Exciton states and optical properties of the CP26

photosynthetic protein

Daniil V. Khokhlov∗, Aleksandr S. Belov, Vadim V. Eremin

Department of Chemistry, Lomonosov Moscow State University, Leninskie gory 1-3,
Moscow, Russia, 119991

Abstract

The photosynthetic complex CP26, one of the minor antennae of the photosys-
tem II, plays an important role in regulation of the excitation energy transfer
in the PSII. Due to instability during isolation and purification, it remained
poorly studied from the viewpoint of theoretical chemistry due to absence of
X-ray crystallography data. In this work, using the recently determined three-
dimensional structure of the complex we study the properties of exciton states in
the complex using quantum chemical approach. Spectral properties, structure
of exciton states and roles of the pigments in the complex and photosystem II
are discussed.
Keywords: Light-harvesting complex, photosystem II, lhcb5, CP26, Spinacia
olearacea, exciton Hamiltonian

1. Introduction

During evolution, higher plants developed a complex photosynthetic appara-

tus containing various types of specialized subunits to provide maximal efficiency
of photosynthesis [1]. An example of such complex organization is the photosys-
5 tem II of the higher plants (PSII) which consists of a reaction center-containing

∗ Correspondingauthor
Email address: daniilkh@yahoo.com (Daniil V. Khokhlov)

Preprint submitted to Computational biology and chemistry November 10, 2017

 core surrounded by a number of light-harvesting complexes called peripheral
antennae. The latter are pigment-containing homologous proteins with a high
degree of similarity encoded by lhcb gene family [2]. Trimeric complexes LHCII,
one of the most abundant peripheral antennae, play the key role in the absorp-
10 tion of light [3]. The transfer of excitation energy from LHCII complexes to
the PSII core is facilitated by a number of smaller complexes [4] called minor
antennae including chlorophyll-containing proteins with molecular masses of 29,
26 and 24 kDa encoded by genes lhcb4 (complex CP29), lhcb5 (complex CP26),
and lhcb6 (CP24), respectively [5, 6]. These complexes have two important
15 functions in PSII. Their main function is the transfer of electronic excitation
energy to the PSII core. In addition, they play a protective role by dissipating
the excessive energy under intense light [7, 8, 9].
The structures of the most complexes of the lhcb family were determined
using X-ray crystallography [10, 11, 12] and cryo-electron microscopy [13] (PSII
20 of the Spinacia olearacea). The complexes of lhca and lhcb families contain
different number of pigments (from 8 to 14 chlorophylls a or b and from 2
to 4 xanthophylls [14, 15]). The binding sites for chlorophyll molecules are
nucleophilic atoms of amino acid residues coordinating magnesium atoms. In
most cases, such residies are histidine, aspartic acid, or glutamic acid. However,
25 their relative positions and exact nature are different in different complexes.
Isolation of these complexes is difficult due to similar physicochemical properties
and low stability. Thus, recombinant proteins are used for most experiments
[16]. Moreover, the pigment composition of the complexes is not constant and
depends on the relative quantities of the pigments during the folding process
30 [17].
The three-dimensional structure for the CP26 complex has been unknown
for a long time. Assumptions about its structure were made basing on indi-
rect methods such as high-performance liquid chromatography and the study
of spectral properties [18, 19]. Chromatography allows one to determine the
35 ratio of different pigments but not their absolute quantities. In earlier experi-
ments, the presence of 9 chlorophylls and 2 carotenoids was suggested for CP26

2
 [20]. Later, point mutations of the chlorophyll binding sites replacing the coor-
dinating amino acids with non-polar ones were used to study the composition
of the complex. A number of mutated complexes based on the lhcb5 gene of
40 Arabidopsis thaliana were constructed using this approach [21]. In that study a
model was proposed for the complex that implied the presence of 9 chlorophylls
and 3 carotenoids; a number of assumptions regarding the energy levels of in-
dividuals pigments was made. A direct study of the PSII supercomplex using
cryo-electron microscopy allowed to obtain the structure of the CP26 complex
45 with 3.2 Å resolution and to clarify the pigment composition. In contrast to
indirect studies, 13 chlorophylls and 3 carotenoids were found in the complex
[13]. Four additional porphyrins were not detected earlier due to the absence of
the specific binding sites for them. Their magnesium atoms are coordinated by
water molecules or, in the case of one chlorophyll, by the carbonyl group of the
50 peptide bond.
Spectral properties of the CP26 complex were first determined for the sample
isolated from spinach, and the ratio of chlorophylls of types a and b in the com-
plex was found to be 3.3 ± 0.1. This result could lead to the correct amount of
pigments, but the authors deemed the presence of 9 chlorophylls in the complex
55 more likely[22]. In the absorption spectrum in the Q-band region (according to
Gouterman model [23]), a single wide peak is present near 676 nm which can be
attributed to the Qy band in the chlorophyll a spectrum. Moreover, the strong
peak corresponding to absorption of chlorophyll b is absent due to the relatively
small amount of chlorophylls b in the complex compared to larger antennae
60 such as LHCII. Linear dichroism (LD) spectrum has a more detailed structure
having not only the main positive peak of the same nature as in the absorption
spectrum but also two small negative peaks near 650 nm corresponding to the
Qy band of chlorophyll b and a peak near 638 nm corresponding to overlapping
Qx bands of chlorophylls a and b. Circular dichroism (CD) spectrum contains
65 three main peaks in the region similar to spectra of other complexes from lhcb
family [22]. In later works, spectral methods were mainly used to study the roles
and spectral contributions of individual pigments within the complex [20, 21].

3
 A model based on the Frenkel excitons is common for description of electronic
states in photosynthetic complexes [24]. Matrix of exciton Hamiltonian Ĥ in
a basis set containing products of wavefunctions of individual pigments can be
represented as the sum of two terms:

Ĥ = Ĥ0 + V̂ , (1)

where the unperturbed Hamiltonian Ĥ0 contains only diagonal elements repre-
senting excitation energies of pigments, and perturbation V̂ contains only non-
70 diagonal ones which are exciton couplings between pigments. Both matrices
can be obtained either from deconvolution of experimental spectra or quantum
chemical calculations, the latter being more reliable for small matrix elements.
Protein in the complex affects both the pigments themselves and their in-
teraction with each other. In the simplest case, the correction to the excitation
75 energies of the individual pigments can be accounted for as the difference in the
protein-pigment interaction energies in different electronic states [25]. However,
such an approach does not take into account the influence of the protein on the
wavefunction of the pigment which is evaluated in vacuo. The hybrid quantum
mechanics/molecular mechanics approach (QM/MM) using classical or polariz-
80 able force fields [26] allows for inclusion of more terms in the pigment-protein
coupling. QM/MM with polarizable force field proved to be useful for modeling
the exciton states in another complex from the lhcb family, namely CP29 [27].
Couplings in the considered systems can be obtained both from the direct cal-
culation of the matrix elements as the integrals on the wavefunctions and from
85 approximate methods such as TrESP (transition ESP) [28]. The latter expands
the ESP approach (charge from electrostatic potential [29]) to the electrostatic
potential of transition. This method is known to combine good accuracy and
acceptable computational complexity.
In this paper, exciton Hamiltonian of the CP26 complex including influence
90 of the polarizable protein medium on the pigments is constructed. Absorption,
linear and circular dichroism spectra are simulated and compared to the exper-
imental ones to confirm the validity of this approach for peripheral antennae of

4
 the photosystem II. Also, possible routes for energy transfer within the complex
are examined using exciton couplings and structures of exciton states.

95 2. Computational methods

2.1. Structure of the CP26 complex

Nuclear geometry for calculation of exciton states was prepared using the
structure from the RCSB data bank (ID:3JCU) obtained by cryo-electron mi-
croscopy [13]. Geometry optimization of the protein was made using molecular
100 mechanics as follows. Protonation states for titrable residues were determined
by the pdb2gmx utility (a part of GROMACS package) using geometric criteria
such as the possibility of formation of salt bridges and hydrogen bonds. This ap-
proach led to standard protonation states for all titrable amino acids. Histidine
residues – HIS81 and HIS221 were in δ-configuration to provide coordination for
105 chlorophyll molecules. The HIS165 residue was also in δ-configuration forming
a hydrogen bond with the ester group of the chlorophyll A610. The protein
was placed in water which was treated as a part of the MM system using the
TIP3P model [30]). The charge of the system was neutralized by randomly
placed sodium ions. Amino acid residues were described using AMBER99 [31]
110 force field. For chlorophylls, an AMBER-compatible parameter set was used [32]
which was based on the parameter set for the components of the PSII proposed
earlier [33]. Force field parameters for xanthophylls were derived from Hessian
matrix using the method of Seminario [34]. GROMACS package [35] was used
for all calculations. The optimized structure of the complex is shown in fig. 1.
115 RMSD for coordinates of heavy atoms in the protein structure was 2.72 Å.
Such significant deviation is due to free movement of the protein terminus and
increased mobility of the protein in water as compared to the entire PSII super-
complex in initial PDB. It should be noted that all specific binding interactions
between chlorophylls’ magnesium atoms and nucleophilic atoms of amino acids
120 have been conserved. Relative positions of the pigments were also unchanged.

5
 a) b)

Figure 1: The structure of the CP26 complex in two projections. Chlorophylls a and b are
shown in blue and green, respectively. Xanthophylls are shown in orange (lutein) and yellow
(neoxanthin). Protein is shown in gray. The image was made with the VMD program [36].

The structure obtained was further used for modeling of the exciton Hamiltonian
and spectral properties.

2.2. Excitation energies

Spectral properties of photosynthetic complexes are determined by two main

125 factors: the interaction of pigments with protein environment and their interac-
tion with each other. Wavefunctions of pigments and, accordingly, their excita-
tion energies are influenced both by presence of a polarizing medium around the
pigments and the heterogeneity of this environment. The first term can be ac-
counted for by using polarizable medium models such as polarizable continuum
130 model (PCM) [37]. However, this approach can not deal with heterogeneity of
the protein requiring use of polarizable force fields.
For this reason, influence of the protein was modeled by QM/MM using po-
larizable force field AMBER02 [38] to describe the molecular mechanics part.
For each chlorophyll molecule in the complex, a separate QM/MM calculation
135 was carried out in the following fashion. The QM part consisted of one chloro-
phyll molecule whose excitation energy was being calculated. Its phytol residue
was replaced by methyl radical to reduce the computational cost. The MM
part contained the protein, the water molecules located at distance less than 20

6
 Å from the magnesium atom of the chlorophyll in QM-part, and all pigments
140 except the one included in the QM-part.
Within the framework of AMBER02, the term corresponding to polariza-
tion of protein is added to the energy functional of the classical force field
AMBER99. The force field utilizes bonded and non-bonded parameters from
AMBER99. Molecular polarizability is represented by atomic polarizabilities
145 from AMBER99 parameters set. Atomic RESP-charges are iteratively recalcu-
lated to exclude the effect of self-polarization, damping functions are not used
during the calculation of induced dipoles [39]. Parameters not included in the
standard set were calculated using AMBER02 approach.
Time-dependent density functional theory (TD-DFT) with hybrid exchange-
150 correlational functional CAM-B3LYP [40] at 6-31G* level was used. This func-
tional is a development of the well-known hybrid functional B3LYP with an im-
proved behavior of the B3LYP functional for the molecules with a large number
of conjugated π-bonds such as porphyrins. CAM-B3LYP was shown to achieve
the accuracy of multiconfigurational perturbation theory (e.g. CASPT2[8,8])
155 in calculations of the excitation energies in the chlorophylls [41]. The quantum
chemical calculations described above were made in GAMESS-US package [42].
The same computation was carried out in vacuo using nuclear geometry
from molecular mechanics optimization to estimate the influence of the protein
medium on the excitation energies of the chlorophylls. In polarizable medium,
160 excitation energies decrease by 0.01-0.03 eV which is consistent with the ex-
pected red shift in photosynthetic complexes of lhcb family and polar solvents
(fig. 2). In addition, the QM/MM calculations with the same system placed
in PCM water using approach described earlier for the CP29 complex [27] were
performed. In this case, red shifts are larger (0.04-0.05 eV depending on the
165 chlorophyll type) than in the system containing only limited number of water
molecules, and these values are consistent with the ones for the CP29 complex
[27] However, in our case these calculations gave an increase of transition dipole
moment (see table S1). The average square of the transition dipole moment
in the absence of PCM water equals 33.9 and 20 D2 for chlorophylls a and b,

7
 Figure 2: Calculated sites energies. Cyan lines correspond to chlorophylls a, green – to
chlorophylls b. Square markers correspond to the energies in vacuo, round marks – to the
energies in the protein environment. Grey horizontal lines are average chlorophyll energies of
the corresponding type.

170 respectively. These values are slightly higher than the experimental estimates
obtained from extrapolation of data for various solvents to permittivity of vac-
uum (21 and 14.7 D2 , respectively) [43]. Addition of the PCM water led to
unfounded increase of the transition dipole moment (up to 74.0 and 47.3 D2 )
and decrease of the excitation energies. Such result can be caused by unphysi-
175 cal behavior of the PCM model for the protein complex which has a significant
negative charge (-8 a.u.). For these reasons, the model without PCM was used
for further calculations.
Frenkel-type exciton Hamiltonian Ĥ was used for the system of pigments.

8
Its matrix element can be expressed as follows:
X X
Hkl = Ei δki δli + Vij δki δlj , (2)
i i6=j

where Ei is the excitation energy of the i-th pigment, Vij is the exciton coupling
between the i-th and the j-th pigment, δij is Kronecker delta. Wavefunctions
of exciton states ψk are linear combinations of the products of wavefunctions of
individual pigments φi :
X
ψk = Cik φi , (3)
i

where expansion coefficients Cik are the contributions of the individual pigments
to the exciton state which can be obtained by diagonalization of exciton Hamil-
tonian matrix. Excitation energies from QM/MM calculation described above
were taken as diagonal elements. Non-diagonal elements were calculated as a
sum of Coulomb interactions between TrESP point charges as follows:
#» #»
XX qki qlj X X dj · (Ri − r#m »)
i m k
Vij = # »i # »j + q # »i , (4)
|Rk − r#m
»|3
k
k l |Rk − Rl | k m

#»
where qki is the TrESP charge of the k-th atom of i-th chlorophyll, Rki is the
#»
position vector of the k-th atom of i-th chlorophyll, djm is the dipole of the m-th
atom of the protein induced by the transition potential of the j-th chlorophyll,
r#m
» is the position vector of the m-th protein atom. The first term corre-
sponds to the interaction between the two sets of TrESP charges in vacuum.
The second one is the correction to the polarizable medium corresponding to
the permittivity of the protein environment. Two fitting parameters were used
in the Hamiltonian to compensate for the errors introduced by quantum chem-
ical methods. First, all excitation energies were reduced by the same value.
The chosen DFT functional describes the excited states quite good but its er-
ror is still large enough as compared to the width of the spectral line of the
CP26 complex. Experimentally determined excitation energies in various sol-
vents were extrapolated earlier [44] to vacuum permittivity to give the estimates
for vacuum. The difference between the average calculated in vacuo excitation

9
 energies of chlorophylls a and b (2.08 and 2.12 eV, respectively) and extrap-
olated in vacuo values (1.91 and 1.99 eV, respectively) can be interpreted as
the TD-DFT error (0.17 and 0.13 eV). We used the same redshift of 0.24 eV
for both types of chlorophylls to obtain the best fit to experimental spectra.
Second, TrESP-charges were multiplied by a factor of 0.854 which is the ratio
between the experimentally determined dipole moment and the calculated one
[27]. Exciton Hamiltonian, its eigenvectors, and stick spectra are reported in
tables S2-S4. In Frenkel exciton model, intensities of lines corresponding to the
electronic transitions depend on the expansion coefficients in eq. 3 since the
~ k can be represented using
transition dipole moment of the k-th exciton state µ
transition dipole moments of individual pigments µ
~ i:

Cik #»
X
~k =
µ µi (5)
i

If the lineshape function for the k-th state Dk (ω) is known, absorption spectrum
lineshape IABS (ω) can be written as follows:

#» |2
X
IABS (ω) ∝ ω Dk (ω)| µ k (6)
k

Linear and circular dichroism spectra can be calculated in a similar way:

#» |2 (1 − 3 cos2 (θ ))
X
ILD (ω) ∝ ω Dk (ω)| µ k k (7)
k

~ j ) · ( #»
µ i × #»
X X
ICD (ω) ∝ ω Dk ~i − R
Cik Cjk (R µ j ), (8)
k i>j
#»
where R i is the position vector of the i-th pigment (located at the magne-
sium atom), θk is the angle between the transition dipole moment and the lipid
180 membrane plane containing the complex.
A well-established approach based on the Redfield theory was used to ob-
tain the lineshape function Dk (ω) [45]. This function accounts for vibrational
sidebands and exciton relaxation and can be represented as follows:
Z ∞
Dk (ω) = < ei(ω−ω̃k0 ) eGk (t)−Gk (0) e−t/τk dt, (9)
0

10
where Gk (t) is the time-dependent function for the k-th exciton state, τk is the
dephasing time and ω̃k0 is the renormalized transition frequency from the ground
state to the k-th exciton state. These parameters depend on the properties of
system and will be discussed below.
The function Gk (t) is related to the spectral density J(ω) as follows:
Z ∞
G(t) = γkk {(1 + n(ω))J(ω)e−iωt + n(ω)J(ω)eiωt }, (10)
0

2
Cil2 , n(ω) is the Bose-Einstein distribution. Empirical spec-
P
where γkl = i Cik
tral density
1 X si 1/2
J(ω) = ω 3 e(−ω/ωi ) (11)
s1 + s2 i=1,2 7!2ωi4

with the parameters s1 =0.8, s2 =0.5, ~ω1 =0.069 meV, ~ω2 =0.24 meV was used
earlier by Renger’s group for bacteriochlorophyll-containing complex B777 [45].
Despite the fact that the complex contained bacteriochlorophylls, this spec-
tral density has been successfully applied to the modeling of the chlorophyll-
containing complexes including antennae from photosystem II [46, 47, 48, 27],
photosystem I [49], and less complex proteins such as Fenna-Matthews-Olson
complex [50] and water-soluble chlorophyll protein [51]. Dephasing time in eq. 9
related to the lifetime broadening can be obtained from the Redfield relaxation
constants kk→l from k-th state to l-th one:

2
τk = P , (12)
k>l kk→l

where kkl depends on the spectral density and includes transition frequency ωkl :

kk→l = 2πγkl ωkl {J(ωkl )(1 + n(ωkl )) + J(ωlk )n(ωlk ))} (13)

Renormalized frequency ω̃k in eq. 9 accounts for exciton-vibrational coupling

in exciton energy εk :
Z ∞
ε
ω̃k = − γkk ~ωJ(ω)dω
~ 0
Z ∞ 2
X ω {J(ω)(1 + n(ω)) + J(−ω)n(−ω))}
− γkl P dω, (14)
−∞ ωkl − ω
k6=l

11
185 where P denotes the principal value of the integral.
Inhomogeneous broadening was taken into account using averaging over dif-
ferent conformations contributing in static disorder of site energies:
Z ∞ Z ∞ K
Y
Iinh (ω) = dω̆1 · · · dω̆K I(ω|ω̆1 , · · · , ω̆K ) P (ω̆i − ωi ) (15)
0 0 i=i

where Iinh and I – lineshape functions (IABS , ICD , and ILD , respectively),
P (ωi − ω̄i ) – normal distribution functions with mean value equal to site energy
ωi and full width at half-maximum 100 cm−1 corresponding to experimental
one for LHCII complex [52].

190 3. Results

In all calculations of the spectra, the temperature was the same as in the
corresponding experimental ones. The spectra of the complex calculated as
described above are shown in fig. 3 (green solid lines). The calculated ab-
sorption spectrum contains main absorption peak near 673 nm. In general, the
195 calculated absorption spectrum demonstrates satisfactory agreement with the
experimental one. However, there is a number of important differences. First,
the absence of a weak peak at 638 nm is due to the fact that the exciton model
does not include the states corresponding to the Qx bands of the individual
chlorophylls. The second and the most important difference is the unexpected
200 broadening of the red sideband caused by a possible underestimation of the
excitation energies of the chlorophylls A609 and A613 which will be discussed
below. The circular dichroism spectrum demonstrates a qualitative agreement
with the experimental ones both in terms of the number of peaks and their posi-
tion. However, obtaining a quantitative agreement with the experimental data
205 is difficult within the used model. This is due to the fact that only the chirality
of the exciton can be taken into account which makes the value of the integral
over the entire spectrum zero. At the same time, in the experimental circular
dichroism spectrum, additional negative contributions from individual pigments
are present which makes it difficult to compare the spectra [27] directly.

12
 Figure 3: Spectral properties of the CP26 complex. Experimental spectra [22] are represented
by black dotted line, calculated spectra – by green solid line, spectra calculated with modified
energies – by cyan solid line.

210 The considerable difference in between the experimental absorption spec-

trum and the calculated one requires further discussion about excitation ener-

13
 gies of the chlorophylls A609 and A613. Two main reasons of such unexpected
behavior can be proposed. First, molecular mechanics optimization is not en-
tirely accurate due to difficulties in global minimum search and an approximate
215 nature of the method. One way to compensate for the minima being not global
is to average the frames from molecular dynamical simulation. Such approach
was used by Jurinovich et al. who used the averaging of 400 uncorrelated config-
urations extracted at every 50 ps of 20 ns trajectory [27]. However, this method
is very time-consuming requiring 5200 TD-DFT calculations for the CP26 com-
220 plex. Second, AMBER02 force field which is the newest available field from
AMBER family (AMBER12 was withdrawn by the authors) does not utilize
damping functions. This may lead to unphysical behavior in the presence of
large charges such as the ones of the deprotonated amino acids.
To check if the simple adjustment of the excitation energies of the chloro-
225 phylls A609 and A613 is enough to produce a correct set of absorption, CD,
and LD spectra for the entire complex, both energies were changed from their
calculated values (2.044 and 2.045) to the average value in the complex (namely,
2.068 eV) thus introducing another correction equal to 24 meV. This correction
is comparable in magnitude with the effect of the environment on the chloro-
230 phyll excitation energy. This correction does not have any clear physical sense,
so it was used only for additional spectra calculations, but not for discussion of
exciton states. The calculated spectra with corrected excitation energies shown
in fig. 3 (cyan solid line) agree well with the experimental ones. Thus, a more
accurate method is required for calculations of the minimum energy-geometry
235 of the complex. However, the polarizable environment model seems to be nec-
essary for the quantitative simulation of spectral properties.

4. Discussion

Direct comparison of the obtained data with the results of deconvolution

of the experimental spectra [20, 21] is not possible because the aforementioned
models implied the presence of nine chlorophylls in the complex. Assuming that

14
 our exciton Hamiltonian is accurate enough we can discuss the pathways of the
excitation energy transfer within the complex. Exciton states (fig. 4) span
over a relatively small range of energies – from 1.79 to 1.87 eV, and they can
be divided into several blocks of states with close excitation energies. Inverse
exciton participation ratio Lk
X
4 −1
Lk = ( Cik ) (16)
i

was used to estimate the delocalization length of the exciton states.

a) b)
Figure 4: a) Exciton couplings in the CP26 complex. Red color corresponds to positive values,
blue – to negative ones. The more saturated the color, the greater is the coupling. b) Exciton
states of the CP26 complex. The more saturated the color, the greater is the contribution of
the pigment to the exciton state.

The states with the highest energy S10-S13 are localized at chlorophylls b
240 as expected.

5. Conclusion

In this paper, theoretical modeling of exciton states and spectral properties

of the CP26 complex was carried out for the first time. This study provided
an insight into the functional architecture of the complex which is important
245 for understanding of the processes taking place in the PSII during the primary
stages of the photosynthesis. Two main groups of exciton states playing different

15
 Figure 5: Spatial distribution of strongly interacting pigments. Chlorophylls a 602, 603, 609,
610, 611, and 612 are shown in violet, chlorophylls b 606, 607, and 608 are shown in red,
another ones are shown in lime color. Lutein molecules are shown in orange, protein – in gray.
The largest exciton couplings and contributing pigments are denoted.

roles were found in the complex. The high-energy group is located mainly on
the chlorophylls b and allows for energy transfer from light-harvesting antennae
to the PSII core. The low-energy group is located in the center of the complex
250 near the xanthophylls and can provide singlet-triplet transfer to the latter in
the NPQ process.

6. Acknowledgement

This research was supported by Russian Foundation for Basic Research

(grant no. 16-03-00736).

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