Excited State Properties of Protochlorophyllide

Analogues and Implications for Light Driven

Synthesis of Chlorophyll

Derren J. Heyes1*, Samantha J. O. Hardman1, David Mansell1, Aisling Ní Cheallaigh1†, John M.

Gardiner1, Linus O. Johannissen1, Gregory M. Greetham2, Michael Towrie2, and Nigel S.

Scrutton1*

1Manchester Institute of Biotechnology and School of Chemistry, The University of Manchester,

131 Princess Street, Manchester M1 7DN, UK

2Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities

Council, Harwell Oxford, Didcot, OX11 0QX, UK

Corresponding Authors

*derren.heyes@manchester.ac.uk, nigel.scrutton@manchester.ac.uk

1

Abstract

Protochlorophyllide (Pchlide), an intermediate in the biosynthesis of chlorophyll, is the substrate

for the light-driven enzyme protochlorophyllide oxidoreductase (POR). Pchlide has excited state

properties that allow it to initiate photochemistry in the enzyme active site that involves

reduction of Pchlide by sequential hydride and proton transfer. The basis of this photochemical

behavior is investigated here using a combination of time-resolved spectroscopies and DFT

calculations of a number of Pchlide analogues with modifications to various substituent groups.

A keto group on ring E is essential for excited state charge separation in the molecule, which is

the driving force for the photoreactivity of the pigment. Vibrational “fingerprints” of specific

regions of the Pchlide chromophore have been assigned, allowing identification of the modes

that are crucial for excited state chemistry in the enzyme. This work provides an understanding

of the structural determinants of Pchlide that are so important for harnessing light energy.

KEYWORDS Pchlide analogues, photochemistry, structural derivatives, time-resolved

spectroscopy, excited state

2

Introduction

The chlorophyll precursor protochlorophyllide (Pchlide) is the major pigment found in

seedlings and etiolated plants and acts as an essential light-activated trigger for the subsequent

formation of chlorophyll and development of the plant.1-3 Pchlide is the substrate and

chromophore for the light-driven enzyme protochlorophyllide oxidoreductase (POR), which

catalyzes the reduction of the C17-C18 double bond to form chlorophyllide (Chlide) and is an

important regulatory step in chlorophyll biosynthesis.1-3 The reaction catalyzed by POR involves

a light-driven hydride transfer from NADPH to the C17 position of the Pchlide molecule,4-5

followed by a thermally-activated proton transfer from a conserved Tyr residue to the C18

position, both reactions involving quantum mechanical tunneling.6

Although the chemical steps in the POR reaction cycle proceed on the microsecond timescale,5

catalysis is dependent on excited-state processes in the Pchlide molecule. Time-resolved studies

on the isolated Pchlide pigment have demonstrated that Pchlide is an intrinsically reactive

molecule with multi-exponential dynamics7-12 that depend strongly on solvent polarity.7-8, 12 The

identification of a number of Pchlide excited state species suggest a multi-phasic quenching of

the Pchlide excited state emission via solvation of an intramolecular charge transfer (ICT) state7-

12 and the subsequent formation of a triplet state on the nanosecond timescale.11-12 Moreover,

time-dependent density functional theory (DFT) calculations have confirmed the ICT character

of the Pchlide excited state in methanol and, in turn, this is thought to induce site-specific

solvation of the photoexcited Pchlide molecule via strengthening of H-bonding interactions.13 A

detailed understanding of the exact mechanism of photochemistry in the ternary enzyme-

substrate complex has proved more challenging although it was recently proposed that excited

state interactions between active site residues and the carboxyl group at the C17 position results

3

in the formation of a ‘reactive’ ICT state.14 This creates a highly polarized C17-C18 double bond,

thereby facilitating the subsequent nucleophilic attack of the negatively charged hydride from

NADPH to the C17 position of Pchlide.14

It is likely therefore that the dipolar character of the ICT state in Pchlide is crucial for

harnessing the light energy to drive POR catalysis and ultimately, the development of the plant.

The highly reactive nature of the excited state is thought to be caused by the presence of a

number of substituent groups on the Pchlide molecule, such as the electron-withdrawing

carbonyl group on ring E,12, 15-16 central Mg ion and the carboxylic acid sidechain at the C17

position, all of which have been shown to be important for enzymatic photoreduction (Figure

1).17 We have now synthesized a number of Pchlide analogues that contain alterations to all of

these positions (compounds A-F in Figure 1) and used a combination of spectroscopic

techniques and computational approaches to understand how the structural changes affect the

photochemical and excited-state properties of the Pchlide molecule that are so crucial for POR

catalysis.

4

Figure 1. The light-driven reduction of the C17-C18 double bond of protochlorophyllide

(Pchlide) to form chlorophyllide (Chlide) is catalyzed by protochlorophyllide oxidoreductase

(POR) and requires NADPH as a cofactor. The dashed box indicates the double bond that is

reduced during the reaction and the red circles show the regions of the Pchlide molecule that

have previously been shown to be important for activity (central Mg, ring E and the side chain at

the C17 position). The structures of all of the Pchlide derivatives described in the present study

are shown underneath with the modifications indicated by dashed red circles.

5

Experimental methods

Synthesis of Pchlide analogues

All chemicals and solvents were purchased from Sigma Aldrich, except where specified, and

were of analytical grade or better. All pigments were synthesized from commercially available

pheophorbide a to yield the target compounds A-F (Figure S1) and were verified by NMR and

mass spectrometry. Chemical syntheses were monitored by thin-layer chromatography using

aluminum foil-coated TLC plates carrying silica gel 60 F254 (0.2 mm thickness; Merck).

Ultraviolet light, and/or phosphomolybdic acid (10 g of phosphomolybdic acid in 100 mL of

absolute ethanol) were used to detect compounds. Purification of compounds was carried out

using column chromatography (Fluka Analytical high-purity grade silica gel, 60 Å pore size,

220−440 mesh particle size, 35−75 μm particle size). NMR spectra were recorded on a Bruker

Avance 400 MHz spectrometer and referenced to the solvent. Coupling constants (J values) are

expressed in Hz and were calculated using iNMR processing software. Proton peaks are

indicated according to their appearance (e.g. singlet, doublet, triplet) or as a multiplet where no

defined multiplicity is observed and/or overlap of multiple signals occurs. Low resolution mass

spectrometry was performed on Waters SQD2 (Q-MS with ES+, ES- and APCI source). High

resolution mass spectrometry was performed on a mixture of Waters QTOF micro (Q-TOF

reflection with ES+ ion source, accurate mass determination <5ppm) and LTQ Orbitrap XL

(Engineering and Physical Sciences Research Council National Mass Spectrometry Facility,

Swansea, UK). Where specified, reactions were carried out in a glovebox (Belle Technology)

under nitrogen atmosphere (< 5ppm oxygen). Deoxygenated solvents were prepared by a freeze

thaw method alternating between vacuum and nitrogen atmosphere where appropriate.

6

An overview of the synthesis of compounds A-F is shown in figure S1 and a full description of

the chemical syntheses, together with NMR and mass spectrometry analyses, can be found in the

supporting information. Briefly, Chlide a (A') was synthesized by magnesium insertion into

pheophorbide a (Inochem Ltd.) in an anaerobic glovebox (Belle Technology) to prevent the

formation of allomerization by-products as previously described,18 using the procedure reported

by Lindsey and Woodford.19 Chlide a to Pchlide a (A) conversion was carried out using 2,3-

dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)20 using a 1:1.1 molar ratio of substrate/DDQ and

a reaction time of 30 minutes. The 1H-NMR showed a downfield shift of signals for H-20 and H-

13b consistent with pyrroline to pyrrole oxidation of ring D. A small portion of A was treated

under acidic conditions to remove the chelating Mg2+. The de-metallated 17,18-

didehydropheophorbide a (B) could be obtained either through treatment with 5% H2SO4 or with

the use of an acid exchange resin (Amberlite IR-150 H+ resin) and was confirmed by the

presence of the N-H peaks in the 1H-NMR spectrum at -2.48 ppm and -3.14 ppm. The Zn

derivative (C) was prepared by Zn insertion into pheophorbide a using Zn acetate21 to produce

(C'), which was subsequently oxidized at the C17-C18 position with DDQ to furnish C.

Three further derivatives were also synthesized from pheophorbide a to provide a range of

compounds with simple modifications to the oxygen containing functional groups in the

molecule. The first modification was the esterification of pheophorbide a using 5% sulphuric

acid in methanol to afford the pheophorbide a methyl ester (D'). Methanolysis was performed to

open the isocyclic ring of pheophorbide a to give the dimethyl ester derivative of chlorin e6

(E').21 Finally, following the procedure described by Tamiaki et al.,22 the ketone of the isocyclic

ring was reduced with NaBH4 to give the 13a-OH compound (F') as a mixture of epimers. It

should be noted that this reaction was unreliable and the desired product formation was

7

accompanied by the formation of a contaminant resulting from concomitant reduction at the site

of the methyl ester, often with over-reduction occurring before full consumption of the starting

material pheophorbide a. This was particularly prevalent during larger scale reactions but

sufficient quantities of F' were obtained for further derivation. The three derivatives D', E' and

F' were then subjected to the above Mg insertion (D'', E'', F'') and DDQ oxidation (D, E, F) as

described for the synthesis of A.

Time-resolved spectroscopy.

Ground state absorbance spectra were recorded using a Cary 50 UV/visible spectrophotometer

(Agilent Technologies). Samples contained each analogue (A-F) at OD ~0.8 in methanol for

time-resolved visible measurements or OD ~0.07 at 430 nm in 2H-methanol for the time-resolved

IR spectroscopy measurements. Visible transient absorption spectroscopy was carried out using a

Ti:sapphire amplifier (hybrid Coherent Legend Elite-F-HE, 1 kHz repetition rate, 800 nm, ~120

fs pulse duration); a non-collinear optical parametric amplifier (light conversion TOPAS White)

was used to generate the pump beam. The 17, 18-didehydropheophorbide a (B) data were

collected using a different Ti:sapphire amplifier system (Spectra Physics Solstice Ace, 6 mJ of

800 nm pulses at 1 kHz with 100 fs pulse duration), a Topas Prime OPA with associated

NirUVis unit which was used to generate the pump beam. In both cases the pump beam was

centred at 430 nm with 1 µJ pulse power and a beam diameter of ~ 150 µm which was

depolarized before the sample. A broad band ultrafast pump-probe transient absorbance

spectrometer ‘Helios’ (Ultrafast systems LLC) was used to collect the ‘fast’ data with a time

resolution of around 0.2 ps. Data were collected for approximately 30 mins per dataset at

randomly arranged time delays ranging between 300 fs and 3 ns. The probe beam consisted of a

white light continuum generated in a sapphire crystal. A broad band sub-nanosecond pump-

8

probe transient absorbance spectrometer ‘Eos’ (Ultrafast systems LLC) was used to collect

‘slow’ data. Data were collected for approximately 30 mins per dataset at randomly arranged

time delays ranging between 0.5 ns 17.2 µs. A 2 kHz white-light continuum fiber laser was used

to generate the probe pulses. The delay between pump and probe was managed electronically.

Samples were magnetically stirred in a 2 mm pathlength quartz cuvette. After correcting the

‘fast’ data for spectral chirp the ‘fast’ and ‘slow’ datasets were combined by scaling the full

‘slow’ dataset by a fixed factor to match the intensity of ground state bleach feature in the ‘fast’

dataset at similar time points (datasets overlap between ~0.5 to 3 ns).

Infra-red transient absorption spectroscopy was carried out at the Ultra facility (Central Laser

Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, UK),

using the time-resolved multiple probe spectroscopy (TRMPS) technique.23 Samples were

contained between two CaF2 windows separated by a teflon spacer to give a pathlength of

approximately 100 µm. An excitation wavelength of 430 nm with 0.5 µJ pulse power and a beam

diameter of ~ 150 µm, set at the magic angle with respect to the probe beam, was used. The

sample was flowed through the cell and the sample holder rastered to avoid sample damage.

Difference spectra were generated relative to the ground state in the spectral window 1500-1800

cm-1 with a spectral resolution of ~3 cm-1. Data were collected for approximately 30 mins per

dataset at randomly arranged time delays ranging between 300 fs and 2 ns.

Fluorescence experiments were carried out using a Ti:sapphire amplifier system (Spectra Physics

Solstice Ace) producing 6 mJ of 800 nm pulses at 1 kHz with 100 fs pulse duration. A portion of

the output of the amplifier was used to pump a Topas Prime OPA with associated NirUVis unit,

which was used to generate the pump beam (~0.4 µJ) centred at 430 nm, with FWHM of ca. 10

nm. A commercial Halcyone (Ultrafast Systems LLC) fluorescence up-conversion spectrometer

9

with CCD detector was used to collect data from 550-750 nm over a timeframe of 0.5-2000 ps

with a time resolution of ~0.5 ps.

Global analysis

The datasets were analyzed globally using the open-source software Glotaran.24 This procedure

reduces the matrix of change in absorbance as a function of time and wavelength to a model of

one or more exponentially decaying time components, each with a corresponding difference

spectrum. Errors quoted with the lifetime values are the standard errors calculated during global

analysis. The lifetimes quoted for the conversion between states also include contributions from

the rates of ground state recovery through both radiative and non-radiative processes.

Differences in experimental conditions (e.g. methanol vs. 2H-methanol) could be expected to

affect the rates of non-radiative relaxation, hence the apparent disparities in some of the rate

constants described here. The purpose of the global analysis was to distinguish the evolution of

spectral features in order to determine the mechanism of intermediate formation rather than

define precise kinetics. The infra-red dataset analysis was performed with the longest lifetime

component fixed from the corresponding analysis of the visible measurements.

DFT Calculations

Energy minimizations and frequency calculations were performed in Gaussian0924 using density

functional theory (DFT) for the ground state and time-dependent DFT (TDDFT) for the first

excited state (Root=1), as previously used to model hydride transfer from NADPH to Pchlide.5

These calculations employed the hybrid DFT B3LYP26 functional with the LANL2DZ basis set

with effective core potential for the metal ions and the 6-31G(d) basis sets for all other atoms.

Energies, dipole moments and solvent reorganization for the ground and first excited states were

10

obtained from single-point calculations using the BP86 functional27 with the triple-ζ valence

basis set with polarization functions Def2-TZVP,28 as previously used for excited state

calculations of PChlide.13 Gas-phase bond dissociation energies for a complex XY were

calculated according to the equation BDE = E(XY) – [E(X) + E(Y)] where E is the potential

energy, and E(XY) includes basis set superposition error calculated by the counterpoise method.

Solvent reorganization energies are the difference of non-equilibrium and equilibrium solvation

contributions to the vertically excited state, calculated in methanol using a polarizable continuum

model. IR spectra were obtained applying Lorentzian peak broadening (with a band width of 10

at ½ peak height) to the calculated values, which was applied prior to the subtraction of ground

state from excited state spectra to obtain the difference spectra.

Results and discussion

Excited state dynamics of Pchlide analogues.

The static absorption spectra of compounds A-F reveal a number of significant differences in

their spectral properties (Figure 2). Substitution of the central Mg2+ ion with Zn2+ (compound C)

has a minimal effect whereas complete removal of the central metal (compound B) results in a

decrease in the distinctive Qy absorbance band at 629 nm. Changing the C17 carboxylic acid to a

methyl ester (compound D) has no effect on the Qy absorbance peak although a slight

broadening of the main Soret absorbance band is observed. However, the most dramatic effect is

found in analogues that contain changes to the C13a keto group of ring E (compounds E and F),

where the red-light absorbing features broaden and decrease in amplitude and there is a

narrowing of the main Soret absorbance band. Hence, this keto group gives Pchlide, and indeed

chlorophylls in general, their characteristic green color and any changes to this region of the

molecule result in a pink / red appearance.

11

Figure 2. Ground state absorbance spectra of Pchlide derivatives A-F in methanol.

Time-resolved spectral changes in the visible region after laser excitation at 430 nm have been

measured for all of the Pchlide analogues in methanol over the complete ps to µs timescale as

described in the experimental methods. Global analysis was then used to model the time-resolved

difference spectra from these measurements by fitting the data to a sequential kinetic model to

yield evolution associated difference spectra (EADS).14 For clarity, only the EADS are shown in

the main manuscript (Figure 3), whereas the raw data, the residuals from the fits and evolution

associated spectra can be found in the supporting information. The excited state dynamics of

Pchlide have been described previously and involve the formation of an excited ICT state from

the initially Sn excited state (~10 ps), solvation of the ICT state (50-300 ps) and decay of the

S1/SICT excited state into a long-lived triplet state on the nanosecond timescale (Figure 3A).7-12

There are only minor differences in the electronic spectra of the different Pchlide excited singlet

12

species, although the triplet has a distinctly blue-shifted bleach at ~629 nm and less intense

excited state absorption features. In methanol the triplet state then relaxes back to the ground

state with a lifetime of approximately 250 ns (Figure 3A). As with Pchlide, the structures of the

time-resolved difference spectra for the Pchlide analogues (Figure 3B-F) also closely resemble

the steady-state absorption spectra. However, the time-dependent spectral changes can only be

fitted to 4 components for 2 of the analogues (compounds D and E), with only minor differences

in the lifetimes of the various excited state species (Figure 3D and E). This suggests that changes

to the C17 carboxylic acid group (compound D) and the opening of ring E (compound E) have

not appreciably altered the excited state properties of the molecule and both analogues relax to

the ground state via the same route (Sn → SICT → solvated SICT → triplet). However, changes to

either the central metal ion (compounds B and C) or to the C13a keto group of ring E (compound

F), do alter the excited state decay kinetics in these analogues (Figure 3B, C and F). In these

cases, the excited state dynamics are best described by fitting to 3 sequentially evolving

exponential functions with time constants that are similar to the τ1, τ3 and τ4 lifetimes observed

for the other Pchlide analogues. This implies that it is likely to be the formation and solvation of

the ICT state that is affected in these analogues, suggesting that the central metal ion and the

C13a keto group are important for charge separation across the Pchlide molecule and/or

hydrogen bonding interactions with the solvent during the excited state dynamics.7-12, 13

13

Figure 3. Evolution associated difference spectra (EADS) resulting from a global analysis of the

time-resolved visible data for Pchlide derivatives A-F in methanol after excitation at 430 nm.

The data were fitted to a sequential model as described in the Experimental Methods. Steady-

state absorption spectra are shown as dashed lines.

Formation and solvation of the ICT state in Pchlide analogues.

The role of the various substituent groups of Pchlide in the formation and solvation of the ICT

state has been investigated further by a combination of fluorescence lifetime measurements and

DFT calculations. Time-resolved fluorescence spectra were recorded for all of the Pchlide

analogues over a range of time points from 0.5 ps to 2 ns after excitation at 430 nm and fitted to

a sequential kinetic model to yield evolution associated spectra (EAS) (Figure 4). All of the

analogues contain 2 major emission peaks as observed previously.9, 12, 13 The time-dependent

14

fluorescence changes for Pchlide could be fitted to 3 distinct lifetimes of 2.4 ps (τ1), 48 ps (τ2)

and 2.5 ns (τ3) (Figure 4A) and have been assigned to vibrational relaxation and reorganization

(τ1), solvation (τ2) and relaxation of the singlet excited state to the ground state (τ3), respectively.

9, 12 Significantly, the red-shift in the fluorescence maximum that is observed between EAS2 and

EAS3 for Pchlide (Figure 4A) has been shown to be caused by solvent reorganization around the

excited ICT state, which leads to a strengthening of the hydrogen bonding network around the

Pchlide molecule.12, 13 Similar red-shifts in the fluorescence maxima are also observed when the

C17 carboxylic acid group is changed to a methylester (compound D) and upon opening of ring

E (compound E), confirming that solvation of the ICT state is not affected in these analogues

(Figure 4D and E). However, the data for those analogues that incorporate changes to the central

metal ion (compounds B and C) and the C13a keto group of ring E (compound F) could only be

fitted with 2 components (Figure 4B, C and F), likely to represent vibrational relaxation and

reorganization (τ1) and, relaxation of the singlet to the ground state (τ2). The EAS for these

analogues also show no significant red-shift in peak position over time, which implies that these

regions of the Pchlide molecule are likely to play a role in the solvation of the ICT state.

15

Figure 4. Evolution associated spectra (EAS) resulting from a global analysis of the time-

resolved fluorescence data for Pchlide derivatives A-F in methanol after excitation at 430 nm.

The data were fitted to a sequential model as described in the Experimental Methods.

Time-dependent DFT calculations have been used to provide more detailed insights into the

role of the central metal ion and the C13a keto group of ring E in the excited state charge

separation across the Pchlide molecule and the subsequent solvation of the ICT state (Table 1).

The magnitude of the dipole across the Pchlide molecule (Figure S57) was calculated in both the

ground and excited states of Pchlide and the Pchlide analogues where the central metal has been

removed (compound B), the central Mg2+ ion has been substituted with Zn2+ (compound C) and

the C13a keto group of ring E has been changed to a hydroxyl group (compound F). In Pchlide

the strength of the dipole moment increases by more than 50 % in the excited state compared to

the ground state (Table 1). Similarly, the ICT character of compounds B and C also increases in

16

the excited state, albeit to a lesser extent than for Pchlide, suggesting that the central metal ion is

not crucial for the dipolar character of the ICT complex. However, changing the C13a keto group

of ring E (compound F) significantly reduces the strength and changes the direction of the dipole

moment in the ground state of the Pchlide molecule (Figure S57). Moreover, the strength of this

dipole moment in compound F does not increase at all in the excited state (Table 1), confirming

that the C13a keto group is essential for excited state charge separation within the Pchlide

molecule. This finding is in agreement with the time-resolved absorbance and fluorescence

measurements for this analogue and explains why one fewer exponentials was required to model

the time-dependent spectral changes. In this case, the τ1 lifetime from the time-resolved

spectroscopy measurements is likely to represent vibrational relaxation and reorganization of the

initially excited Sn state to an S1 state with no ICT character.

Table 1. Calculated dipole moment in Debyes (D) of the ground and excited states and the

solvent reorganization energy for compounds A, B, C and F.

Pchlide analogue A B C F

Ground state dipole (D) 6.43 6.67 6.42 3.05

Excited state dipole (D) 9.85 9.55 9.53 2.96

Relative difference in dipole (%) 53.2 43.2 48.3 -2.9

Solvent reorganization energy (kJ mol-1) 9.72 7.43 8.51 0.18

The fact that the increase in the dipolar character of the excited Pchlide molecule is similar

upon changing the central Mg2+ ion does not help to explain the differences in the excited state

decay kinetics for compounds B and C. The most likely explanation is that the site-specific

solvation of the excited ICT state is affected in these analogues. Time-dependent DFT

calculations show that the solvent reorganization energy for the ICT state of Pchlide (compound

17

A) is 9.72 kJ mol-1 (Table 1), which corresponds to a time constant of ~49 ps for the solvent

reorganization step (estimated from the Arrhenius equation with a prefactor of 1012). This is in

strong agreement with the time constants obtained from the time-resolved absorbance and

fluorescence measurements (57 and 48 ps, respectively) for this reorganization process. The level

of solvent reorganization is calculated to be lower when the central Mg2+ ion is removed

(compound B) or replaced by Zn2+ (compound C), although the solvent reorganization energy is

still significant in these analogues (Table 1). However, it is important to note that these values,

which are calculated using implicit solvation, do not take into account specific H-bonds or

coordination of solvent molecules with the Pchlide molecule, whereas Zhao and Han have

previously shown that only specific H-bond interactions between the Pchlide and methanol are

strengthened during the solvation step.13 Importantly, there is a strong coordination bond

between the central Mg2+ ion of Pchlide and the oxygen of a coordinating methanol molecule,

which becomes shorter and stronger in the excited state.13 In addition, this coordinated methanol

molecule has been shown to form a H-bonding chain by bridging another methanol molecule,

which also strengthens considerably in the S1 state.13 Including these two methanol molecules for

compounds A and C (Figure S58 and Table S1) increases the change in dipole and solvent

reorganization energy upon excitation, meaning that the solvent reorganization energy

(calculated from implicit solvation) is slightly underestimated. It is likely that these specific

bonds to solvent are affected in analogues that contain changes to the central metal ion

(compounds B and C), and it is the bonding network between the Mg2+ and neighboring

methanol molecules that is responsible for most of the spectral changes in the visible region upon

solvation of the ICT state. Certainly, it is the case that on completely removing the Mg2+ ion

(compound B) there is no metal-coordinating solvent molecule and this specific H-bonding

18

network will be much weaker. Also, substitution of the central Mg2+ ion with Zn2+ (compound C)

results in a weaker coordination bond with the oxygen of the coordinating methanol molecule

and therefore, weaker binding to these two methanol molecules, with dissociation energies

calculated to be 151.1 kJ mol-1 for Mg2+ and 119.5 kJ mol-1 for Zn2+.

Vibrational “fingerprints” of Pchlide modes.

Time-resolved spectral changes in the mid-IR region, measured over 2 ns, show comparable

kinetics to the visible data but importantly, provide vibrational “fingerprints” of specific regions

of the Pchlide chromophore (Figure 5 and supporting information). This is important for

mapping the coupling of any vibrational modes to the excited state chemistry and subsequent

catalytic steps in POR. As previously suggested12, 15, 29 the major negative peak at ~1690 cm-1 and

positive peak at ~1625 cm-1 represent the C13a=O carbonyl frequency as these features are

absent in analogues that contain changes to the C13a keto group of the isocyclic ring

(compounds E and F) (Figure 5E and F). As the majority of the IR signals originate from this

C13a keto group, very few spectral changes are observed for compounds E and F following the

decay of the fast component in a few ps, providing further evidence that the ICT state is strongly

dependent on the C13a=O. Similar to the time-resolved visible measurements the data for

compounds A and D can be modeled with 4 component lifetimes (Figure 5A and D), whereas

changes to the central metal ion (compounds B and C) yields data that are best fitted to 3

components (Figure 5 B and C). In all cases the first lifetime, representing vibrational relaxation

and reorganization of the initially excited Sn state, involves a decrease in the amplitude of the

negative feature at ~1740 cm-1. In compounds A and D the solvation of the ICT state (EADS3) is

accompanied by an increase in signals in the 1500-1600 cm-1 region, which have been assigned

19

to in-plain porphyrin ring vibrations (C=C and C=N modes).29 It should also be noted that when

the central Mg2+ is replaced by Zn2+ (compound C) minor changes to the 1500-1600 cm-1 region

can still be observed in EADS2. This provides further indication that there is a reduction in the

level of any solvent reorganization around this analogue, which makes it difficult to kinetically

resolve the steps involving the formation and solvation of the ICT state. The S1/ICT state is then

converted to the long-lived triplet state on the nanosecond timescale.12

Figure 5. Evolution associated difference spectra (EADS) resulting from a global analysis of the

time-resolved IR data for Pchlide derivatives A-F after excitation at 430 nm. The data were fitted

to a sequential model as described in the supplementary information.

20

In order to assign more of the IR modes to specific regions of the Pchlide molecule the initial

S1 excited state species (EADS1) have been fitted with a combination of Gaussian components

for all of the analogues (Figure 6). In most cases the spectral features associated with the

C13a=O carbonyl group can be modeled with 2 separate sub-populations, with the negative peak

at ~1690 cm-1 resolved into 2 distinct bands centered at 1689 cm-1 and 1706 cm-1 and the positive

peak at ~1625 cm-1 resolved into 2 distinct bands centered at 1606 cm-1 and 1634 cm-1. These are

likely to represent differences in the solvent coordination or local environment around this

group12 and the frequencies of both species are influenced by changes to other regions of the

Pchlide molecule (compounds B-E). The negative feature at 1740 cm-1, previously proposed to

arise from C=O modes of the substituents at the C13b and C17 positions,15 is almost identical to

Pchlide when the C17 carboxylic acid group is changed to a methylester (compound D).

However, the signal is downshifted by ~13 cm-1 upon opening of the ring E (compound E) and

the peak, therefore, can be assigned entirely to the methylester at the C13b position. In addition,

this band is only slightly sensitive to changes to the central metal ion (compounds B and C) or

the C17 carboxylic acid (compound D) but removal of the C13a=O group (compound F)

downshifts the C13b ester band by ~8 cm-1, confirming that it is coupled to other vibrational

modes in the ring E. Although these groups are the most intense IR markers in this region, due to

their close proximity to the conjugated electronic macrocycle, the in-plane porphyrin ring

vibrations in the 1500-1600 cm-1 region are influenced by alterations to the central metal ion. The

C17 carboxylic acid group is located further away from the main macrocycle and therefore,

changing this group to a methylester (compound D) only has a minor effect on the IR difference

spectrum upon electronic excitation.

21

Figure 6. Fitting of EADS1 from the global analysis of the transient IR absorption data for

Pchlide derivatives A-F to a sum of Gaussian functions. The EADS (black dots) have been fitted

with a sum (black line) of Gaussian functions (negative shown as blue lines, positive as red

lines). Positions and FWHM (in brackets) are indicated. Peaks can be assigned to the C13a

carbonyl (1569-1706 cm-1), C13b methylester (1730-1754 cm-1) and C=C of delocalized

porphyrin skeleton modes (below 1572 cm-1).

DFT frequency calculations of ground and excited state structures in the gas phase confirm the

experimental assignments, as described in the supplementary information. The excited state

frequency calculations can be subtracted from those calculated in the ground state to generate

difference spectra, which confirm that the major signals arise from the ring E C=O groups

(Figure S30). The two major vibrations at 1814 and 1805 cm-1 correspond to coupled vibrations

from the C13b ester and C13a=O carbonyl groups, respectively, whereas all features at lower

22

frequencies correspond to bulk ring vibrations. The C17 carboxylic acid group is vibrationally

coupled to the C13b ester group but there is virtually no change in this peak upon excitation,

confirming that its contribution to the experimental data is negligible.

Conclusions

Previous studies have shown that Pchlide is an intrinsically reactive molecule in the excited

state and it is these unique photochemical properties that allow it to harness sunlight to trigger

POR catalysis.7-12, 13 This in turn initiates the formation of the photosynthetic apparatus required

for plant development.1-3 An ICT state is the major driving force behind the excited state

reactivity of the molecule7-12 and a combination of time-resolved spectroscopies and DFT

calculations has now shown that the dipolar nature of this species is caused by the presence of

the carbonyl group at the C13a position of ring E. The ICT state does not form when this group

is removed, suggesting that it is essential for charge separation across the Pchlide molecule upon

excitation. The direct attachment of this carbonyl group to the main conjugated porphyrin

macrocycle is likely to be important for increasing its electron-withdrawing properties to

facilitate ICT formation. The formation of the excited ICT state leads to a further strengthening

of the hydrogen bonding interactions with the solvent in a solvation process that appears to be

impaired when the central Mg2+ ion is removed or substituted by Zn2+. Significantly, these

primary photochemical processes are crucial for seedlings and dark-grown plants to capture light

energy to ultimately trigger plant development.1-3

Time-resolved IR measurements and DFT calculations have allowed the assignment of

vibrational fingerprints of the Pchlide molecule. The C13b ester and C13a=O carbonyl groups

are the main IR markers of the chromophore and these vibrational modes are coupled in the

excited state. In addition to ring vibrations from the main porphyrin macrocycle, the frequencies

23

of both groups are also influenced by changes to other regions of the Pchlide molecule. These

assignments provide direct confirmation of previous models for the involvement of specific

vibrational modes in the excited state dynamics of Pchlide.12, 15 Our work also supports the model

that was recently proposed for the excited state chemistry in the ternary enzyme-substrate

complex, where excited state interactions between active site residues and the carboxyl group at

the C17 position create a highly polarized C17-C18 double bond to facilitate POR-catalyzed

hydride transfer from NADPH.14 These findings will now be crucial for mapping the coupling of

any vibrational modes to the hydride and proton transfer chemistry in POR and for understanding

the role of specific regions of the Pchlide molecule in POR catalysis.

ASSOCIATED CONTENT

Supporting Information. Detailed experimental methods, NMR spectra and mass spectrometry

of target compounds, raw transient absorption and fluorescence data, residuals of the fits of the

global analysis for all of the datasets.

AUTHOR INFORMATION

Notes

† Current Address: AstraZeneca UK, Charter Way, Silk Road, Macclesfield, Cheshire, SK10

2NA, UK

The authors declare no competing financial interests.

24

ACKNOWLEDGMENT

This work was funded by the UK Engineering and Physical Sciences Research Council (Grant

EP/J020192). NSS is an Engineering and Physical Sciences Research Council Established Career

Fellow. Time-resolved absorption and fluorescence measurements were performed at the

Ultrafast Biophysics Facility, Manchester Institute of Biotechnology, as funded by BBSRC

Alert14 Award BB/M011658/1. The time-resolved infrared measurements were carried out

through program access support of the UK Science and Technology Facilities Council (STFC).

The EPSRC National Mass Spectrometry Service, Swansea are thanked for mass spectrometric

analyses.

REFERENCES

1. Heyes, D. J.; Hunter, C. N. Making Light Work of Enzyme Catalysis:

Protochlorophyllide Oxidoreductase. Trends Biochem. Sci. 2005, 30, 642-649.

2. Masuda, T.; Takamiya, K. Novel Insights into the Enzymology, Regulation and

Physiological Functions of Light-Dependent Protochlorophyllide Oxidoreductase in

Angiosperms. Photosynth. Res. 2004, 81, 1-29.

3. Scrutton, N. S.; Groot, M. L.; Heyes, D. J. Excited State Dynamics and Catalytic

Mechanism of the Light-Driven Enzyme Protochlorophyllide Oxidoreductase. Phys. Chem.

Chem. Phys. 2012, 14, 8818-8824.

4. Heyes, D. J.; Heathcote, P.; Rigby, S. E. J.; Palacios, M. A.; van Grondelle, R.; Hunter,

C. N. The First Catalytic Step of the Light-Driven Enzyme Protochlorophyllide Oxidoreductase

Proceeds Via a Charge Transfer Complex. J. Biol. Chem. 2006, 281, 26847-26853.

25

5. Heyes, D. J.; Sakuma, M.; de Visser, S. P.; Scrutton, N. S. Nuclear Quantum Tunneling

in the Light-Activated Enzyme Protochlorophyllide Oxidoreductase. J. Biol. Chem. 2009, 284,

3762-3767.

6. Menon, B. R. K.; Waltho, J. P.; Scrutton, N. S.; Heyes, D. J. Cryogenic and Laser

Photoexcitation Studies Identify Multiple Roles for Active Site Residues in the Light-driven

Enzyme Protochlorophyllide Oxidoreductase. J. Biol. Chem. 2009, 284, 18160-18166.

7. Dietzek, B.; Maksimenka, R.; Siebert, T.; Birckner, E.; Kiefer, W.; Popp, J.; Hermann,

G.; Schmitt, M. Excited-State Processes in Protochlorophyllide a a Femtosecond Time-Resolved

Absorption Study. Chem. Phys. Lett. 2004, 397, 110-115.

8. Dietzek, B.; Kiefer, W.; Hermann, G.; Popp, J.; Schmitt, A. Solvent Effects on the

Excited-State Processes of Protochlorophyllide: A Femtosecond Time-Resolved Absorption

Study. J. Phys. Chem. B 2006, 110, 4399-4406.

9. Dietzek, B.; Kiefer, W.; Yartsev, A.; Sundstrom, V.; Schellenberg, P.; Grigaravicius, P.;

Hermann, G.; Popp, J.; Schmitt, M. The Excited-State Chemistry of Protochlorophyllide a: A

Time-Resolved Fluorescence Study. ChemPhysChem 2006, 7, 1727-1733.

10. Dietzek, B.; Tschierlei, S.; Hermann, G.; Yartsev, A.; Pascher, T.; Sundstrom, V.;

Schmitt, M.; Popp, J. Protochlorophyllide a: A Comprehensive Photophysical Picture.

ChemPhysChem 2009, 10, 144-150.

11. Dietzek, B.; Tschierlei, S.; Hanf, R.; Seidel, S.; Yartsev, A.; Schmitt, M.; Hermann, G.;

Popp, J. Dynamics of Charge Separation in the Excited-State Chemistry of Protochlorophyllide.

Chem. Phys. Lett. 2010, 492, 157-163.

26

12. Sytina, O. A.; van Stokkum, I. H. M.; Heyes, D. J.; Hunter, C. N.; van Grondelle, R.;

Groot, M. L. Protochlorophyllide Excited-State Dynamics in Organic Solvents Studied by Time-

Resolved Visible and Mid-Infrared Spectroscopy. J. Phys. Chem. B 2010, 114, 4335-4344.

13. Zhao, G. J.; Han, K. L. Site-Specific Solvation of the Photoexcited Protochlorophyllide a

in Methanol: Formation of the Hydrogen-Bonded Intermediate State Induced by Hydrogen-Bond

Strengthening. Biophys. J. 2008, 94, 38-46.

14. Heyes, D. J.; Hardman, S. J. O.; Hedison, T. M.; Hoeven, R.; Greetham, G. M.; Towrie,

M.; Scrutton, N. S. Excited-State Charge Separation in the Photochemical Mechanism of the

Light-Driven Enzyme Protochlorophyllide Oxidoreductase. Angew. Chem. Int. Ed. 2015, 54,

1512-1515.

15. Colindres-Rojas, M.; Wolf, M. M. N.; Gross, R.; Seidel, S.; Dietzek, B.; Schmitt, M.;

Popp, J.; Hermann, G.; Diller, R. Excited-State Dynamics of Protochlorophyllide Revealed by

Subpicosecond Infrared Spectroscopy. Biophys. J. 2011, 100, 260-267.

16. Sytina, O. A.; van Stokkum, I. H. M.; Heyes, D. J.; Hunter, C. N.; Groot, M. L.

Spectroscopic Characterization of the First Ultrafast Catalytic Intermediate in

Protochlorophyllide Oxidoreductase. Phys. Chem. Chem. Phys. 2012, 14, 616-625.

17. Klement, H.; Helfrich, M.; Oster, U.; Schoch, S.; Rudiger, W. Pigment-Free NADPH :

Protochlorophyllide Oxidoreductase from Avena Sativa L - Purification and Substrate

Specificity. Eur. J. Biochem. 1999, 265, 862-874.

27

18. Heyes, D. J.; Hardman, S. J. O.; Mansell, D.; Gardiner, J. M.; Scrutton, N. S. Mechanistic

Reappraisal of Early Stage Photochemistry in the Light-Driven Enzyme Protochlorophyllide

Oxidoreductase. PLoS One 2012, 7, e45642.

19. Lindsey, J. S.; Woodford, J. N. A Simple Method for Preparing Magnesium Porphyrins.

Inorg. Chem. 1995, 34, 1063-1069.

20. Schoch, S.; Helfrich, M.; Wiktorsson, B.; Sundqvist, C.; Rudiger, W.; Ryberg, M.

Photoreduction of Zinc Protopheophorbide-b with NADPH-Protochlorophyllide Oxidoreductase

from Etiolated Wheat (Triticum aestivum l). Eur. J. Biochem. 1995, 229, 291-298.

21. Helfrich, M.; Schoch, S.; Lempert, U.; Cmiel, E.; Rudiger, W. Chlorophyll Synthetase

Cannot Synthesize Chlorophyll a'. Eur. J. Biochem. 1994, 219, 267-275.

22. Tamiaki, H.; Watanabe, T.; Miyatake, T. Facile Synthesis of 13(1)-Oxo-Porphyrins

Possessing Reactive 3-Vinyl or 3-Formyl Group, Protochlorophyll-a/d Derivatives by 17,18-

Dehydrogenation of Chlorins. J. Porphyrins Phthalocyanines 1999, 3, 45-52.

23. Greetham, G. M.; Sole, D.; Clark, I. P.; Parker, A. W.; Pollard, M. R.; Towrie, M. Time-

Resolved Multiple Probe Spectroscopy Rev. Sci. Instrum. 2012, 83, 103107.

24. Snellenburg, J. J.; Laptenok, S. P.; Seger, R.; Mullen, K. M.; van Stokkum, I. H. M.

Glotaran: A Java-Based Graphical User Interface for the R Package TIMP. J. Stat. Softw. 2012,

49, 1-22.

25. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman,

J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A. et al Gaussian 09, Gaussian,

Inc.: Wallingford, CT, USA, 2009.

28

26. Becke, A. D. Density-Functional Thermochemistry .3. The Role of Exact Exchange. J.

Chem. Phys. 1993, 98, 5648-5652.

27. Perdew, J. P., Density-Functional Approximation for the Correlation Energy of the

Inhomogeneous Electron Gas. Phys. Rev. B 1986, 33, 8822-8824.

28. Weigend, F.; Ahlrichs, R. Balanced Basis Sets of Split Valence, Triple Zeta Valence and

Quadruple Zeta Valence Quality for H to Rn: Design and Assessment of Accuracy. Phys. Chem.

Chem. Phys. 2005, 7, 3297-3305.

29. Hanf, R.; Tschierlei, S.; Dietzek, B.; Seidel, S.; Hermann, G.; Schmitt, M.; Popp, J.

Probing the Structure and Franck-Condon Region of Protochlorophyllide a Through Analysis of

the Raman and Resonance Raman Spectra. J. Raman Spectrosc. 2009, 41, 414-423.

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