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
*[email protected], [email protected]
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.
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