to as PΦB1, is shown in Fig. 2, and corresponds to a protonated chromophore.17,18,20,23,24

In this model, the thioether linkage, the propionate side chains of pyrroles B and C, and the methyl group of each pyrrole are replaced by hydrogens.

It is here appropriate to emphasize that the present study neglects the effect of the surrounding protein on the photochemistry of PΦB.

Even though this effect may be considerable, we are herein aiming at estimating relative reactivities of different isomerization pathways (on which similar influence from the protein to some extent may be expected), rather than estimating accurate absolute reactivities.

The C15-Z,syn to C15-E,anti isomerization was modeled as follows.

First, the suitability of using PΦB1 in the modeling was explored by comparing calculated wavelengths of maximum absorption (vertical S1 excitation energies) for PΦB1 in Z,syn and E,anti configurations with the corresponding λmax as obtained for three larger models adding a) the missing propionate side chains of pyrroles B and C (PΦB2) b) the missing methyl group of each pyrrole (PΦB3) c) the missing propionate side chains of pyrroles B and C and the missing methyl group of each pyrrole (PΦB4).

In addition, optimized S0 geometric parameters for PΦB1, PΦB2, PΦB3, and PΦB4 were compared.

Next, basis set effects on excitation energies were investigated by calculating vertical excitation energies for the S1 and S2 states of PΦB1 in Z,syn, E,syn, Z,anti, and E,anti configurations using the 6-31G(d), 6-31G(d,p), 6-31+G(d), and 6-311+G(d,p) basis set.

Subsequently, zero-point vibrational energy (ZPVE) corrections and thermal corrections to Gibbs free energies (Gcorr) for optimized S0, S1, and S2 structures of PΦB1 (Z,syn, E,syn, Z,anti, and E,anti) were obtained through B3LYP/6-31G(d) and CIS/6-31G(d) frequency calculations, respectively.

Finally, S0, S1, and S2 PES for PΦB1 taking into account variation in C13C14−C15C16 and C14−C15C16−C17 dihedrals were computed employing 99 ‘grid points’, each combining one value from C13C14−C15C16 = {−0, −30, −60, −75, −90, −105, −120, −150, −180°} with one value from C14−C15C16−C17 = {0, 30, 45, 60, 75, 90, 105, 120, 135, 150, 180°}.

At each point, B3LYP/6-31G(d) and CIS/6-31G(d) geometry optimizations were carried out relaxing all other degrees of freedom.

In order to obtain more accurate excited-state energies, TD-B3LYP/6-31G(d) single point calculations were then performed on the optimized CIS geometries.

On the basis of the initial ‘benchmark’ calculations (commented upon in detail below), neither ZPVE nor thermal corrections for these grid point were accounted for.

It should be noted that the use of DFT for optimizing ground-state structures and CIS − a Hartree–Fock (HF)-based method − for optimizing excited-state structures to some extent results in an unbalanced description of the PES.

A more suitable approach in this respect would have been to either optimize the former (S0) at the HF level of theory, or to optimize also the latter (S1 and S2) using DFT.

However, since B3LYP in general improves significantly upon HF-derived ground-state molecular properties, and since CIS offers an efficient methodology for the calculation of excited-state geometries, we believe that the actual approach undertaken constitutes the best compromise between computational accuracy and efficiency.

Results and discussion

A. Evaluation of computational model and procedures

The results from the calculations comparing ground-state geometric parameters and wavelengths of maximum absorption for PΦB1, PΦB2, PΦB3, and PΦB4 are presented in Table 1.

From the dihedral angles, we first and foremost note that the dipyrrolic moiety comprising rings C and D regardless of computational model and configuration is non-planar.

Even though the Z,syn, E,syn, Z,anti, and E,anti designations strictly speaking refer to configurations with the C14−C15C16−C17 and C13C14−C15C16 dihedrals equal to 180 and 0, 0 and 0, 180 and −180, and 0 and −180°, respectively, these designations will for the sake of simplicity refer to geometry-optimized configurations in this section.

Moreover, we observe (cf. PΦB1 and PΦB2) that none of the bond lengths, all of which one may expect to be of relevance for the isomerization, are sensitive to the exclusion of the propionate side chains of pyrroles B and C. This is also reflected by PΦB1 and PΦB2 having very similar λmax (0.01 eV difference for both Z,syn and E,anti).

As for the influence of the methyl groups (cf. PΦB1 and PΦB3), a somewhat larger effect is observed.

Yet, the absolute mean-deviation between the two sets of bond lengths is fairly small (<0.01 Å), as are the differences in λmax between PΦB1 and PΦB3 (0.05 eV for both Z,syn and E,anti).

Even though an absolute comparison of calculated λmax, representing the absorption by the chromophore in the gas phase, with experimental spectra, representing the absorption of phytochrome in vitro, for obvious reasons cannot be made, it is encouraging to note that the maximum absorption of the E,anti configuration throughout is red-shifted relative to that of Z,syn, which is in accord with the maximum absorption of Pfr being bathochromically shifted relative to that of Pr.5

We furthermore note that the calculated red-shifts essentially remain unaltered (0.07, 0.07, 0.07, and 0.10 eV for PΦB1, PΦB2, PΦB3, and PΦB4, respectively) upon enlargement of the computational model, and that these are in reasonable agreement with the experimental value of 0.18 eV.

Table 1 also lists HF ground-state and CIS excited-state bond lengths for PΦB1.

The observed shortening and lengthening (relative B3LYP) of nominal double and single bonds, respectively, is a well-known deficiency of HF when applied to conjugated systems.

By comparing HF and CIS bond lengths, we see that the bond alternation is less pronounced in the S1 state.

This is a consequence of this state originating from a one-electron HOMO → LUMO π−π* transition.

The most notable geometric feature of the S2 state (in comparison with HF S0) is the inversion of the C13C14 and C14–C15 bonds lengths, which is a result of this state including contributions from two π−π* transitions.

Since CIS due to the inclusion of only singly excited configurations in general seriously overestimates excitation energies for states having pronounced double-excitation character (e.g., the S2 state of PΦB1), it should be stressed that the S2 geometries likely are less accurate than the S1 ones.

Even though the fact that TD-DFT in general offers a significant improvement over CIS in reproducing transition energies for low-lying valence-excited states40,42 – including to some extent also those having appreciable double-excitation character43 – motivates the construction of excited-state PES using CIS geometries and TD-DFT energies, it is nevertheless important to emphasize that the S2 PES presented below should be regarded as preliminary.

The observation that no inversion of the C13C14 and C14–C15 bonds is displayed in the S2 state of the E,anti configuration can probably be attributed to the aforementioned shortcoming associated with the CIS methodology.

By subjecting the optimized S1 geometries of PΦB1 to TD-B3LYP/6-31G(d) single point calculations, wavelengths of maximum emission were obtained as well (Table 1).

Depending on phytochrome size, the maximum fluorescence of Pr in vitro is located in the 672–692 nm (1.79–1.85 eV) region,12i.e., 0.03–0.09 eV below the absorption maximum at 660 nm (1.88 eV).5

Encouragingly enough, the calculated 0.09 eV Stokes shift (difference between vertical excitation and emission energies) for Z,syn PΦB1 accounts rather satisfactorily for this feature of the photochemistry of phytochrome.

The results from the calculations investigating basis set effects on electronic energies of PΦB1 are given in Table 2 together with calculated ZPVE and thermal corrections.

It is seen that using a larger basis set than 6-31G(d) has only a minor (≤0.4 kcal mol−1) effect on relative ground-state electronic energies.

Increasing the basis set does not significantly alter vertical excitation energies either, which is in accordance with recent TD-DFT studies on the absorption of the highly conjugated astaxanthin chromophore.44,45

At the 6-311+G(d,p) basis set level, the S1 and S2 excitation energies are lowered by 0.04 and 0.02–0.03 eV, respectively, with respect to the 6-31G(d) values.

The relative insensitivity of vertical excitation energies to the inclusion of diffuse s and p-functions on heavy atoms reflects the ‘valence-excited state’ nature of S1 and S2.

Considering differences in vertical excitation energies between different configurations (of particular relevance for the present work), basis set effects on both states throughout do not exceed 0.01 eV.

Turning to the results from the frequency calculations, we note that ZPVE corrections and Gcorr for the optimized ground-state structures differ within 0.3 and 0.2 kcal mol−1, respectively, between the different configurations.

As for the optimized excited-state structures, the dependence of ZPVE correction (within 0.5 (S1) and 0.2 (S2) kcal mol−1) and Gcorr (within 1.2 (S1) and 0.6 (S2) kcal mol−1) on conformation is not pronounced either.

These results indicate that the energetics of the PΦB1 isomerization can be assessed on the basis of electronic energies only.

Even though the effects of neglecting ZPVE corrections and Gcorr for the individual states of course may add up when the states are considered simultaneously, the conclusions to be drawn from the present work are based on electronic energy differences that, we believe, are sufficiently large to marginalize such effects.

Vertical S1 and S2 excitation energies for PΦB1 in Z,syn, E,syn, Z,anti, and E,anti configurations were in addition computed at the CIS/6-31G(d)//HF/6-31G(d) level of theory.

For both states, which respectively have HOMO → LUMO and {HOMO − 1 → LUMO/HOMO → LUMO + 1} character within the framework of CIS as well, this yielded considerably higher transition energies than did the TD-B3LYP/6-31G(d)//B3LYP/6-31G(d) calculations (0.8 and 2.3 eV higher for S1 and S2, respectively).

This finding is in accord with previously reported calculations on the electronic spectra of hexamethylpyrromethene indicating that CIS grossly overestimates excitation energies for pyrrole compounds.33

In summary, the results reported in Table 1 suggest that the approximation imposed by not including the thioether linkage, the propionate side chains, and the methyl groups in the computational model is sound, and also that PΦB1 is a reasonable model in that the calculated absorption red-shift induced upon Z,synE,anti isomerization, as well as the calculated Stokes shift for the Z,syn configuration, agree rather well with the corresponding experimental shifts as obtained for phytochrome.

The results given in Table 2, in turn, suggest that using a larger basis set than 6-31G(d) for calculating electronic energies is superfluous, and that the accounting for ZPVE and thermal corrections can be omitted.

Finally, it should be noted that our conclusion that the computational procedure is suitable for calculating the relevant isomerization PES of course is directly dependent on the validity of the benchmark calculations also for distorted phytochromobilins along the isomerization path.

B. Potential energy surfaces

In the first part of this section, we will report S0 and S1 PES for PΦB1, and thereby propose a mechanism for the Z,synE,anti isomerization involving these two states only.

In the second part, we will present different cuts of the S2 PES constituting further support for such a two-state model.

The S0 and S1 PES, and their difference, are shown in Fig. 3.

Considering first the S0 PES, we note that thermal ZE isomerization of both the Z,syn and the Z,anti configuration is prevented by a high-energy barrier (>35 and >32 kcal mol−1, peaking at β = 75°), as expected.

The barriers for EZ isomerization are of a similar magnitude (>34 and >32 kcal mol−1 for E,syn and E,anti).

We furthermore observe that light does not seem to be an absolute requirement for synanti isomerization as such, as indicated by a barrier of ∼8 and ∼9 kcal mol−1 (peaking at α = −90°) for thermal isomerization of the E,syn and Z,syn configuration, respectively.

The barriers for antisyn isomerization are likewise low (∼7 kcal mol−1 for both E,anti and Z,anti).

Turning to the S1 PES, we recall that, as outlined in Section I, the primary photochemical reaction in phytochrome in all likelihood involves a ZE isomerization of the chromophore.

Moreover, it has been argued that also the synanti isomerization is part of the primary photochemical reaction and that the two isomerizations occur simultaneously in a concerted fashion,5,22 whereas others have suggested that synanti is initiated thermally upon completion of ZE,19 thus favouring a stepwise process.

Hence, it is interesting to note that – while photochemical ZE isomerization of the Z,syn configuration is associated with a flat energy profile (maximum at β = 135° lying ∼4 kcal mol−1 above Z,syn) and a subsequent energy minimum region (at β = 90° lying ∼7 kcal mol−1 below Z,syn) enabling the system to decay to the S0 PES and evolve to E,syn – the reaction path corresponding to a concerted Z,synE,anti photoisomerization has an unfavourable energy profile.

For example, the central {α = −90°, β = 90°} structure, the 9 structures covering the {−105 ≤ α ≤ −75°, 75 ≤ β ≤ 105°} region, and the 25 structures covering the {−120 ≤ α ≤ −60°, 60 ≤ β ≤ 120°} region lie ∼30, ∼17–32, and ∼11–32 kcal mol−1 above Z,syn, respectively.

Furthermore, no S1 → S0 decay channel seems to exist along this path (cf. Fig. 3, S1−S0 PES).

These findings strongly suggest that the photoactivation of phytochrome is less likely mediated by a concerted Z,synE,anti photoisomerization than by a stepwise ZE, synanti mechanism involving a photochemical ZE isomerization (∼4 kcal mol−1 barrier) followed by a thermal synanti isomerization (∼8 kcal mol−1 barrier).

The S1 PES also suggests that an alternative mechanism (that has not been discussed in the experimental literature) involving consecutive synanti and ZE photoisomerizations could be energetically feasible.

The estimated barriers for these isomerizations are ∼6 and ∼3 kcal mol−1, respectively, which effectively means that the calculations do not unambiguously show that photochemical ZE followed by thermal synanti is the most probable route to the activation of phytochrome.

Nevertheless, of the scenarios actually discussed in the experimental literature,5,19,22 the calculations clearly show that photochemical ZE followed by thermal synanti is energetically preferable over a concerted Z,synE,anti photoisomerization.

The decay process by which the excited system returns to the ground state is a key mechanistic element of photochemical reactions.

By means of CASSCF calculations, it has been shown that the decay channel in a number of systems corresponds to a conical intersection of the ground and excited-state potential energy surfaces.25,46,47

Given that single-reference DFT methods in general fail to explicitly locate conical intersections, the possibility that the decay along the photochemical Z,synE,syn path is governed by an actual S0/S1 crossing cannot be excluded.

This uncertainty is however irrelevant for distinguishing Z,synE,syn from Z,synE,anti as the most probable photoisomerization pathway.

The S1−S0 PES shows that the two states are the closest in energy at {α = 0°, β = 75°} (∼11 kcal mol−1 apart) and {α = −180°, β = 75°} (∼12 kcal mol−1 apart).

It has been suggested that the primary photochemical reaction in phytochrome takes place within picoseconds.10,48

This is slower than the photoisomerizations underlying the biological activity of the rhodopsin family of proteins, which take place in the sub-picosecond regime and hence are essentially barrierless.

Still, the barrier for PΦB photoisomerization should be very low.

Even though the prediction that ∼4 kcal mol−1 are needed for excited Z,syn to reach the decay channel is compatible with an isomerization occurring within a photochemically reasonable period of time, it is clear that the calculations, due to, e.g., neglected interactions with the surrounding protein and limited computational accuracy, overestimate the barrier for photoisomerization.

Furthermore, a more elaborate quantum chemical treatment would require the explicit localization of excited-state stationary points and minimum energy path calculations.25–27

In order to, in turn, put the prediction that ∼8 kcal mol−1 are required for thermal E,synE,anti isomerization into an experimental perspective, it can be noted that an overall energy barrier of 17–20 kJ/mol (∼4–5 kcal mol−1) for Pr → Pfr conversion has been derived through the monitoring of changes in Pr fluorescence in vitro.49

Even though the calculations once again appear to provide an overestimation, we believe that – considering the various approximations employed – the fair agreement between theory and experiment is rather encouraging.

Finally, as for the S2 PES, we note that while an equal number (99) of S1 and S2 geometry optimizations were initiated, roughly one third of the latter were not successfully completed.

However, we are here primarily interested in the shape of the S2 PES along the Z,synE,syn isomerization coordinate.

Rather than presenting a S2 PES covering a lesser range than S1, we therefore focus our attention on the Z,synE,syn cut of S2.

This particular cut, as well as that along the Z,antiE,anti coordinate, is shown in Fig. 4 together with the corresponding cuts of S1.

We note that there are no signs of an S2/S1 avoided crossing along the Z,synE,syn isomerization coordinate, as evidenced by the facts that the S2–S1 energy gap increases (from ∼11 to ∼36 kcal mol−1 at β = 90°) as the system evolves towards the decay channel, and that the S2 state throughout retains its character.

The same features are observed along the Z,antiE,anti coordinate.

These results indicate that the two-electronic state model involving only S0 and S1 previously assigned to, e.g., the retinal photoisomerization in rhodopsins25–27 is applicable to the PΦB photoisomerization in phytochrome as well.

As emphasized in Section IIIA, the S2 PES may be qualitatively incorrect due to the inadequacy of CIS in treating the PΦB S2 state.

Therefore, the proposed two-state model for PΦB photoisomerization should be regarded as a tentative model, rather than as a rigorously derived one.


In order to gain some insight into the intrinsic mechanism governing the PΦB C15-Z,syn → C15-E,anti isomerization responsible for the photoactivation of phytochrome, we have in the present study subjected a simplified PΦB model to quantum chemical calculations.

By computing ground and excited-state PES for rotations around the C14–C15 and C15C16 bonds, it is found that a stepwise ZE, synanti mechanism is energetically much more favourable than a concerted Z,synE,anti process.

More specifically, on the basis of the calculated PES and in light of experimental observations,19 it is suggested that the primary photochemical reaction involves a ZE isomerization only, and that the subsequent synanti isomerization occurs thermally.

In addition, it is proposed that the ZE isomerization proceeds solely through interactions between the S0 and S1 electronic states.

While the elucidation of the intrinsic isomerization mechanism for an isolated chromophore in the gas-phase constitutes a natural first goal for quantum chemical studies, the ultimate goal is to be able to consider also (some of) the interactions with the surrounding protein.

Of particular interest are then the molecular mechanisms responsible for the lowering of energy barriers, and whether the protein favours another reaction pathway than that proposed in this work.

Future X-ray crystallographic structures of phytochrome will facilitate such studies.