Article

ISSN 0006-2979, Biochemistry (Moscow), 2023, Vol. 88, No. 10, pp. 1580-1595 © Pleiades Publishing, Ltd., 2023.

Published in Russian in Biokhimiya, 2023, Vol. 88, No. 10, pp. 1908-1925.

1580

Femtosecond Dynamics of Excited States

of Chlorophyll Tetramer in Water-Soluble

Chlorophyll-Binding Protein BoWSCP

Dmitry A. Cherepanov

1,2,a

*, Konstantin V. Neverov

3,4

, Yuriy N. Obukhov

Yulia V. Maleeva

, Feodor E. Gostev

, Ivan V. Shelaev

1,2

, Arseny V. Aybush

Michail S. Kritsky

, and Victor A. Nadtochenko

1,5,b

Semenov Federal Research Center for Chemical Physics, Russian Academy of Sciences, 119991 Moscow, Russia

Belozersky Research Institute of Physical and Chemical Biology, Lomonosov Moscow State University,

119992 Moscow, Russia

Bach Institute of Biochemistry, Federal Research Center

“Fundamentals of Biotechnology” of the Russian Academy of Sciences, 119071 Moscow, Russia

Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia

Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

e-mail: tscherepanov@gmail.com

e-mail: nadtochenko@gmail.com

Received June 22, 2023

Revised September 22, 2023

Accepted September 22, 2023

Abstract— The paper reports on the absorption dynamics of chlorophylla in a symmetric tetrameric complex of the wa-

ter-soluble chlorophyll-binding protein BoWSCP. It was measured by a broadband femtosecond laser pump-probe spec-

troscopy within the range from 400 to 750nm and with a time resolution of 20fs-200ps. When BoWSCP was excited in

the region of the Soret band at a wavelength of 430nm, nonradiative intramolecular conversion S

→S

was observed with a

characteristic time of 83±9fs. When the complex was excited in the region of the Q

band at 670nm, relaxation transition

between two excitonic states of the chlorophyll dimer was observed in the range of 105±10fs. Absorption spectra of the ex-

cited singlet states S

and S

of chlorophylla were obtained. The delocalization of the excited state between exciton-coupled

Chl molecules in BoWSCP tetramer changed in time and depended on the excitation energy. When BoWSCP is excited in

the Soret band region, an ultrafast photochemical reaction is observed. This could result from the reduction of tryptophan

in the vicinity of chlorophyll.

DOI: 10.1134/S0006297923100139

Keywords: chlorophyll a, WSCP proteins, femtosecond laser spectroscopy, excited state spectrum, exciton dynamics, intra-

molecular conversion

Abbreviations: BoWSCP, water-soluble chlorophyll-binding

protein (WSCP) from Brassica oleracea var. botrytis; Chl,chlo-

rophyll.

* To whom correspondence should be addressed.

INTRODUCTION

Analysis of the photochemical reactions of energy

transfer and charge separation in photosynthetic pig-

ment–protein complexes is challenging due to their com-

plicated molecular organization and a large number

of exciton-coupled chlorophyll (Chl) molecules. Fur-

thermore, interpretating the absorption dynamics of

photosynthetic complexes registered by femtosecond

laser spectroscopy is a challenging methodological is-

sue [1, 2]. Water-soluble chlorophyll-binding pro-

teins (WSCP) from higher plants represent an ideal

model system to study the mechanism of photochem-

ical reactions [3-6]. These proteins are water-soluble,

photostable and thermally stable, and can be effective-

ly modified and produced through genetic and protein

engineering methods. In a plant cell these proteins are

FEMTOSECOND DYNAMICS OF CHLOROPHYLL TETRAMER 1581

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

located outside the thylakoid membranes of chloroplasts

and do not contribute to photosynthesis. Their physio-

logical functions are thought to be related to the activity

of the cells anti-stress systems [4, 7, 8].

Compared to the photosynthetic pigment–protein

complexes, WSCP protein structure is relatively simple.

WSCP holoforms are water-soluble homotetramers with

a molecular weight of 69-80 kDa. Each complex contains

up to four noncovalently bound Chl molecules. Depend-

ing on homology of the primary apoprotein structure,

WSCP proteins are subdivided into classesI and II, and

the latter has subclassesIIa and IIb by different affinity

of apoproteins for Chla and b [6,9]. It was shown that

upon photoexcitation of Chl in WSCP, energy migration

occurs both within the dimer and between dimer pairs

[10, 11]. This makes classII WSCP a promising model

for studying the mechanisms of pigment–pigment and

pigment–protein interactions, which will enable re-

search into the mechanisms of excitation energy transfer

between pigments [12-14] and interaction of the excited

pigments with surrounding proteins [15-17].

In this paper, a broadband femtosecond laser pump-

probe spectroscopy was used for studying photochemi-

cal processes in a symmetric tetrameric complex of the

water-soluble chlorophyll-binding protein BoWSCP

(subclassIIa) from Brassica oleracea var. botrytis, which

mainly binds Chla, i.e., its pigment composition can be

considered as almost homogeneous [5]. The tetrameric

Chl complex in BoWSCP is in the form of two dimers,

and the angle between porphyrine nuclei of pigments

in the dimer is about 30° [18]. Taken together, the two

dimers in a holoprotein macromolecule form a non-co-

valent tetrameric structure, where Chl dimer pairs are

connected by phytol tails [19]. The exciton interac-

tion energy of monomers in the dimer is approximate-

ly100 cm

–1

, while the interaction between dimers is an

order of magnitude weaker [13, 20]. It has recently been

shown that Chl forming part of this structure manifests

photocatalytic activity in redox processes [21].

This work aims to quantitatively analyse the pro-

cesses of nonradiative intramolecular conversion from

the third excited state to the lowest one, and electron-

ic transitions between exciton substates of the Q

band

of the Chl a tetramer when BoWSCP is excited in the

region of the Soret band and the Q

band, respective-

ly. Spectral characteristics are considered, which allow

the determination of the degree of exciton delocalization

between Chl molecules in the BoWSCP tetrameric com-

plex and the detection of electron transfer reactions in-

volving excited Chl molecules.

MATERIALS AND METHODS

BoWSCP apoprotein preparations were obtained

by expression of genes encoding this protein incorpo-

rated in a plasmid, by which cells of Escherichia coli

BL21(DE3) producer strain were transformed, in ac-

cordance with the protocol [5, 6, 22]. Due to six histi-

dine residues (6xHis) on N-terminal domains of apo-

proteins, their preparations were purified using affinity

chromatography on a Ni-agarose column with a subse-

quent preparation dialysis. Preparation purity was test-

ed using denaturing polyacrylamide gel electrophoresis

[5, 6]. Self-assembly of BoWSCP holoforms was per-

formed in vitro by joint incubation of apoproteins and

isolated thylakoid membranes of spinach [4-6, 8, 22].

Taking into account high thermal stability of WSCP

protein tetramers, the obtained holoform preparations

were heated (95°C, 10 min) to remove unnecessary pro-

teins. The final purification of BoWSCP was then car-

ried out on the Ni-agarose column [1,23]. The purified

preparation was dialysed against 10 mM Tris-HCl buf-

fer, pH 8.0. Quality of BoWSCP tetramer preparations

was tested using native polyacrylamide gel electropho-

resis, absorption spectroscopy, fluorescence spectros-

copy and circular dichroism spectroscopy. Denaturating

and native electrophoresis of proteins was carried out in

Mini PROTEAN Tetra System (Bio-Rad, USA). Pro-

tein content in samples was measured using the Brad-

ford assay [24]. The absorption spectra of the samples

were registered using Cary-50 (Agilent, USA) and

Shimadzu-1601PC (Shimadzu, Japan) spectrophotom-

eters. Circular dichroism (CD) spectra were registered

using Chirascan circular dichroism instrument (Applied

Photophysics, USA). Signal amplitude (Δε) was con-

verted into units of ellipticity(θ) for circular dichroism

spectra in accordance with [23].

Preparations for femtosecond spectroscopy were

concentrated by centrifugation in Amicon Ultra-15 cen-

trifugal devices with 30 or 50kDa filters. Concentration

was achieved by centrifugation of BoWSCP prepara-

tions (several times) in 10 mM Tris-HCl buffer, pH 8.0,

with addition of up to 150 mM NaCl or 10% glycerine

to prevent protein aggregation in the highly concentrat-

ed solution. The concentrate was diluted with the same

buffer with NaCl or glycerine to obtain 1ml of solution

with OD

673

= 5.

Ultrafast absorption changes ΔA(λ

) were record-

ed by the femtosecond pump-probe laser spectroscopy

in the spectral range of 400 ≤ λ ≤ 780 nm and time de-

lays from 20 fs to 200 ps. The experimental setup and

measurement methodology were described earlier [25].

The excitation of the BowSCP was achieved by fem-

tosecond pulses with a maximum at 430 nm (duration

40 fs, energy 20 nJ) and 670 nm (duration 16 fs, energy

15 nJ). Absorption changes were enquired by the broad-

band pulses at the polarization angles of 0, 54.7, and

90 degrees relative to the exciting pulse polarization.

The difference absorption spectra in the range of 400-

780 nm were registered using the CCD-camera (Roper

Scientific SPEC-10, USA), connected to the polychro-

CHEREPANOV et al.1582

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

mator (Acton SP-300, USA). The experiments were car-

ried out at a temperature of 6°C in a circulating opti-

cal cell 0.5 mm thick, the diameter of optical windows

0.2 mm, the optical density of the sample 10 cm

−1

The rate of sample circulation (9 ml/min) was high

enough to avoid multiple excitations of the same sample

volume. The spectra were corrected by the group delay

dispersion, as previously described [26, 27]. Deconvo-

lution of the instrumental function accounting for the

coherent artifact in the region t = 0 was carried out ac-

cording to the method proposed in the works [26, 28].

For this purpose, the absorption changes caused by

nonlinear interactions of overlapping pump and probe

pulses (coherent spike) were approximated by the basic

gaussian function(1):

G(t) = 1/

√

2πd

exp[−½(t/d)

], (1)

and its derivatives in time G′ and G′′, where the width

of the pump pulse d was determined by the absorption

changes of the pure solvent [29]. The signal related to

changes in the electronic states of pigments was approx-

imated by convolution of the basic Gaussian function

G(t) and the step function of Hevisaid H(t) (2):

χ(t) = G(t)*H(t) = ½Erfc[−t/

√

2πd

], (2)

as well as convolutions of G(t) and exponential func-

tions(3):

ε(t,τ

) = G(t)*exp(−t/τ

) =

= ½exp[½(d/τ

)

− t/τ

]Erfc[(d/τ

− t/d)/

√

2].

(3)

The amplitudes A

(λ

) were determined by linear

regression and the characteristic times τ

by nonlinear

minimization.

The mathematical analysis of the absorption dynam-

ics included approximation of spectral-temporal ma-

trices ΔA(λ

) by a linear combination of n discrete

exponential functions(4):

Q[τ](λ,t) =

(λ)⋅exp(−t/τ

)+D

n+1

(λ), (4)

under the assumption that the characteristic times

τ={τ

} do not depend on the probe wavelength λ and,

therefore, can be considered as “global” parameters de-

termined by nonlinear minimization of the normalized

discrepancy (5):

R[τ] = (L⋅M−n)

−1

ΣΣ

[ΔA(λ

)−

−Q[τ](λ

)]

(5)

The wavelength-dependent amplitudes D

(λ), which

were calculated by the linear regression method for each

exponential functionk, represent decay associated spec-

k = 1

l = 1

m = 1

tra(DAS). When the optimal values {τ

} of characteristic

times are found, the standard errors of their estimation

can be evaluated using the Jacobi matrixJ(6):

δQ

[τ]

δτ

. (6)

Here the index j runs over L × M experimental

points of the spectral-temporal matrix ΔA(λ

) [30].

The numerical assessment of Jacobian can be obtained

by decomposing the expression Q

[τ] in a Taylor series of

the first order (7):

δQ

[τ]

δτ

≈

[τ +Δτe

]−Q

[τ −Δτe

]

2Δτ

. (7)

Here τ is a set of optimal values of characteristic

times{τ

}, e

is the k

single vector, and Δτ is a small in-

crease of τ (the value of 0.015τ was used for a numerical

assessment). Standard errors σ

of characteristic times

estimation can be obtained from the diagonal elements

of the covariance matrix C=R[τ]×(J

−1

(8):

√

. (8)

The values of σ

are standard deviations character-

izing the uncertainty of{τ

} assessments. According to

the Student’s t-distribution, 95% reliability is obtained

with the doubling of standard deviations, so the trust in-

tervals are τ

±2σ

, respectively.

RESULTS

Using the method proposed by Hughes etal. [20],

the pigment composition of the obtained BoWSCP ho-

loform preparations was determined by decomposing

its absorption spectrum in the Soret band region (360-

500 nm) into relative contributions of Chl a and Chl b

using the absorption spectra of individual pigments

in 90% acetone [31]. Figure 1a shows comparison of

BoWSCP absorption spectrum with the spectra of Chl a

and Chl b. In the absorption spectrum of free Chl a, the

Soret and Q

absorption bands with peaks at approxi-

mately 430 and 663 nm have a comparable amplitude;

in the spectrum of Chl b, these bands are located closer

to each other at 458 and 645 nm, respectively, and the

amplitude of the Soret band is increased, whereas that

of the Q

band is reduced in relation to the Chl a bands.

The presence of Chl b in the BoWSCP spectrum is de-

tected based on a small “arm” in the region of 470 nm,

while absorption in the interval of 380-430 nm is related

mainly to Chl a. Comparison of the spectra of BoWSCP

and free Chl a shows a significant effect of surround-

ing protein in the region of 360-450 nm. Firstly, Chl a

bands are shifted to the red by ~8 nm. Secondly, the in-

tensities of the two main transitions of Chl a in the Soret

region (the B

band with the peak at 430 nm corresponds

FEMTOSECOND DYNAMICS OF CHLOROPHYLL TETRAMER 1583

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 1. Determination of the contributions of Chla and Chlb to the absorption spectrum of BoWSCP. a)Comparison of the absorption spectrum

of BoWSCP holoform in the buffer solution(1) with the spectra of Chla(2) and Chlb(3) in 90% acetone (based on the open spectral database

[31]). b)Modelling of BoWSCP spectrum(1) using Chla(2) and Chlb(3) spectra modified in accordance with the equation(9). In the re-

gion of 500-720nm, the amplitude of Chla spectrum is increased by 15%. Relative contributions of Chla and Chlb to the model spectrum(4)

are 89% and 11%, respectively.

to the S

→S

transition, and the B

band with the peak

at 380 nm corresponds to the S

→S

transition [32]) are

changed significantly in favour of the band with the peak

at ~380 nm. The electronic structure of the absorption

spectrum of Chl a in the Soret region cannot be de-

scribed properly by the Gouterman four-orbital mod-

el [33], because there are non-Gouterman states be-

tween the excited B

and B

states, mixed with vibronic

substates of the B

band. Consequently, energy and

dipole power of these states depend on dielectric sur-

rounding of the pigment [32, 34, 35].

As follows from Fig.1a, the relative probability of

→S

transition of Chl a in BoWSCP is reduced, while

the probability of S

→S

transition is increased compared

to the properties of free Chl a in solution. For this rea-

son, the BoWSCP absorption spectrum was modelled by

the empirical expression representing a sum of modified

absorption spectra of Chl a and Chl b, and taking into

account the redistribution of Chl a bands intensity in

the region of 360-500 nm:

A(λ) = α⋅A

Chla

(λ − δ

)⋅[1 − γ⋅tanh((λ − λ

/Δ)] +

+ β⋅A

Chlb

(λ − δ

(9)

Here, α and β are the relative fractions of Chl a and

Chl b in the BoWSCP tetramer, A

Chla

and A

Chlb

are the

spectra of Chl a and Chl b in 90% acetone, δ

and δ

show how the spectra of Chl a and Chl b in BoWSCP

are shifted relative to their spectra in the solution.

The empirical function γ·tanh((λ−λ

)/Δ) describes

changes in the ratio of the B

and B

bands of Chl a

intensities in the spectral interval centered at the wave-

lengthλ

and the width Δ. Values of these parameters

determined by non-linear minimization amounted

to: α = 89.2%; β = 10.8%; δ

= 9.2 nm; δ

= 1.4 nm;

γ = 0.67; λ

= 413 nm; Δ = 50 nm; the model spectrum

CHEREPANOV et al.1584

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 2. Location of chlorophyll molecules in the BoWSCP tetramer-

ic complex according to the X-ray crystallographic structure PDBID:

6s2z [18], where Chlb is replaced by Chla. Arrows show the directions

of vectors μ

(k = 1,…,4) characterizing the S

→S

transition dipole

moments of four Chl monomers, and dipole moments of four exciton

states E

of the tetrameric complex.

Fig. 3. The absorption spectrum (a), its second(b) and fourth(c) derivatives of BoWSCP holoform (dots). Solid lines represent a model spectrum,

and dashed lines show its main Gaussian components. Vertical dashed lines show the maxima of the fourth derivative of the absorption spectrum.

is shown in Fig. 1b by a thin solid line. Relative contri-

butions of Chl a and Chlb were equal to 89% and 11%,

respectively. The ratio between Chla/b pigments of 8 : 1

corresponds to the range of values 6-10 : 1 obtained in

various papers for BoWSCP native protein [3].

Absorption of BoWSCP in the Q

band region

(660-700 nm) is related primarily to Chl a, while the

Chl b contribution is negligible in this interval (Fig. 1b).

The BoWSCP spectral properties are adjusted by the

molecular structure of the pigment–protein complex:

four identical subunits form a tetramer in which three or-

thogonal axes of rotational symmetry C

(X)C

(Y)C

(Z)

define the coordinate system XYZ [18]. Four chloro-

phyll molecules in the hydrophobic locus form a dou-

ble dimer: interactions between dimers are much weak-

er than interactions between monomers within a dimer

(Fig.2).

The effect of exciton interaction between Chl mol-

ecules within the dimer predominates in the region of

660-700 nm, while the interaction between dimers is

much weaker. The shape of BoWSCP absorption spec-

trum in the Q

band region (Fig. 3a) indicates the presence

FEMTOSECOND DYNAMICS OF CHLOROPHYLL TETRAMER 1585

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 4. Absorption changes of BoWSCP at the wavelengths of 450nm(a) and 670nm(b), induced by the pulse at 670nm with a duration of 16fs

(the black solid line). The coherent artifact was approximated by superposition of the Gaussian function G(t) (Eq.1) and its derivatives G′ and

G′′ (the dashed line), while the BoWSCP absorption changes were modelled by a combination of functions χ(t) and ε(t,τ

) (the dot-dash line),

with the parameter value d=24fs. The BoWSCP absorption response was obtained by subtraction of the coherent artifact (the red solid line).

of two exciton bands with peaks at 672 and 684 nm,

corresponding to the minima of the second derivative

(Fig. 3b) and maxima of the fourth derivative (Fig. 3c).

Using the method proposed for analyzing WSCP spec-

tra [20], the parameters of Chl a exciton interaction

were determined by decomposing the BoWSCP absorp-

tion spectrum in the Q

(0,0) band region into Gauss-

ian components. For an appropriate approximation

of the Q

(0,0) band with intramolecular vibrational

modes of the Chl tetrapyrrole macrocycle taken into

account, additional Gaussian components were used in

the high-energy region of the Q

(0,0) band [20, 36, 37].

The relative intensity of the main E

band peaking at

672 nm was 79%, while the contribution of the sec-

ond E

band peaking at 684 nm was about 21%. Spec-

tral characteristics of the E

state were not determined,

and the E

state has zero intensity in the approximation

ofpoint dipoles.

Broadband femtosecond laser pump-probe spec-

troscopy was used for measuring the absorption dynam-

ics of BoWSCP holoform, induced by excitation in the

region of wavelengths of 430 and 670 nm. Excitation in

these spectral regions is related mainly to Chl a (quan-

tum yield is >98%) and only marginally to Chl b (<2%).

CHEREPANOV et al.1586

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 5. Transient absorption spectra of BoWSCP at time delays of 50fs(a) and 200ps(b) induced by excitation at 430nm(1) and 670nm(2).

The spectra are scaled in the Soret band region of 400-500nm at the delay of 200ps. The polarization angle is 54.7°. The inset shows the anisotro-

py coefficient in the Q

band region

Figure 4 shows the coherent artifact (coherent spike)

arising at short time delays due to nonlinear interactions

of overlapping pump and probe pulses. The contribution

of the coherent spike was divided from the absorption

changes of the pigments according to the procedure

described in “Materials and Methods”. The BoWSCP

absorption changes, starting from the time delay t = 0,

were assessed by deconvolution of the resulting signal

inaccordance with equations(2) and(3).

Figure 5a shows transient absorption spectra of

BoWSCP at a time delay of 50 fs, induced by the puls-

es at 430 nm (1,the solid line) and 670 nm (2,the dot-

dash line). The 430 nm excitation is related mainly to

the Chl transition S

→S

into the third excited singlet

state (the B

band), while the 670 nm excitation produc-

es the S

state (the Q

band). This assignment is con-

firmed by a negative anisotropy observed in the region

of the Q

band upon excitation at 430 nm, where the

anisotropy coefficient is close to the theoretical limit

of–0.1 (see the inset in Fig.5a).

The amplitude of the Q

band bleaching in the

BoWSCP transient absorption spectra in Fig. 5 is

3-5times higher than that of the Soret band, while the

amplitude of the Q

band in the BoWSCP linear absorp-

tion spectrum in Fig. 1a is comparable to that of the

Soret band. A transient absorption spectrum observed

FEMTOSECOND DYNAMICS OF CHLOROPHYLL TETRAMER 1587

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 6. Decay associated difference spectra of BoWSCP in the range of 100 fs(a) and 10 ps(b) after excitation at 430nm (1,3) and 670nm (2,4).

Characteristic times: 1)83fs, 2)105fs, 3)19ps, 4)14ps. The marks on the graphs in the Q

band area show the confidence intervals calculated

using equation(8).

in the femtosecond measurements is the sum of three

contributions. First is the spectrum of bleaching, which

results from the disappearance of the ground state S

and its form corresponds to the inversed linear absorp-

tion spectrum. Second is the absorption spectrum of the

emerging excited state S

, and its contribution is always

positive. Third is the spectrum of the reverse S

→S

tran-

sition, stimulated by emission, and for the lowest excited

state its shape is close to the spontaneous fluorescence

spectrum; its contribution is always negative. The main

difference between the spectra (1) and (2) in Fig. 5a

is visible in the Q

band region, where the stimulated

emission of the S

→S

transition is observed, which does

not exist during the lifetime of the S

state produced by

the excitation at 430 nm. The less considerable differ-

ence between the spectra (1) and (2) in the region of

420-500 nm can be due to the stimulated emission of

the S

→S

transition.

Figure 5b shows transient absorption spectra at

a time delay of 200 ps using the same notations: 1) –

excitation at 430 nm, 2) – 670 nm. Within this time

range the processes of S

→S

intramolecular conver-

sion and thermal equilibration between the four ex-

citon states E

of the tetrameric complex should be

completed. The observed final spectra are close in the

entire range, except for the Q

band, where the bleaching

amplitude after the excitation at 670 nm is much larg-

er than that of the bleaching induced by the excitation

at 430nm.

The most significant BoWSCP absorption changes

are related to the amplitude of the Q

band bleaching

after excitation at 430 nm. Its quantitative characteris-

tics were determined by decomposing the absorption

dynamics into exponential components (decay associ-

ated spectra, DAS, see “Materials and Methods”); de-

composition revealed spectral transitions in the range

of 100fs and 10ps (Fig.6).

Figure 6a shows the difference decay spectra for

the femtosecond range. After excitation at 430 nm the

main spectral changes occur with the characteristic

time of 83 ± 9 fs. They can be attributed to the nonra-

diative intramolecular S

→S

conversion, as a result of

which the stimulated emission disappears in the Soret

band region (450-480 nm) but appears in the region of

the Q

band (670-690 nm). When BoWSCP is excited

in the Q

band region at 670 nm, clearly distinguishable

small spectral changes occur with a characteristic time

of 105 ± 10 fs, and they can be attributed to the relax-

ation transition between the two excitonic substates E

and E

of the Q

band of Chl a dimer.

CHEREPANOV et al.1588

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

In the picosecond range, significant absorption

changes occur with a characteristic time of ~16 ps.

The DAS calculated for excitation at 430 nm (charac-

teristic time 19 ± 3 ps) and 670 nm (characteristic time

14 ± 2 ps) are close throughout the entire spectral range

(Fig. 6b) and are associated with insignificant absorp-

tion recovering in the region of the Q

band with a max-

imum around 675 nm. Possible interpretations of these

changes will be considered further.

To provide quantitative characteristics of photo-

chemical processes observed by means of femtosecond

measurements in the system of excitonically coupled

pigments, transient absorption (TA) was decomposed

into three components: ground state bleaching (GSB),

excited state absorption (ESA), and stimulated emis-

sion(SE). The probability of light absorption with the

quantum energy ħω

by a separate pigment molecule

is proportional to square of the dipole moment |μ|

for

its transition from the ground state to the excited state

(dipole strength of the transition). For Chl a monomer

in protein, the dipole moment of S

→S

transition is

equal to 5.5 D [38]. The pump pulse causes transition

of ΔN molecules in the sample from the ground state

to the excited state. The probe pulse is split into two

beams: one beam passes through a non-excited sam-

ple, and another beam passes through the excitation

region. The difference in beams intensity determines

the difference absorption. In the excitation region the

number of molecules in the ground state is decreased

by ΔN. Therefore, absorption at the frequency ω

in this

region is proportional to the transition dipole strength,

ΔA

GSB

∝ |μ|

. At the same time, there is light emission of

the reverse S

→S

transition stimulated by the light wave,

its probability is also proportional to the dipole moment

strength, so the absorption at the frequency ω

is addi-

tionally decreased by the same value, ΔA

∝ |μ|

. For an

ideal two-level system, the ratio β = ΔA

/ΔA

GSB

is equal

to one [39]; for Chl a in various solvents the estimate

was β≈0.9 [40,41].

For excitonically coupled pigments the situation is

way more complex. The dipole moment of excitonically

coupled pigments represents a linear sum of the dipole

moments of monomers [42]. In the BoWSCP crystal-

lographic structure [18], the dipole moments of mono-

mers μ

are almost parallel to the crystallographic axisX,

while projections of the moments to axes Y and Z are

much smaller in terms of absolute values [13]. In the first

approximation they may be assumed to be equal to zero.

Projections of the monomer dipole moment to crystallo-

graphic axes are shown schematically in Fig.2.

Quantitative description of light interaction with

Chl molecules in the BoWSCP tetramer raises a question

of basic nature: is the emerging excited state localized

within the dimer (the coupling energy of ~100 cm

–1

or is it delocalized across the entire tetrameric complex

(the coupling energy of ~10 cm

–1

)? The femtosecond

measurements enable determining the degree of excited

state delocalization, i.e., the number of Chl molecules

effectively involved in the exciton coupling.

For a symmetric dimer, the dipole moments of the

transitions to two possible excited states E

and E

−

are

equal [43] (10):

√2

(μ

± μ

). (10)

It should be noted that the excitonic interaction of

monomers does not change the total dipole strength of

the complex: |M

+ |M

−

= μ

+ μ

, therefore, the in-

tegral amplitude of the linear absorption spectrum re-

mains constant. Due to the geometry of the BoWSCP

tetramer, the dipole strength of the transition to the E

state is equal to |M

≅ μ

+ μ

= 2μ

, while the dipole

strength of the transition to the E

−

state is close to zero;

therefore, when BoWSCP is excited by a broadband

pulse centered at a wavelength of 670 nm (half-width of

50 nm, time duration of 16 fs), in the initial distribution

of arising exciton states, the E

state significantly pre-

dominates.

In this case, the total amplitude of bleaching ΔA

in the initial transition spectrum near the exciton split-

ting band at frequency ω

is (11):

ΔA

= ΔA

GSB

+ ΔA

∝ (1 + β)2μ

≈ 4μ

. (11)

In the rest of the spectral range, the bleaching of

the ground state is proportional to the dipole strength of

the free monomer (12):

ΔA

GSB

(ω) ∝ μ

. (12)

For a symmetric tetramer, the transition dipole

moments of four possible exciton states are found by a

unitary transformation [42], the moment with the larg-

est amplitude is equal to (13):

(μ

+ μ

− μ

). (13)

In this case, the total bleaching amplitude of the

transition spectrum near the exciton splitting band

is(14):

ΔA

∝ 8μ

. (14)

In general, the number of excitonically coupled

molecules, n

excited

, between which the excited state is

delocalized, can be determined by the ratio of the total

bleaching amplitude of the transition spectrum at the

frequency of the exciton transition ω

and the amplitude

of the linear spectrum, which does not take into account

the selective nature of the complex excitation (15):

excited

= ½ΔA

/ΔA

GSB

. (15)

FEMTOSECOND DYNAMICS OF CHLOROPHYLL TETRAMER 1589

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

Fig. 7. Decomposition of the BoWSCP transient absorption spectrum (1–Transient Absorption, TA) into components of ground state bleach-

ing (2–Ground State Bleaching, GSB), excited state absorption (3–Excited State Absorption, ESA) and bleaching in the stimulated emission

region (4–Bleach, BL). Transient absorption spectra were obtained at a delay of 50fs after excitation at 430nm(a) and 670nm(b). The bleach-

ing spectrum(2) was normalized based on the TA spectrum(1) in the region of 400-500nm, taking into account approximation of the excited

state spectrum by a cubic polynomial function(×). The model bleaching spectrum in the region of 650-700nm near the stimulated emission

(4) in the range ΔA

=ΔA

GSB

+ΔA

was represented by a sum ΔA

=n

excited

·(ΔA

GSB

+ΔA

) of the linear absorption spectrum and its mirror re-

flection relative to the maximum of the absorption band Q

peak and the Stokes shift Δλ. The model spectrum amplitude n

excited

was determined

by linear regression using polynomial approximation of the excited state spectrum(○). The thick gray line shows the result of such decomposition.

When BoWSCP is excited at 670 nm, the amplitude

of the ground state bleaching ΔA

GSB

can be determined

from the Soret band. In Fig. 7 dots show the transition

spectra of BoWSCP at a time delay of 50 fs after exci-

tation at wavelengths of 430nm(a) and 670 nm(b).

In the region of 400-500 nm, the transition spec-

tra (1) are close in shape to the absorption spectrum

of BoWSCP (2), with the exception of the region of

450-480 nm, where absorption of Chl b and stimu-

lated emission of Chl a from the S

state predominate.

Assuming that the absorption spectra of the excited

states S

and S

are smooth functions in the specified

wavelength range and can be correctly described by

third-degree polynomials P

(λ), the transition spectra

were approximated by the expression (16):

ΔA

(λ) = ξ ⋅ΔA

GSB

(λ) + P

(λ), (16)

in which the scaling factor ξ and the coefficients of

the cubic polynomial P

were found by the standard

CHEREPANOV et al.1590

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

solution to the linear regression problem. The absorp-

tion spectra of excited states in the region where the

stimulated emission is absent are found as the differ-

ence ΔA

ESA

(λ) = ΔA

(λ) − ξ⋅ΔA

GSB

(λ). Figure 7 shows

the obtained bleaching spectra of the ground state(2),

the polynomial functions P

 (×), and the absorption

spectra of excited states(3).

The model bleaching spectrum near the Qy

band(4) was represented by the sum of the linear ab-

sorption spectrum taken with the opposite sign ΔA

GSB

(λ),

and its mirror reflection relative to the maximum of the

Qy band ΔA

(λ). Since the position of the 0-0 transi-

tion remained unknown, the value of the Stokes shift Δλ

of the SE spectrum was found by nonlinear minimiza-

tion of the total model spectrum (17):

ΔA

(λ) = n

excited

⋅ξ⋅[ΔA

GSB

(λ) +

+ ΔA

GSB

(λ

+ Δλ − λ)] + P

(λ).

(17)

The effective number of excitonically coupled mol-

ecules n

excited

and the coefficients of the polynomial func-

tionP

were found by linear regression. Gray thick line

in Fig. 7 presents the result of this decomposition. When

excited at 430 nm, the observed Q

band bleaching

amplitude ΔA

is only slightly larger than the ground

state bleaching amplitude ΔA

GSB

, while when excited at

670 nm, the ΔA

amplitude is three times the ampli-

tudeΔA

GSB

Figure 8a shows time dependence of the bleach-

ing amplitude of the Soret band (1, 2) and the Q

band(3, 4), induced by BoWSCP excitation at 430 nm

(solid lines) and 670 nm (dot-dash lines). Absorption

changes in the time range of 100 fs with excitation at

430 nm (3) reflects stimulated emission which appears

as a result of electronic transition S

→S

, with a charac-

teristic time of 80 fs. As it was noted above, the bleach-

ing amplitude of the Q

band after excitation in the re-

gion of 430 nm remains much lower throughout the time

interval than the amplitude of this band after excitation

at 670 nm. The high bleaching amplitude of Q

band af-

ter excitation at 670 nm throughout the time interval is

another feature: its amplitude is three times higher than

that of the Soret band.

Figure 8b shows changes in the effective number

excited

of exciton-coupled Chl molecules in the course

of time, which is calculated according to the equa-

tion(15) for two BoWSCP excitation variants. Between

these molecules, the excited state of the tetramer is de-

localized. For short delays in the case of excitation at

670 nm, n

excited

is close to 2, i.e., BoWSCP acts as an ex-

citon-coupled dimer. In case of excitation at 430 nm at

short delays, n

excited

is close to 0.5. As it was noted above,

this means that the excited state S

prevails in these

conditions.

Finally, Fig. 8c shows changes in the anisotropy

coefficient, r = (ΔA

− ΔA

⊥

) / (ΔA

+ 2ΔA

⊥

), calculat-

ed in the Q

band region for two excitation variants.

The changes in the Q

band bleaching amplitude at

the timescale of ~100 fs are almost unrelated to the an-

isotropy changes.

DISCUSSION

WSCP proteins represent a uniquely organized

model system of exciton-coupled chlorophyll mole-

cules [11], which allows research into the mechanisms

Fig. 8. Changes in the main parameters characterizing the excited state

of BoWSCP tetrameric complex in the course of time. a) Bleaching

amplitude of the Soret band(1,2) and the Q

band(3,4) after exci-

tation at 430nm(1,3) and 670nm(2,4). b)The effective number of

excitonically coupled Chl molecules calculated by to the equation(15)

for excitation at 430nm(1) and 670nm(2). c)The anisotropy coeffi-

cient calculated in the Q

band region for excitation at 430nm(1) and

670nm(2).

FEMTOSECOND DYNAMICS OF CHLOROPHYLL TETRAMER 1591

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

of excitation energy transfer between pigments [12-14]

and the interaction of excited pigments with surround-

ing proteins [15-17]. Energy exchange between the

electron subsystem and nuclei vibration triggers the

evolution of electronic states [44, 45]. The efficiency

of energy exchange between exciton states and phonon

modes depends on spectral density of nuclei vibration-

al states. To determine this experimentally, spectral hole

burning [46-48] and time-resolved fluorescence spec-

troscopy [10] were used. Chl a vibron bands comprise

over 40 modes within the range of 1500 to 260 cm

−1

[49-51]. The dipole–dipole interaction of adjacent

Chl a molecules in the BoWSCP tetramer obtained by

joint decomposition of absorption and circular dichro-

ism spectra is ~100 cm

−1

[20], which allows considering

the delocalized excited states of pigments in terms of the

exciton theory [42]. The value of exciton interaction of

dimers of WSCP tetramer calculated using the X-ray

structure of the complex is ~10vcm

−1

[13], which allows

describing energy transfer between dimers using Förster

theory [52].

Measurement of absorption dynamics of WSCP

proteins of subclasses IIa (BoWSCP, from Brassica

oleracea) and IIb (LvWSCP, from Lepidium virginicum)

in the femtosecond range had been carried out in the Q

band region using pump-probe spectroscopy [14] and

2D spectroscopy [12, 13]. Theiss et al. measured absorp-

tion changes in the BoWSCP complex contained 20-30%

Chl b, and in that system, transfer of excitation from

Chl b to Chl a with a characteristic time of 400 fs was

registered [14]. That process was considered as an exci-

ton relaxation of heterodimer Chl a-Chl b, but later the

exciton relaxation of the heterodimer was shown to oc-

cur faster than in 100 fs, and processes with the time of

250-400 fs were ascribed to the energy transfer between

dimers [12]. The methods of 2D spectroscopy were used

for studying absorption dynamics of LvWSCP and

BoWSCP homotetramers containing 100% Chl a [13].

In both systems monoexponential absorption dynamics

with characteristic times of 70 and 100 fs, respectively,

were observed in the subpicosecond scale; these pro-

cesses were interpreted as the E

→E

relaxation to the

lowest exciton state [13]. Calculations with the modified

Redfield theory predict that equilibrium between exci-

ton states should be achieved within this time range [15].

The spectral transition with the time of 110 fs, observed

in case of BoWSCP excitation at 670 nm (Fig. 6a, spec-

trum2) matches the previously obtained data.

In this work the absorption dynamics of Chl a te-

trameric complex was first measured in the wide spec-

tral range of 400-750 nm with a time resolution of 20 fs.

This made is possible to register the intramolecular con-

version of Chl a from the excited state S

to S

, to ana-

lyze relaxation dynamics of exciton-coupled molecules

of Chl a, and to provide quantitative characteristics of

distribution between the E

and E

excitonic states relat-

ed to the Q

band of Chl a dimer. All that was possible

within a single experimental system.

Quantitative comparison of absorption dynamics in

the Soret and Q

band regions is required for properly

interpreting the data obtained with large photosynthetic

complexes. For instance, the bleaching amplitude of

Chl a in the Q

band region in primary transient ab-

sorption spectra of photosystemI from cyanobacterium

Thermosynechococcus elongatus in case of excitation in

the far-red region [53], and of Chl a and Chl f in similar

spectra of photosystem I from cyanobacterium Fisch-

erella thermalis PCC 7521 [54] is 3-6 times higher than

the bleaching amplitude of Soret bands. Meanwhile,

in similar spectra of photosystem I from cyanobacteri-

um Synechocystis sp. PCC 6803 the bleaching amplitude

of the Q

band is only slightly higher than that of the

Soret band [55]. Van Stokkum et al. assumed that the

high amplitude of the Q

band in the primary spectra

of photosystem I is an evidence for quenching of most

excited states by preoxidized chlorophyll of the special

pair P700 [56]. However, the light-harvesting complex-

es of photosystem I of cyanobacteria T. elongatus and

F. thermalis PCC 7521 contain exciton-coupled dimers

and trimers of Chl with maximum absorption in the far-

red region, as distinct from Synechocystissp. PCC 6803

[57-60]. The results obtained in this paper regarding the

impact of exciton coupling on the bleaching amplitude

of the Q

band (Fig.8) explain the observed effects by

structural features of the light-harvesting complexes.

Presented in Fig. 8, the dependencies of the bleach-

ing amplitude of the Soret band and the Q

band togeth-

er with the determined n

excited

coefficient representing the

number of exciton-coupled Chl molecules provide sev-

eral conclusions about the physical and chemical prop-

erties of BoWSCP excited states.

1. In case of BoWSCP excitation in the Q

band

region, the highest excitonic state E

with the peak at

672nm is predominantly formed. The thermal equilibri-

um between the E

and E

states (peak at about 684 nm)

is established at the characteristic time τ

= 105 ± 10 fs.

However, the contribution of the high-energy E

state

prevails over the low-energy E

state in the entire time

interval of hundreds of picoseconds (the relative contri-

bution is ≥80%). The population inversion can be due

to a considerable contribution of entropy to the differ-

ence in free energy of the E

and E

states. This impli-

cates a strong electronic interaction between BoWSCP

monomers, which takes place in case of considerable

overlapping of wave functions of excited states. In such

strongly coupled dimers, resonant charge-transfer states

arise, which determine a strong electron-phonon cou-

pling [61,62].

2. When BoWSCP is excited in the Soret band

region, the electronic state S

(also designated as B

)

is predominantly produced, which is transformed to

the S

state by a process of intramolecular conversion

CHEREPANOV et al.1592

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

with the characteristic time of 83 ± 9 fs. However, the

stimulated emission in the later state is characterized by

the amplitude which is almost halved compared to the

equilibrium S

state formed after excitation at 670 nm.

The decreased stimulated emission amplitude can re-

sult from an ultrafast photochemical reaction of Chl

oxidation and reduction of a tryptophan residue, “com-

peting” with the intramolecular conversion reaction.

In this case a reverse recombination reaction of Chl

Trp

−

ion-radical pair can take more than 200 ps. The pho-

toreduction of tryptophan Chl

→Chl

Trp

−

was registered

for LvWSCP by spectral hole burning at the temperature

of 4 K in an hour timescale and with a low quantum

yield [47]. It can thus be assumed that spectral changes

with the characteristic time τ

= 16 ± 3 ps (Fig. 6b) can

result from acceleration of this photochemical reaction

at the temperature of 279 K.

3. An increased bleaching amplitude of the Q

band

in relation to the Soret band was observed in transient

absorption spectra of photosystemI of various cyanobac-

teria with specific excitation of long-wavelength chloro-

phyll forms [53, 54, 63]. Long-wavelength chlorophyll

forms in the light-harvesting antenna and the reaction

center of photosystem I arise due to formation of exciton-

ically coupled Chl dimers and trimers [60, 64, 65], which

obviously has the molecule structure similar to the archi-

tecture of chlorophyll molecules in the WSCP complex.

Contributions. D.A.Ch. analysis of spectral mea-

surement results; K.V.N., Yu.N.O., and Yu.V.M. pro-

duction, recovery, and biochemical characterization of

BoWSCP preparations; F.E.G., I.V.Sh., and A.V.A. de-

velopment of a mathematical apparatus, methods and

performing femtosecond measurements; M.S.K. and

V.A.N. concept development. All authors made a con-

siderable contribution to the manuscript.

Funding. This research was funded by the Russian

Science Foundation (grant no.21-74-20155).

Acknowledgments. The authors thank A. L. Dob-

ryakov for assistance in developing the coherent arti-

fact approximation method and setting the femtosecond

device. In this paper the equipment (the femtosecond

device) provided by the Center for Collective Use of

N. N. Semenov Federal Research Center for Chemi-

cal Physics, Russian Academy of Sciences “Analysis of

Chemical and Biological Systems and Natural Materi-

als: Mass Spectral Microscopy and Femtosecond Laser

Microscopy and Spectroscopy” (registration number:

506694). The expression vector pET-24b with the em-

bedded BoWSCP gene was kindly provided by Prof.

Dr. Harald Paulsen (Institute of Molecular Physiology,

Johannes Gutenberg University Mainz, Germany)

Ethics declarations. The authors declare no conflict

of interests in financial or any other sphere. This article

does not contain any studies with human participants

oranimals performed by any of the authors.

REFERENCES

1. Charlton, A., and Zachariou, M. (2007) Immobilized

metal ion affinity chromatography of histidine-tagged

fusion proteins, Methods Mol. Biol., 421, 137-149,

doi:10.1007/978-1-59745-582-4_10.

2. Cherepanov, D. A., Semenov, A. Y., Mamedov, M. D.,

Aybush, A. V., Gostev, F. E., Shelaev, I. V., Shuvalov,

V.A., and Nadtochenko, V.A. (2022) Current state of the

primary charge separation mechanism in photosystemI of

cyanobacteria, Biophys. Rev., 14, 805-820, doi: 10.1007/

s12551-022-00983-1.

3. Murata, T., Toda, F., Uchino, K., and Yakushiji, E. (1971)

Water-soluble chlorophyll protein of Brassica oleracea var.

botrys (cauliflower), Biochim. Biophys. Acta Bioenerg.,

245, 208-215, doi:10.1016/0005-2728(71)90023-5.

4. Satoh, H., Nakayama, K., and Okada, M. (1998) Molec-

ular cloning and functional expression of a water-soluble

chlorophyll protein, a putative carrier of chlorophyll mol-

ecules in cauliflower, J.Biol. Chem., 273, 30568-30575,

doi:10.1074/jbc.273.46.30568.

5. Takahashi, S., Yanai, H., Nakamaru, Y., Uchida, A.,

Nakayama, K., and Satoh, H. (2012) Molecular cloning,

characterization and analysis of the intracellular local-

ization of a water-soluble chl-binding protein from brus-

sels sprouts (Brassica oleracea var. gemmifera), Plant Cell

Physiol., 53, 879-891, doi:10.1093/pcp/pcs031.

6. Takahashi, S., Yanai, H., Oka-Takayama, Y., Zanma-

Sohtome, A., Fujiyama, K., Uchida, A., Nakayama, K.,

and Satoh, H. (2013) Molecular cloning, characterization

and analysis of the intracellular localization of a water-sol-

uble chlorophyll-binding protein (WSCP) from Virginia

pepperweed (Lepidium virginicum), a unique WSCP that

preferentially binds chlorophyll b in vitro, Planta, 238,

1065-1080, doi:10.1007/s00425-013-1952-7.

7. Satoh, H., Uchida, A., Nakayama, K., and Okada, M.

(2001) Water-soluble chlorophyll protein in Brassicace-

ae plants is a stress-induced chlorophyll-binding protein,

Plant Cell Physiol., 42, 906-911, doi:10.1093/pcp/pce117.

8. Horigome, D., Satoh, H., Itoh, N., Mitsunaga, K., Oon-

ishi, I., Nakagawa, A., and Uchida, A. (2007) Structural

mechanism and photoprotective function of water-soluble

chlorophyll-binding protein, J. Biol. Chem., 282, 6525-

6531, doi:10.1074/jbc.M609458200.

9. Maleeva, Y. V., Neverov, K. V., Obukhov, Y. N., and

Kritsky, M. S. (2019) Water soluble chlorophyll-bind-

ing proteins of plants: structure, properties and func-

tions, Mol. Biol. (Mosk), 53, 998-1011, doi: 10.1134/

S0026898419060120.

10. Schmitt, F. J., Trostmann, I., Theíss, C., Pieper, J.,

Renger, T., Fuesers, J., Hubrich, E. H., Paulsen, H.,

Eichler, H. J., and Renger, G. (2008) Excited state dy-

namics in recombinant water-soluble chlorophyll pro-

teins (WSCP) from cauliflower investigated by transient

fluorescence spectroscopy, J.Phys. Chem.B, 112, 13951-

13961, doi:10.1021/jp8024057.

FEMTOSECOND DYNAMICS OF CHLOROPHYLL TETRAMER 1593

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

11. Renger, G., Pieper, J., Theiss, C., Trostmann, I.,

Paulsen,H., Renger, T., Eichler, H.J., and Schmitt, F.J.

(2011) Water soluble chlorophyll binding protein of high-

er plants: Amost suitable model system for basic analy-

ses of pigment–pigment and pigment–protein interac-

tions in chlorophyll protein complexes, J.Plant Physiol.,

168, 1462-1472, doi:10.1016/j.jplph.2010.12.005.

12. Alster, J., Lokstein, H., Dostál, J., Uchida, A., and Zig-

mantas, D. (2014) 2D spectroscopy study of water-soluble

chlorophyll-binding protein from lepidium virginicum,

J.Phys. Chem.B, 118, 3524-3531, doi:10.1021/jp411174t.

13. Fresch, E., Meneghin, E., Agostini, A., Paulsen, H., Car-

bonera, D., and Collini, E. (2020) How the protein envi-

ronment can tune the energy, the coupling, and the ultra-

fast dynamics of interacting chlorophylls: the example of

the water-soluble chlorophyll protein, J.Phys. Chem. Lett.,

11, 1059-1067, doi:10.1021/acs.jpclett.9b03628.

14. Theiss, C., Trostmann, I., Andree, S., Schmitt, F. J.,

Renger, T., Eichler, H. J., Paulsen, H., and Renger, G.

(2007) Pigment–pigment and pigment–protein interac-

tions in recombinant water-soluble chlorophyll proteins

(WSCP) from cauliflower, J.Phys. Chem.B, 111, 13325-

13335, doi:10.1021/jp0723968.

15. Renger, T., Trostmann, I., Theiss, C., Madjet, M. E.,

Richter, M., Paulsen, H., Eichler, H.J., Knorr, A., and

Renger, G. (2007) Refinement of a structural model of a

pigment–protein complex by accurate optical line shape

theory and experiments, J. Phys. Chem. B, 111, 10487-

10501, doi:10.1021/jp0717241.

16. Friedl, C., Fedorov, D. G., and Renger, T. (2022) Towards

a quantitative description of excitonic couplings in photo-

synthetic pigment-protein complexes: Quantum chemistry

driven multiscale approaches, Phys. Chem. Chem. Phys.,

24, 5014-5038, doi:10.1039/d1cp03566e.

17. Lahav, Y., Noy, D., and Schapiro, I. (2021) Spectral tun-

ing of chlorophylls in proteins – electrostatics vs. ring

deformation, Phys. Chem. Chem. Phys., 23, 6544-6551,

doi:10.1039/d0cp06582j.

18. Agostini, A., Meneghin, E., Gewehr, L., Pedron, D.,

Palm, D.M., Carbonera, D., Paulsen, H., Jaenicke, E.,

and Collini, E. (2019) How water-mediated hydrogen

bonds affect chlorophyll a/b selectivity in water-solu-

ble chlorophyll protein, Sci. Rep., 9, 18255, doi:10.1038/

s41598-019-54520-4.

19. Bednarczyk, D., Dym, O., Prabahar, V., Peleg, Y., Pike,

D. H., and Noy, D. (2016) Fine tuning of chlorophyll

spectra by protein-induced ring deformation, Angew. Che-

mie Int. Ed., 55, 6901-6905, doi:10.1002/anie.201512001.

20. Hughes, J. L., Razeghifard, R., Logue, M., Oakley, A.,

Wydrzynski, T., and Krausz, E. (2006) Magneto-op-

tic spectroscopy of a protein tetramer binding two exci-

ton-coupled chlorophylls, J.Am. Chem. Soc., 128, 3649-

3658, doi:10.1021/ja056576b.

21. Obukhov, Y. N., Neverov, K. V., Maleeva, Y. V., and

Kritsky, M.S. (2023) Chlorophyll a dimers bound in the

water-soluble protein BoWSCP photosensitize the reduc-

tion of cytochromec, Dokl. Biochem. Biophys., 509, 60-64,

doi:10.1134/S1607672923700126.

22. Takahashi, S., Uchida, A., Nakayama, K., and Satoh,H.

(2014) Three-step photoconversion of only three sub-

units of the water-soluble chlorophyll-binding protein te-

tramer from Chenopodium album, ProteinJ., 33, 337-343,

doi:10.1007/s10930-014-9565-y.

23. Kelly, S. M., Jess, T. J., and Price, N. C. (2005) How to

study proteins by circular dichroism, Biochim. Biophys.

Acta Proteins Proteomics, 1751, 119-139, doi: 10.

1016/

j.bbapap.2005.06.005.

24. Bradford, M. M. (1976) Arapid and sensitive method for

the quantitation of microgram quantities of protein utiliz-

ing the principle of protein-dye binding, Anal. Biochem.,

72, 248-254, doi:10.1016/0003-2697(76)90527-3.

25. Cherepanov, D. A., Shelaev, I. V., Gostev, F. E., Mame-

dov, M.D., Petrova, A.A., Aybush, A.V., Shuvalov, V.A.,

Semenov, A.Y., and Nadtochenko, V.A. (2017) Mecha-

nism of adiabatic primary electron transfer in photosys-

temI: Femtosecond spectroscopy upon excitation of reac-

tion center in the far-red edge of the QYband, Biochim.

Biophys. Acta Bioenerg., 1858, 895-905, doi: 10.1016/

j.bbabio.2017.08.008.

26. Dobryakov, A. L., Pérez Lustres, J. L., Kovalenko, S. A.,

and Ernsting, N. P. (2008) Femtosecond transient ab-

sorption with chirped pump and supercontinuum probe:

Perturbative calculation of transient spectra with gener-

al lineshape functions, and simplifications, Chem. Phys.,

347, 127-138, doi:10.1016/j.chemphys.2007.11.003.

27. Golubeva, E. N., Zubanova, E. M., Melnikov, M. Y.,

Gostev, F. E., Shelaev, I. V., and Nadtochenko, V. A.

(2014) Femtosecond spectroscopy and TD-DFT calcu-

lations of CuCl

2−

excited states, Dalt. Trans., 43, 17820-

17827, doi:10.1039/C4DT01409J.

28. Dobryakov, A. L., Kovalenko, S. A., Weigel, A., Ṕrez-

Lustres, J.L., Lange, J., Müller, A., and Ernsting, N.P.

(2010) Femtosecond pump/supercontinuum-probe spec-

troscopy: Optimized setup and signal analysis for sin-

gle-shot spectral referencing, Rev. Sci. Instrum., 81,

113106, doi:10.1063/1.3492897.

29. Kovalenko, S. A., Dobryakov, A. L., Ruthmann, J., and

Ernsting, N. P. (1999) Femtosecond spectroscopy of

condensed phases with chirped supercontinuum prob-

ing, Phys. Rev. A At. Mol. Opt. Phys., 59, 2369-2384,

doi:10.1103/PhysRevA.59.2369.

30. Šimůnek, J. and Hopmans, J.W. (2002). 1.7 Parame-

ter Optimization and Nonlinear Fitting, in Methods of

Soil Analysis (eds Dane, J. H. and Clarke Topp, G.),

pp. 139-157, doi:10.2136/sssabookser5.4.c7.

31. Clementson, L. A., and Wojtasiewicz, B. (2019) Data-

set on the absorption characteristics of extracted phyto-

plankton pigments, Data Br., 24, 103875, doi: 10.1016/

j.dib.2019.103875.

32. Sirohiwal, A., Berraud-Pache, R., Neese, F., Izsák, R.,

and Pantazis, D. A. (2020) Accurate computation of the

absorption spectrum of chlorophyll a with pair natural

CHEREPANOV et al.1594

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

orbital coupled cluster methods, J. Phys. Chem.B, 124,

8761-8771, doi:10.1021/acs.jpcb.0c05761.

33. Gouterman, M. (1961) Spectra of porphyrins, J. Mol.

Spectrosc., 6, 138-163, doi:10.1016/0022-2852(61)90236-3.

34. Bricker, W. P., Shenai, P. M., Ghosh, A., Liu, Z., En-

riquez, M. G. M., Lambrev, P.H., Tan, H.S., Lo, C.S.,

Tretiak, S., Fernandez-Alberti, S., and Zhao, Y. (2015)

Non-radiative relaxation of photoexcited chlorophylls:

theoretical and experimental study, Sci. Rep., 5, 13625,

doi:10.1038/srep13625.

35. Götze, J. P., Anders, F., Petry, S., Witte, J.F., and Lok-

stein, H. (2022) Spectral characterization of the main

pigments in the plant photosynthetic apparatus by theory

and experiment, Chem. Phys., 559, 111517, doi: 10.1016/

j.chemphys.2022.111517.

36. Umetsu, M., Wang, Z. Y., Kobayashi, M., and Noza-

wa, T. (1999) Interaction of photosynthetic pigments

with various organic solvents. Magnetic circular di-

chroism approach and application to chlorosomes, Bio-

chim. Biophys. Acta Bioenerg., 1410, 19-31, doi: 10.1016/

S0005-2728(98)00170-4.

37. Shipman, L. L., Cotton, T. M., Norris, J. R., and Katz,

J. J. (1976) An analysis of the visible absorption spec-

trum of chlorophylla monomer, dimer,and oligomers in

solution, J.Am. Chem. Soc., 98, 8222-8230, doi:10.1021/

ja00441a056.

38. Cherepanov, D. A., Milanovsky, G. E., Aybush, A. V., and

Nadtochenko, V.A. (2023) Dipole moment of the S0-S1

chlorophyll a transition in solvents with varied refraction

index, Russ.J. Phys. Chem.B, 17, 584-593, doi:10.1134/

S1990793123030181.

39. Berera, R., van Grondelle, R., and Kennis, J. T. M. (2009)

Ultrafast transient absorption spectroscopy: principles

and application to photosynthetic systems, Photosynth.

Res., 101, 105-118, doi:10.1007/s11120-009-9454-y.

40. Cherepanov, D. A., Gostev, F. E., Shelaev, I. V., Aibush,

A. V., Mamedov, M.D., Shuvalov, V.A., Semenov, A.Y.,

and Nadtochenko, V.A. (2020) Visible and near infrared

absorption spectrum of the excited singlet state of chlo-

rophyll a, High Energy Chem., 54, 145-147, doi:10.1134/

S0018143920020058.

41. Cherepanov, D. A., Petrova, A. A., Mamedov, M. D.,

Vishnevskaya, A. I., Gostev, F. E., Shelaev, I. V., Aybush,

A. V., and Nadtochenko, V. A. (2022) Comparative ab-

sorption dynamics of the singlet excited states of chlo-

rophylls a and d, Biochem., 87, 1179-1186, doi: 10.1134/

S000629792210011X.

42. Davydov, A. S. (1962) The Theory Of Molecular Excitons,

McGraw-Hill, New York, NY.

43. Kasha, M., Rawls, H. R., and El-Bayoumi, M. A. (1965)

Theexciton model in molecular spectroscopy, Pure Appl.

Chem., 11, 371-392, doi:10.1351/pac196511030371.

44. Scholes, G. D., and Ghiggino, K. P. (1994) Rate ex-

pressions for excitation transfer I. Radiationless transi-

tion theory perspective, J. Chem. Phys., 101, 1251-1261,

doi:10.1063/1.467817.

45. Renger, T., and Marcus, R. A. (2002) On the relation of

protein dynamics and exciton relaxation in pigment-pro-

tein complexes: an estimation of the spectral density and

a theory for the calculation of optical spectra, J. Chem.

Phys., 116, 9997-10019, doi:10.1063/1.1470200.

46. Pieper, J., Rätsep, M., Trostmann, I., Schmitt, F. J.,

Theiss, C., Paulsen, H., Eichler, H.J., Freiberg, A., and

Renger, G. (2011) Excitonic energy level structure and pig-

ment-protein interactions in the recombinant water-solu-

ble chlorophyll protein. II.Spectral hole-burning experi-

ments, J. Phys. Chem. B, 115, 4053-4065, doi: 10.1021/

jp111457t.

47. Kell, A., Bednarczyk, D., Acharya, K., Chen, J., Noy, D.,

and Jankowiak, R. (2016) New insight into the water-sol-

uble chlorophyll-binding protein from Lepidium virgini-

cum, Photochem. Photobiol., 92, 428-435, doi: 10.1111/

php.12581.

48. Adolphs, J., Berrer, M., and Renger, T. (2016) Hole-burn-

ing spectroscopy on excitonically coupled pigments in

proteins: theory meets experiment, J. Am. Chem. Soc.,

138, 2993-3001, doi:10.1021/jacs.5b08246.

49. Gillie, J. K., Lyle, P. A., Small, G. J., and Golbeck,

J. H. (1989) Spectral hole burning of the primary elec-

tron donor state of PhotosystemI, Photosynth. Res., 22,

233-246, doi:10.1007/BF00048302.

50. Pieper, J., Voigt, J., and Small, G.J. (1999) Chlorophyll

a Franck–Condon factors and excitation energy transfer,

J.Phys. Chem.B, 103, 2321-2322, doi:10.1021/jp984460e.

51. Peternian, E. J. G., Pullerits, T., van Grondelle, R., and

van Amerongen, H. (1997) Electron-phonon coupling

and vibronic fine structure of light-harvesting com-

plexII of green plants: temperature dependent absorption

and high-resolution fluorescence spectroscopy, J. Phys.

Chem.B, 101, 4448-4457, doi:10.1021/jp962338e.

52. Förster, T. (1948) Intermolecular energy migration

and fluorescence [in German], Ann. Phys., 437, 55-75,

doi:10.1002/andp.19484370105.

53. Cherepanov, D. A., Shelaev, I. V., Gostev, F. E., Aybush,

A. V., Mamedov, M.D., Shuvalov, V.A., Semenov, A.Y.,

and Nadtochenko, V.A. (2020) Generation of ion-radical

chlorophyll states in the light-harvesting antenna and the

reaction center of cyanobacterial photosystem I, Photo-

synth. Res., 146, 55-73, doi:10.1007/s11120-020-00731-0.

54. Cherepanov, D. A., Shelaev, I. V., Gostev, F. E., Aybush,

A. V., Mamedov, M.D., Shen, G., Nadtochenko, V. A.,

Bryant, D. A., Semenov, A.Y., and Golbeck, J.H. (2020)

Evidence that chlorophyll f functions solely as an anten-

na pigment in far-red-light photosystemI from Fischerel-

la thermalis PCC7521, Biochim. Biophys. Acta Bioenerg.,

1861, 148184, doi:10.1016/j.bbabio.2020.148184.

55. Shelaev, I. V., Gostev, F. E., Mamedov, M. D., Sarkisov,

O. M., Nadtochenko, V.A., Shuvalov, V.A., and Semen-

ov, A. Y. (2010) Femtosecond primary charge separation

in Synechocystis sp. PCC 6803 photosystem I, Biochim.

Biophys. Acta Bioenerg., 1797, 1410-1420, doi: 10.1016/

j.bbabio.2010.02.026.

FEMTOSECOND DYNAMICS OF CHLOROPHYLL TETRAMER 1595

BIOCHEMISTRY (Moscow) Vol. 88 No. 10 2023

56. Van Stokkum, I. H. M., Müller, M. G., Weißenborn, J.,

Weigand, S., Snellenburg, J. J., and Holzwarth, A. R.

(2023) Energy transfer and trapping in photosystem I

with and without chlorophyll-f, IScience, 26, 107650,

doi:10.1016/J.ISCI.2023.107650.

57. Pålsson, L.-O. O., Flemming, C., Gobets, B., van Gron-

delle, R., Dekker, J.P., and Schlodder, E. (1998) Energy

transfer and charge separation in photosystemI: P700 oxi-

dation upon selective excitation of the long-wavelength an-

tenna chlorophylls of Synechococcus elongatus, Biophys.J.,

74, 2611-2622, doi:10.1016/S0006-3495(98)77967-6.

58. Gobets, B., Kennis, J. T. M., Ihalainen, J. A., Braz-

zoli, M., Croce, R., Stokkum, I. H. M. van, Bassi, R.,

Dekker, J.P., van Amerongen, H., Fleming, G.R., and

van Grondelle, R. (2001) Excitation energy transfer in di-

meric light harvesting complexI: acombined streak-cam-

era/fluorescence upconversion study, J. Phys. Chem. B,

105, 10132-10139, doi:10.1021/jp011901c.

59. Riley, K. J., Reinot, T., Jankowiak, R., Fromme, P., and

Zazubovich, V. (2007) Red antenna states of photosystemI

from cyanobacteria Synechocystis PCC 6803 and Ther-

mosynechococcus elongatus: single-complex spectroscopy

and spectral hole-burning study, J. Phys. Chem.B, 111,

286-292, doi:10.1021/jp062664m.

60. Gisriel, C., Shen, G., Kurashov, V., Ho, M.Y., Zhang, S.,

Williams, D., Golbeck, J.H., Fromme, P., and Bryant,

D.A. (2020) Thestructure of PhotosystemI acclimated

to far-red light illuminates an ecologically important accli-

mation process in photosynthesis, Sci. Adv., 6, eaay6415,

doi:10.1126/sciadv.aay6415.

61. Young, R. M., and Wasielewski, M. R. (2020) Mixed elec-

tronic states in molecular dimers: Connecting singlet fis-

sion, excimer formation, and symmetry-breaking charge

transfer, Acc. Chem. Res., 53, 1957-1968, doi: 10.1021/

acs.accounts.0c00397.

62. Casanova, D. (2018) Theoretical modeling of sin-

glet fission, Chem. Rev., 118, 7164-7207, doi: 10.1021/

acs.chemrev.7b00601.

63. Petrova, A. A., Casazza, A. P., Shelaev, I. V., Gostev,

F. E., Aybush, A. V., Nadtochenko, V. A., Semenov,

A. Y., Santabarbara, S., and Cherepanov, D. A. (2023)

Role of pheophytin a in the primary charge separation of

photosystem I from Acaryochloris marina: femtosecond

optical studies of excitation energy and electron trans-

fer reactions, Biochim. Biophys. Acta Bioenerg., 1864,

148984, doi:10.1016/j.bbabio.2023.148984.

64. Akhtar, P., and Lambrev, P. H. (2020) On the spectral

properties and excitation dynamics of long-wavelength

chlorophylls in higher-plant photosystem I, Biochim.

Biophys. Acta Bioenerg., 1861, 148274, doi: 10.1016/

J.BBABIO.2020.148274.

65. Karapetyan, N. V., Bolychevtseva, Y. V., Yurina, N. P.,

Terekhova, I. V., Shubin, V. V., and Brecht, M. (2014)

Long-wavelength chlorophylls in photosystemI of cyano-

bacteria: origin, localization, and functions, Biochemistry

(Moscow), 79, 213-220, doi:10.1134/S0006297914030067.