Exciton interactions in self-organised bacteriochlorophyll

a - aggregates

J. Linnanto,* J. A. I. Oksanen and J. E. I. Korppi-Tommola

Department of Chemistry, University of Jyvaskyla, P.O. Box 35, FIN-40351, Jyvaskyla, Finland.E-mail: Linnanto@tukki.jyu.fi

Received 24th July 2001, Accepted 28th January 2002First published as an Advance Article on the web 20th May 2002

Exciton interactions of self-organised bacteriochlorophyll a - aggregates in non-polar solution linked via waterand dioxane have been studied. Absorption and CD spectra of the aggregates show large red shifts typical ofJ-aggregates. Femtosecond excitation of the Qy band of the aggregates is followed by wavelength dependentnon-exponential picosecond relaxation and anisotropy decay takes place in subpicosecond time scale. To explainthese observations exciton theory and semi-empirical MO/CI calculations, that constitute the basis of theCIEM-approach developed by Linnanto et al. (J. Phys. Chem. B, 1999, 103B, 8739) was used. Structural modelsof aggregates were created by using the molecular mechanics method. Absorption and CD spectra of the modelstructures were calculated from excitonic wavefunctions. A stable J-type helical structure of BChl a–wateraggregate with a diameter of about 20 nm, in agreement with experiment, was obtained. Calculations for thisstructure produced the experimental absorption and CD spectra of the BChl a–water aggregate correctly. ForBChl a–dioxane aggregates several stable H-type linear structures were calculated and blue shifted absorptionand CD spectra with respect to the monomer Qy transition were predicted. Almost a perfect match of the shapeof the calculated CD spectrum with the experimental spectrum suggests that solvent interaction not included inthe calculations is mostly responsible for the red shift observed experimentally for the dioxane aggregates.The results are discussed with reference to molecular interactions of BChl’s in solution and in light harvestingantenna of photosynthetic bacteria.

Introduction

Aggregates of chlorophylls (Chl) and bacteriochlorophylls(BChl) have been studied extensively in the past as they maybe considered as model systems to aggregates of these chromo-phores in photosynthetic bacteria and plants.1 Several spectro-scopic methods have been used to study such bindingmechanisms.2,3 A common feature of Chl’s and BChl’s innon-polar solutions is self-assembly, that takes place sponta-neously or when small amounts of polar solvent is added insolution. In the latter case aggregation induces large red shiftsof the Qy absorption band and strong CD signals are observedwith concomitant dramatic reduction of the fluorescencequantum yield and shortening of the fluorescence lifetime.Fundamental questions arise: (i) what are the molecular inter-actions that keep the monomers together and determine thestructure of the aggregate; (ii) can we predict spectral proper-ties, if we can describe binding in the aggregates; (iii) wheredoes excitation energy go in the aggregates?Electron microscopy and neutron scattering methods have

revealed tubular structures of Chl and BChl aggregates in solu-tion4,5 with diameters ranging from 5 to 20 nm and lengthsfrom a few hundred up to a few thousand nanometers.1,5,6,7

No crystal structures of self-assembled Chl or BChl aggregatesare available. Structure of crystalline ethyl chlorophyllide a –2�H2O (ref. 8) has been used as a model structure of chloro-phylls in several theoretical studies.Aggregates of Chl a and Chl b are functional chromophores

in photosynthetic complexes of algae and plants.1 Structuraldata on the photosynthetic PSI and PSII complexes9,10 suggestfairly long distances between chromophores and irregular orien-tation of the chromophores in these protein complexes. Weakmolecular interactions between individual chlorophylls and a

particular chlorophyll and its local protein environment deter-mine the spectral properties and excitation energy transfer ratesand direction of these complexes. The protein environment ofPSI includes also carotenoids and a large number of watermole-cules. The structure reveals special water bound chlorophyllaggregates in PSI.10 To understand optical transitions in suchcomplex systems, reliable computational methods are neededfor estimataion of weak local molecular interactions and meth-ods have to be developed that can handle systems containinghundreds of chromophores. Aggregation of Chl a in hydrocar-bon solution has been studied extensively over the years.3,11,12 Amodel structure of chlorophyll–dioxane aggregate with twointernal tetramers have been proposed to explain the experimen-tal absorption and fluorescence polarization spectra.13 For Chla–water aggregate, a tube-like structure consisting of helicalstrings and a hydrophilic interior has been proposed. It was con-cluded that both chormophore–chromophore interaction andexcess water inside the tube contribute to the experimentallyobserved red shift. Trimeric Chl a–water aggregate with struc-ture very similar to that in the helix,13 has now been observedin the crystalline structure of PSI of cyanobacteria.10

Aggregates of BChls (BChl c, d and e) are present in chloro-somes of green bacteria.6,14–16 These bacteriochlorophyll tubu-lar assemblies which contain a large number (10 000 to 20 000)of chromophores, are stacked on top of each other. Tubes arefrom 100 to 200 nm long and have varying diameters from 5to 15 nm depending on the chromophore and the environment.Typical feature of chlorosomes is that they may contain severalhomologues of the same BChl. Chlorosomes show large spec-troscopic shifts (from 60 to 80 nm) of the monomer Qy transi-tion as compared to the monomer absorption at about 660 nm.The shift is very similar to that observed for the Chl a–wateraggregates and BChl a–water aggregates in solution. Reversible

DOI: 10.1039/b106692g Phys. Chem. Chem. Phys., 2002, 4, 3061–3070 3061

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aggregation and similar spectroscopic shifts are observed forpure BChl c17,18 and BChl e19 aggregates in solution.Aggregates of BChl a are much less studied, than those of

Chl a, BChl c and BChl d. This is somewhat surprising sinceBChl a is the functional chromophore in the photosyntheticpurple and green bacteria.20 The dimerization of BChl a in car-bon tetrachloride solution has been studied.21 Aggregates ofBChl a have been claimed to have a low aggregation numberin formamide–water mixture, in Triton X-100, in benzeneand in carbon tetrachloride.22–25 BChl a aggregates with largeaggregation number can be prepared in non-polar solutions byusing a bifunctional interpigment ligand.26 Small-angle neu-tron diffraction results indicate that BChl a–water aggregatesin hydrocarbon solution have tubular structures with a radiusof 10.5 nm and Qy absorption shifted from monomer value of773 nm to 865 nm (Fig. 1).5 The Qy absorption band of BChla–dioxane aggregate is red-shifted to 815 nm with a shoulderat 845 nm (Fig. 1).7,27,28

Aggregates of BChl a perform an important task in purplebacteria, they trap sun’s light for the use of photosynthesis.Aggregates are located in special light harvesting complexeswhere BChl a monomers are organised into ring-shaped struc-tures with characteristic number of monomers in each ring.29

Such aggregates of BChl a are found in the LH2 antenna ofRhodopseudomonas (Rps.) acidophila and Rhodospirillum(Rs.) molischianum and in the LH1 antenna of Rps. viridis.The large spectroscopic shifts of the B850 rings of the LH2and the chromophore ring of the LH1 antenna are very similarto those found for Chl a–water, BChl a–water, BChl c and daggregates in solution. It is likely that the molecular interac-tions responsible of these large shifts are of the same originboth in protein and in solution, strong chromophore–chromo-phore and probably weaker environment–chromophoreinteractions.30 In LH2 complexes aggregates with weakinteractions are present in the B800 ring. In this case local pro-tein environment determines the spectroscopic shift observedexperimentally.31 The situation is very similar in solutions ofmonomeric chromophores. Recently, we have shown that sol-vent binding to Chl’s or BChl’s induces energy level shifts ofthe excited states of the dyes, especially for the states wheremagnesium atom has high electron density.32 The interactionis weak, but strong enough to induce considerable spectro-scopic shifts, especially in the Qx and Soret regions. A spec-trum of a chromophore in solution or in protein may beconsidered as an ensemble of a large number of transitionsenergetically perturbed by local molecular interactions. Suchperturbations give rise the inhomogeneous line width.To understand the relationship between molecular-level

interactions and spectroscopic properties of dye molecules in

various environments quantum chemical methods have beenused extensively. One of the first quantum chemical formalismsfor computation of spectral properties of chlorophyll andbacteriochlorophyll dimers included the classical four-levelmodel.33 Exciton calculations of the absorption spectra ofaggregates of Chl a and BChl a containing from 2 to 10 mono-mers have been reported.34 Since this pioneering work compu-tational possibilities have dramatically improved and quantumchemical methods have been applied to several photosyntheticlight harvesting systems containing BChl a.30,31

In the present paper we have used three different approachesin an effort to try to understand inter molecular interactionsbetween bacteriochlorophyll a molecules in self-assembledaggregates and to predict their absorption and CD spectra insolution. As there exists no crystal structures of BChl a aggre-gates model structures were generated by using molecularmechanics optimisation. Such an approach has been used topredict structures and spectroscopic properties of Chl a –water, Chl a – dioxane and BChl d aggregates.13,35 To calculatespectra of aggregates, the traditional dipole–dipole interactionexciton model (DDEM), configuration interaction excitonmodel (CIEM) and quantum chemical semi-empiricalZINDO/S CI method were applied. The calculations suggestthat molecular interactions in the two aggregates studied theBChl a–water and BChl a–dioxane are very different. In theformer a J-like structure with strong chromophore–chromo-phore interaction was observed. In the latter, minimum energyH-type model structures (also three-dimensional) could notexplain the experimentally observed red shift, suggesting thatthe environmental shift must be the main factor that inducesthis shift.

Experimental

BChl a was dissolved in dry 3-methyl pentane (Fluka Chemica,>99%) to obtain about a 1� 10�4 mol�1 solution. The solventwas dried by passing it through a column filled with activatedaluminium oxide and stored over 4 A molecular sieves. Toavoid light induced oxidation of BChl a 3-methyl pentanewas bubbled with dry nitrogen for 15 min before sample pre-paration and solutions were kept in the dark. Dioxane andwater were added in solutions with a microliter Hamilton syr-inge. Aggregates were formed when BChl a : water or dioxaneratio was from 1/30 to 1/60. Gentle stirring of the solutionshelped aggregate formation.Absorption spectra were recorded by using a Jasco 7800

UV/vis spectrometer and 1 mm path length. A Jasco J-715CD spectrometer was used for CD measurements. For absorp-tion, CD and femtosecond experiments solutions havingabsorbancies between 0.5 to 1.0 were used. For fluorescenceexcitation few tens of milliwatts of CW output at 760 nm froma Coherent MIRA 900 Ti–sapphire laser pumped by a 15 WCoherent Innowa argon ion laser was used. The fluorescencespectrum was recorded with a spectrometer consisting of animaging monochromator (Acton Research Corporation, Spec-tra Pro 300i) and a CCD detector (Princeton Instruments,TEA/CCD-1024-E/1, 256� 1024 pixels). An amplified femto-second Ti–sapphire laser with a pulse duration of about 250 fswas used for single colour transient absorption measurements.The details of the femtosecond spectrometer used are givenelsewhere.36 The kinetic traces were deconvoluted by usingGlobal Analysis software from the University of Illinois,USA. All measurements were performed at room temperature.

Computational methods

The monomer structures of BChl a and dioxane were fullyoptimised at the semi-empirical PM3 (ref. 37) level on a

Fig. 1 Experimental absorption spectra in the Qy region of (a) mono-meric BChl a, lmax ¼ 772 nm; (b) BChl a–dioxane aggregate,lmax ¼ 770 and 815 nm; (c) BChl a–water aggregate, lmax ¼ 780 and867 nm at room temperature. Monomer spectrum was recorded inacetone, while aggregate spectra were recorded in 3-mehtylpentane,where small amounts of dioxane or water was added.

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Silicon Graphics Indigo2 workstation by using SPARTAN38

(Version 5.0) software. The calculated geometry of Bchl awas then used as a starting geometry in the study of differentaggregate structures by using QUANTA (release 3.2) softwareon a Silicon Graphics PERSONAL IRIS 4D/35 TG+ work-station. The standard parameters of QUANTA were usedwith the exception of the bond length between all the nitro-gens and the magnesium atom in the porphyrin ring beingset to 2.086 A. This value was taken from the X-ray structureof ethyl chlorophyllide a dihydrate.8 The aggregate structureswere minimised by using a molecular mechanics methods withthe CHARMm force field and Newton Raphson39 optimisa-tion method. Single point ZINDO/S40–42 semi-empirical CIScalculations for the monomers, dimers, trimers, and tetramerswere performed by using HyperChem43 software running on apentium PC.

The Hamiltonian

Consider a system of N molecules that form an aggregate with-out exciton–phonon coupling with only one relevant molecularelectronic transition from the ground to the excited state andonly one particle-hole excitation. The Hamiltonian is givenin second quantized form as44

HH ¼XNn¼1

enBBþnBBn þ

1

2

XNm6¼nm¼1

Jnm BBþmBBn þ BBþ

n BBm

� �; ð1Þ

where en ¼ Den+Dn+ dn , Den is the electronic excitationenergy of a single molecule n, Dn is so called environmentalshift,45

Dn ¼XNm6¼n

1n0mh jHHnm 1n0mj i �XNm 6¼n

0n0mh jHHnm 0n0mj i ð2Þ

where Hnm contains electron–electron, electron–nuclear andnuclear–nuclear interaction terms between molecules n andm, and the states defined below. dn is the surrounding effect(e.g. solvent, protein, etc.), Jnm is the matrix element of theinteraction operator between the molecules m and n, and theBn operators are Pauli creation (B+), and annihilation (B)operators for excitons at molecule n.46 The eigenstate of theground state of the aggregate |0iagg has the form

0j iagg ¼ 01; 02; . . . ; 0Nj i ¼YNi¼1

0ij i; ð3Þ

and the exciton state |Cfi

Cf

�� �¼

XNn¼1

cfn 1nj iagg; ð4Þ

where the one-exciton state |1niagg has the form

1nj iagg ¼Yn�1

i¼1

0ij i" #

� 1nj i �YN

i¼nþ1

0ij i" #

¼ BBþn 0j iagg: ð5Þ

The matrix element of the interaction operator between themolecules m and n is usually described by using the well-knowndipole–dipole interaction approximation. In this case thematrix element has the form:

Jnm � Jdipnm ¼ 1

4pe� ~mmn �~mmm

R3nm

� 3ð~mmn � ~RRnmÞð~mmm � ~RRnmÞR5

nm

" #ð6Þ

where ~mmn is effective transition dipole moment vector in mole-cule n (ref. 47 and 48)

~mmn ¼eþ 2

3~mm0n ; ð7Þ

and ~mmn0 is transition dipole moment vector for vacuum (e ¼ 1

[e0]), ~RRnm is the position vector between the two dipoles, Rnm isthe distance between these two dipoles and e is the dielectricconstant for the transition frequency.48

In the traditional dipole–dipole interaction exciton model(DDEM) the matrix element Jnm is Jnm

dip [eqn. (6)]. In the con-figuration interaction exciton model (CIEM) the matrix ele-ment of the interaction operator is described by using twodifferent terms depending on the distance between the twointeracting molecules, as indicated by eqn. (8). When the dis-tance Rnm is less or equal than R0 the value of the matrixelement Jnm is estimated by using a quantum chemicalconfiguration interaction method and the supermoleculeapproach. At larger distances dipole–dipole matrix elementsare used. In this work R0 is set to 15 A.

Jnm ¼Jdipnm þ Jextra

nm � JCInm; Rnm � R0

Jdipnm ; Rnm > R0

8<: ð8Þ

Calculation of absorption and CD spectra

For calculation of absorption and CD spectra of model struc-tures of bacteriochlorophyll a–dioxane and bacteriochloro-phyll a–water aggregates, three approaches were adopted. Inthe first the Qy band positions and intensities were calculatedby using the structural parameters of the aggregates from com-puted structures and the dipole–dipole approximation. Exci-tonic energies were computed by diagonalizing the H matrixof eqn. (1). The diagonal elements Hii of H were taken as theS0!S1 transition energies of monomeric Bchl a molecule(773 nm), and off-diagonal elements Hij of H were calculatedby using eqn. (6) and the structural parameters of the aggre-gate. The value of transition dipole vector was taken as 6.13D directed along N(A)–N(C) atoms of BChl a and dielectricconstant was set to e ¼ 1 [e0]. The intensities of the f:th absorp-tion and CD bands were calculated by using eqns. (9) and (10),respectively

m2f ¼XNn;m¼1

~mmnj j~mmmj j mmn � mmm½ cfncfm ð9Þ

Rf ¼ 1:7�10�5XNn;m¼1

nn ~mmnj j~mmmj jRnm RRnm � mmm � mmn½ � �

cfncfm ð10Þ

where ~mmn is effective transition dipole moment vector in mole-cule n, mmn is a unit vector in the direction of the transition, ~RRnm

is the position vector between the two dipoles, Rnm is distancebetween the dipoles, nn is the energy (in cm�1) of the transitionon nth molecule, and cfn is nth element of the eigenvector forthe fth exciton state.49 In the second approach the Qy bandpositions and intensities were calculated by using the samestructural parameters as in the first case, but estimating thenearest neighbour interaction energies with the semi-empiricalZINDO/S CIS method according to eqn. (8). The diagonalelements of the exciton matrix were kept at monomer transi-tion energies. The intensities were calculated by making useof eqn. (9). In the third approach transition energies and oscil-lator strengths (f) of dimers, trimers, and tetramers of eachaggregate were calculated by using the semi-empiricalZINDO/S CIS method. All possible singly excited configura-tions from HOMO-15 to LUMO+15 were included in the cal-culation. Linear scaling of the calculated and experimentalmonomer transition energies was used to make comparisonsof the transition energies between different complexes possible.Theoretical stick spectra of Figs. 7 and 10 show transitionswith f values greater than 0.01.

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Results and discussion

Spectroscopic results

A Gaussian deconvolution of the absorption spectrum of BChla–dioxane aggregate gives absorption bands in the Qy regionat 772, 815 and 845 nm. The first band is assigned to BChl amonomer. The latter two bands are due to BChl a–dioxaneaggregates. This assignment is supported by the appearanceof the CD spectrum of the solution. A non-conservative spec-trum is observed with almost no rotational strength at 772 nm,a negative peak at 817 nm and strong positive peaks at 830 and850 nm (Fig. 2). Excitation at 760 nm results in a strong emis-sion at 775 nm due to the monomer and a much weaker andbroader emission around 850 nm (Fig. 3). The weak emissionis observed also with 778 and 798 nm excitations, while excita-tion at 810 to 860 nm gives no detectable fluorescence. Arough estimation of the quantum yield of the weak emissionband as compared to the monomer emission band suggeststhat the lowest emitting state of the aggregate decays in about200 ps.Single colour femtosecond transient absorption measure-

ments give three isotropic decay components of BChl a–diox-ane aggregate 1 ps, 10 ps and a long residual component ofabout 250 ps (Fig. 4a) at 815 and at 836 nm. The fastest com-

ponent has an amplitude in the range of from 70 to 95% whilethe 10 ps component has an amplitude from 5 to 25% depend-ing on the excitation wavelength and time window of the mea-surement (Fig. 4b). The amplitude of the longest componentincreases from 5% to about 10% as excitation wavelengthchanges from 815 to 836 nm (Fig. 4a). The anisotropy decaytime at 815 nm is very fast, roughly 500 fs (Fig. 4b).We assign the fast 1 ps and 10 ps components to relaxation

processes from higher excitonic states to the lowest emittingexcited state of the aggregate. The fast decay times most prob-ably also contain a contribution from ultrafast excitationenergy flow towards geometrical defects in the aggregate thatmay serve as local energy traps. The longest resolved >250ps component corresponds to the emission lifetime of theaggregate, in good qualitative agreement with the estimate ofthe lifetime from quantum yield calculation. As further sup-port for this assignment is the increase of the amplitude of longlifetime component as excitation and probe wavelengths shifttowards red. The very fast anisotropy decay about 500 fs isassigned to loss of anisotropy due to exciton relaxation andprobable simultaneous excited state absorption.

Model structures of BChl a–dioxane aggregates

The monomer structures of BChl a and dioxane were opti-mised by using the semi-empirical PM3 method. In the mini-mum energy conformation in vacuum the phytyl tail wastwisted above the porphyrin plane.50 The PM3 minimised geo-metry of BChl a (except that the magnesium atom was centred,see computational methods) was used as a starting geometry inthe molecular mechanics optimisations. Several dimer struc-tures, including efforts to build J-type dimers, were tried.Finally, three energetically most favourable structures of BChla–dioxane dimers (Fig. 5) were used in further calculations. Inall these dimers the dioxane molecule binds directly to themagnesium atoms of the adjacent BChl’s. This agrees withthe experimental finding that Chl a–dioxane and Chl b–diox-ane form colloidal aggregates but pheophytin–dioxane inhydrocarbon solution does not.11 Also, infrared spectroscopicresults suggest that the dioxane molecule binds directly to themagnesium atom of the porphyrin in Chl a–dioxane com-plexes. Table 1 lists Mg–Mg distances, dihedral and twistangles, and binding energies of these dimers.In the lowest energy dimer, which we call a sandwich dimer,

the phytyl chains of the BChl’s are oriented parallel in thespace between the stacked porphyrin rings (Fig. 5c). The dis-tance between the oxygen atoms of dioxane and Mg atomsof BChl’s is approximately 2.17 A and the distance betweenMg–Mg atoms in the dimer is about 7.13 A. In the sandwichdimer the Qy transition dipole vectors of monomeric bacterio-chlorophyll a molecules, directed along N(A)–N(C) atoms ofthe porphyrin, are almost parallel. If the phytyl tails of thesandwich dimer are straightened out, energy increases by afew kJ’s, but the basic geometry of the dimer remains. In realhydrocarbon solution the phytyl tails are most probablyoriented towards the solvent. In the next stable dimer, whichwe call an open dimer, the phytyl tails of monomers areoriented in opposite directions (Fig. 5a). In this dimer the por-phyrin rings are not necessarily parallel, and the tilt finallydetermines the three-dimensional structure of larger aggre-gates. The distance between Mg–Mg atoms in the open dimeris about 6.78 A and the Qy transition dipole vectors of the twoBChl a molecules form an angle about 80 degrees with respectto each other. In the third dimer structure, which we call anarray dimer, the phytyl tails of monomers are almost paralleland they point towards the solvent (Fig. 5b).To build larger aggregates, the sandwich dimer (Fig. 5c),

where the phytyl tails were forced out of the space betweenthe porphyrin planes, was used as a building block. In largeraggregates the dioxane molecule binds two adjacent sandwich

Fig. 2 Absorption and CD spectra of BChl a–dioxane aggregate.Observe the weak CD signal around the monomer absorption.

Fig. 3 Fluorescence spectrum of BChl a–dioxane aggregate. 760 nmCW output from a Ti–sapphire laser was used for excitation.

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dimers together, forming an angle (angle y defined in Fig. 5) ofabout 170 between the sandwich dimers. The one-dimensionalaggregate model consists of two repeating dimers—one with astructure shown in Fig. 5c (sandwich) and the other with astructure shown in Fig. 5a (open). Fine-tuning of the anglesy and f of the dimers in the aggregate structure produced dif-ferent overall structures of the aggregates, the shape varyingfrom a stick to a ring. Similar behaviour was obtained forChl a–dioxane aggregates.13 According to calculations thestick BChl a–dioxane aggregate is energetically most favour-able. It is quite possible that such stick-structures may leadto formation of needle-like crystals, which have been observedfor Chl a and Chl b crystallised from dioxane.4 In the stickaggregate porphyrin rings are almost parallel, the rings ofsandwich and open dimers are tilted at the most about 5–10

and 10–20 (angle f), respectively. The distance betweenMg–Mg atoms of sandwich and open dimers of the aggregaterange between 6.97–7.14 A and 6.89–7.05 A, respectively. Athree-dimensional model structure shown in Fig. 6 containsfive one-dimensional BChl a–dioxane stick aggregates. This

cluster (120 BChl a’s) has an energy minimum and it can beeasily extended along the long axis and also further one-dimen-sional stacks may be added to the structure. We believe that itis a good model for the description of formation of needle crys-tals observed experimentally for Chl a and Chl b. It is to benoted that J-type aggregation is present in this 3D structure,but this interaction is much weaker than that between theBChl’s in the stack and hence it can not account for the experi-mentally observed red shift.

Simulation of spectra of BChl a–dioxane aggregates

In hydrocarbon solution the characteristic monomer Qy

absorption band of BChl a appears at 772 nm (Fig. 1a). Add-ing dioxane in solution creates a BChl a–dioxane adduct thatshows the monomer band at 772 and two new red-shiftedbands at 817 and 845 nm (Fig. 1b), the red shifts being 730and 1136 cm�1, respectively.28 The experimentally observedshifts may be compared to the computed spectra. For the

Fig. 4 (a) Magic angle single colour decay curves of the BChl a–dioxane aggregate with 815 and 836 nm excitations; (b) anisotropy decay withpump and probe wavelengths at 816 nm.

Fig. 5 Model structures for BChl a–dioxane dimers: (a) open dimer;(b) array dimer; (c) sandwich dimer. For structural details see Table 1and text.

Fig. 6 Three dimensional model structure for BChl a–dioxane aggre-gate as predicted by molecular mechanics calculations. The simulationcontained five H-type stacks, each containing 24 BChl a’s and 23 diox-anes. Single stacks are shown on the left and right hand sides.

Table 1 Geometrical parameters and bonding energies of BChl a–

dioxane dimers. For structures of the dimers see Fig. 5

Structure Mg–Mg distance R/A f/ y/ Binding energy/kJ mol�1

A 6.8 0 90 71.8

B 6.9 50 90 52.1

C 7.1 0 180 106.3

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one-dimensional (BChl a–dioxane)n stick structure the excitoncalculations (Table 2 and 3) give the primary absorption bandsof the Qy transition at 750, 748, 746, 746, 746, 745, 744, 750,746, and 744 nm within the DDEM model and at 779, 747,747, 748, 748, 746, 746, 746, 746, and 745 nm with the CIEMmodel for n ¼ 2, 3, 4, 5, 6, 7, 8, 9, 10, and 36, respectively.Very little oscillator strength resides on other transitions.Neither approach gives the expected red shifts. However, theshape of the calculated CD spectrum matches (the CIEMmodel) the experimental spectrum (Fig. 2) almost perfectly.The spectrum (not shown) has to be shifted by 800 cm�1 tomatch the experimental CD spectrum with maxima at817(�), 830(+) and 850(+) nm. We think that the discrepancybetween the calculated and the experimental spectra arisespartly from the environmental shift [egn. (2)] of the BChl a–dioxane aggregate. The shift terms were not included in thecalculations and they are difficult to estimate theoretically.

The discrepancy between the calculated and experimental spec-tra may also partly arise from special interaction of dioxanewith monomeric BChl a in the aggregate that was not includedin the calculation, as Qy transition energy of monomeric BChla in the diagonal of the exciton matrix was used. BChl a–diox-ane interaction may be included in the calculation by takingthe diagonal element as a ‘site ’ monomer Qy transition energyof BChl a, where two dioxane molecules bind to the magne-sium atom of BChl a. Fig. 7a gives a ‘ site ’ energy of BChla–dioxane2 at 783 nm. Such value gives slightly better agree-ment of the calculated and observed spectrum but does notexplain the discrepancy fully. The missing red shift must comefrom the fact that the aggregates have three-dimensional struc-tures in real solution. Extension of the structure in threedimensions (Fig. 6) brings in J-type interactions between BChla’s of adjacent stacks. However, the distances between thenearest neighbour BChl a’s remain quite large because of sterichindrances. For this reason J-type couplings are weak andinduced red shifts are small. However, the three-dimensionalstructure can have an effect on the diagonal elements of theexciton matrix, i.e. both in the environmental [eqn. (2)] andin the surrounding [eqn. (1)] shifts. The simulated spectrumof the aggregate of Fig. 6 is broadened, but remains blueshifted with main oscillator strength from 730 to 765 nm anda weak doublet located at 790 and 820 nm. We have simulatedseveral different one-dimensional structures of BChl a–diox-ane, Chl a–dioxane13 and Chl a–pyrazine aggregates,51 andin all these cases the calculated Qy absorption band positionsbecome blue-shifted in contradiction to experimental findings.Obviously, further calculations of these weakly bound three-dimensional aggregates are needed.At this stage we point out that self-assembly of chlorophyll

and bacterioclorophyll aggregates depends strongly on the sol-vent in which they are formed. By choosing the right solventmonomeric, dimeric or larger aggregates may be created.3

For large aggregates solvent and molecular properties of thelinker molecules determine the physical structure of the aggre-gate. Similar interactions must be functional in photosyntheticprotein complexes, with the difference that individual proteinsites determine the degree of aggregation and arrangement ofchromophores in the protein. It may turn out that environ-mental and surrounding shifts become important, when spec-tra of large chlorophyll assemblies of PSI and PSII aresimulated.

Table 2 Wavelengths and relative oscillator strengths of Qy bands of

(BChl a–dioxane)n aggregates (n ¼ 2, ..., 10) in vacuum using the

DDEM method

l/nm

Fa

Dimer 750 798

1.00 0.08

Trimer 748 773 800

1.00 0.41 0.02

Tetramer 746 755 792 802

1.00 0.67 0.03 0.02

Pentamer 746 754 773 794 802

1.00 0.80 0.38 0.01 0.02

Hexamer 746 749 755 790 799 803

1.00 0.67 0.44 0.04 0.02 0.01

Heptamer 745 747 755 773 792 800 804

1.00 0.20 0.45 0.13 0.03 0.00 0.02

Octamer 744 746 753 758 788 794 801 805

1.00 0.05 0.75 0.04 0.03 0.01 0.01 0.01

Nonamer 744 746 750 756 773 790 796 802 806

0.98 0.18 1.00 0.14 0.15 0.03 0.00 0.01 0.01

Decamer 743 746 747 754 760 786 792 800 803 807

0.41 1.00 0.41 0.48 0.09 0.02 0.01 0.01 0.01 0.01

Table 3 Wavelengths and relative oscillator strengths of Qy bands of

(BChl a–dioxane)n aggregates (n ¼ 2, ..., 10) in vacuum using the

CIEM method

l/nm

Dimer 779 788

1.00 0.08

Trimer 747 784 824

1.00 0.54 0.17

Tetramer 747 783 785 824

1.00 0.72 0.29 0.12

Pentamer 746 748 784 822 826

0.86 1.00 0.54 0.37 0.25

Hexamer 746 748 783 784 822 826

0.78 1.00 0.48 0.58 0.28 0.19

Heptamer 746 747 749 784 820 824 828

1.00 0.33 0.20 0.29 0.12 0.19 0.10

Octamer 746 747 749 784 784 820 824 828

1.00 0.25 0.22 0.37 0.17 0.12 0.16 0.08

Nonamer 746 747 747 750 784 820 823 825 828

1.00 0.28 0.87 0.07 0.31 0.06 0.29 0.18 0.05

Decamer 746 747 747 750 784 784 820 823 825 828

1.00 0.36 0.92 0.08 0.40 0.26 0.07 0.28 0.17 0.05

Fig. 7 (a) Calculated stick spectra of BChl a–dioxane aggregateobtained by the ZINDO/S method, from top to bottom: monomerBChl a–dioxane2 , lmax ¼ 783 nm, sandwich dimer and open dimer;(b) from top to bottom: trimer, lsl-tetramer and sls-tetramer. For dis-cussion of spectra see text.

3066 Phys. Chem. Chem. Phys., 2002, 4, 3061–3070

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In the third approach transition energies of one-dimensionalmodel structures were calculated by using the semi-empiricalZINDO/S method. The estimated absorption spectra ofmonomer, dimers, trimer, and tetramers are shown in Figs.7a and 7b. The estimated Qy band position of calculated BChla–dioxane2 monomer (Fig. 7a, top spectrum) is at 783 nm. TheQy band positions of BChl a–dioxane dimer are at 755 and 789nm for the sandwich structure (Fig. 7a, middle spectrum), at728 and 824 nm for the open dimer (Fig. 7a, bottom spec-trum), a strong doublet at 726 and 776 nm and weaker bandat 835 nm for the trimer (Fig. 7b, top spectrum), a strongdoublet at 720 and 735 nm and weaker doublet at 820 and840 nm for the sls-tetramer (the letters sls refer qualitativelyto two different magnesium–magnesium distances in the tetra-mer, l ¼ longer, s ¼ shorter distance, Fig. 7b, bottom spec-trum), a strong doublet at 728 and 765 nm and two weakerbands at 782 and 836 nm for the lsl-tetramer (Fig. 7b, middlespectrum). The weaker doublets predicted for both tetramersare red shifted with respect to the monomer Qy band positionand their wavelengths are near the experimental values of 815and 845 nm. Yet the calculated oscillator strengths of thesetransitions are small as compared to the strongest doubletspredicted at 720 and 776 nm. The fact that we get two doubletsfor the two different tetramers present periodically in themodel structures is in accord with the appearance of thenon-conservative CD spectrum that may be an indication oftwo species present in solution.It is clear that all of the three approaches used to calculate

the spectrum of a one-dimensional BChl a–dioxane aggregatefailed to produce the experimental shift, the quantum chemicalZINDO/S CIS method produced the best results. The mainreason for discrepancy lies most probably in inadequacy ofone-dimensional structure to describe the real aggregate andthat the environmental and surrounding shifts were not ade-quately accounted for in the BChl a–dioxane simulation.

Structure of BChl a–water aggregate

The simulation of the structure of BChl a–water aggregate wasstarted by using a building unit, where only one water mole-cule binds with two bacteriochlorophyll a molecules. The basicdimer model structure was taken from the crystal structure of

ethyl chlorophyllide a dihydrate.8 Construction of a largeraggregate by using dimers as building blocks resulted in one-dimensional structure of BChl a–water aggregate, where thewater molecule binds to the central Mg atom (5 co-ordination)of one BChl a and to the keto oxygen of the next BChl a (oxogroup at position C-131) (Fig. 8). The Mg–O (water) distancewas about 2.18 A and the O (keto)–H (water) distance wasabout 2.26 A. The nearest neighbour magnesium–magnesiumdistances in the aggregate vary from 8.7 to 9.1 A. Perpendicu-lar distance between the porphyrin planes is about 3.2 A.Increasing the aggregate size, the one-dimensional structureturns into a helix having a diameter of 22 nm, a rise of 75nm per full turn (Fig. 9) and about 120 BChl a monomers ina single turn. The calculated helix diameter is very close tothe experimental value of 21 nm reported for tube-like BChla–water micelles in hydrocarbon solution.5 In the helical struc-ture, the angle between the Qy transition dipole vectors ofnearest neighbour BChl a’s is about 5. The helical string struc-ture may be used to build tube-like aggregates assumed to bepresent in solution.5 We estimate that 24 helixes are neededto form a tube, where oxygen atoms of BChl a’s point towardsthe interior of the tube. Such a hydrophilic interior is capableof storing excess water experimentally needed for formation ofthe aggregate.

Simulation of spectra of BChl a–water aggregates

As shown in Fig. 10 (top spectrum), binding of water to themagnesium of BChl a shifts the Qy transition only by 2 nm.In case of dioxane the shift was five times larger. It is thenexpected that for BChl a water–aggregate solvent effects arenot that important as in the dioxane case. The formation ofBChl a–water adduct in hydrocarbon solution is indicated bya strongly red-shifted Qy absorption band at 867 nm at roomtemperature (Fig. 1c). The total red shift from the Qy mono-mer absorption is about 1436 cm�1. For the one-dimensional(Bchl a–H2O)n helical structure our calculations (Tables 4and 5) give the primary absorption bands of the Qy transitionsat 796, 808, 817, 820, 823, 824, 826, 827, 828, and 832 nm withdipole–dipole interaction and at 807, 824, 834, 839, 843, 846,848, 849, 850, and 856 nm by using the CIEM model forn ¼ 2, 3, 4, 5, 6, 7, 8, 9, 10, and 40, respectively. In the CIEM

Fig. 8 A close-up of the structure of the BChl a–water aggregate. Observe co-ordination of (1) water oxygen to the central magnesium atom, (2)water hydrogen to the Ring E keto carbonyl. For structural parameters see text.

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case the nearest neighbour interaction energy of JnmCI ¼ 550

cm�1, obtained from ZINDO/S CIS (15,15) calculations,was used.The corresponding values of Shipman et al.34 are 848, 871,

883, 889, 894, 897, 899, 900, 901, and 908 nm for n ¼ 2, 3,4, 5, 6, 7, 8, 9, 10, and 1, respectively, and those of Strouse52

796, 809, 817, 822, 825, 827, and 836 nm for n ¼ 2, 3, 4, 5, 6, 7,and 1, respectively. In calculations of Strouse monomer Qy

transition energy of 770 nm and transition dipole strength of4.87 D were used. Shipman et al. introduced an environmentalshift in their calculations; they used Hii ¼ 12 788 cm�1 (782nm) for i ¼ 1 and Hii ¼ 11 954 cm�1 (837 nm) for i� 2 andtransition dipole strength of 6.05 D, which explains the ‘over-shooting’ results. In the present calculations transition dipolestrength was 6.13 D (for vacuum), e ¼ 1.0 [e0], and the mono-mer Qy transition energy was set to 773 nm as the solvent shiftwas small.Within the dipole–dipole approximation our values are simi-

lar to those of Strouse, but less red-shifted than those of Ship-man et al.. The one-dimensional model structure of the (BChla–water)40 aggregate gives a shift of 951 cm�1 of the Qy band.This is only two-thirds of the experimentally observed shift of1436 cm�1. Using the CIEM model gives clearly morered-shifted value of 1288 cm�1 of the Qy band for the (BChl

a–water)40 aggregate, already close to the experimental shift.The main result from the two simulations is that at close dis-tances dipole–dipole approximation clearly fails to describethe chromophore–chromophore interaction of BChl a’s in theaggregate. The CIEM approach takes into account quantum

Fig. 9 Overall structure of (BChl a–water)96 aggregate. The phytil tails are oriented on one side of the helix. The helix has a diameter of 22 nmand a rise of 75 nm per turn. Twenty-four such helixes can form a tube with hydrophobic interior.

Fig. 10 Calculated stick spectra of BChl a–water aggregates obtainedby ZINDO/S CIS (16,16) method, from top to bottom. (a) BChl a–water, lmax ¼ 774 nm; (b) dimer, lmax ¼ 807 nm; (c) trimer,lmax ¼ 838 nm; (d) tetramer, lmax ¼ 861 nm.

Table 4 Wavelengths and relative oscillator strengths of Qy bands of

(BChl a–water)n aggregates (n ¼ 2, ..., 10) in vacuum using the DDEM

method

l/nm

Dimer 747 796

0.00 1.00

Trimer 740 768 808

0.02 0.00 1.00

Tetramer 735 753 784 817

0.00 0.04 0.00 1.00

Pentamer 734 748 767 792 820

0.00 0.00 0.07 0.00 1.00

Hexamer 733 743 756 779 800 823

0.00 0.01 0.00 0.07 0.01 1.00

Heptamer 732 739 751 767 786 806 824

0.00 0.00 0.01 0.00 0.06 0.01 1.00

Octamer 732 736 747 757 777 792 812 826

0.00 0.00 0.00 0.02 0.00 0.05 0.00 1.00

Nonamer 732 735 745 754 767 781 796 814 827

0.00 0.00 0.00 0.00 0.03 0.00 0.06 0.00 1.00

Decamer 731 735 742 749 758 775 788 801 816 828

0.00 0.00 0.00 0.00 0.00 0.02 0.00 0.08 0.00 1.00

Table 5 Wavelengths and relative oscillator strengths of Qy bands of

(BChl a–water)n aggregates (n ¼ 2, ..., 10) in vacuum using CIEM

l/nm

Dimer 741 807

0.00 1.00

Trimer 730 770 824

0.03 0.00 1.00

Tetramer 725 751 790 834

0.00 0.05 0.00 1.00

Pentamer 723 741 769 804 839

0.00 0.00 0.06 0.00 1.00

Hexamer 721 734 756 784 815 843

0.00 0.01 0.00 0.07 0.00 1.00

Heptamer 720 730 747 769 795 822 846

0.00 0.00 0.01 0.00 0.07 0.00 1.00

Octamer 719 727 740 758 780 804 828 848

0.00 0.00 0.00 0.02 0.00 0.07 0.00 1.00

Nonamer 719 725 736 751 769 789 811 832 849

0.00 0.00 0.00 0.00 0.02 0.00 0.08 0.00 1.00

Decamer 718 724 733 745 760 778 797 817 835 850

0.00 0.00 0.00 0.01 0.00 0.02 0.00 0.08 0.01 1.00

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chemical interactions between the nearest neighbour BChl a’sand the difference between our calculated and the experimentalshift is only 148 cm�1. In one dimensional water aggregateBChl a–BChl a interaction seems to determine the spectralshift and the importance of the three-dimensional structureand environmental shift seem to be smaller than in the caseof BChl a–dioxane aggregates.The stick spectra of BChl a–water monomer and three small

BChl a–water aggregates, dimer, trimer and tetramer, using theZINDO/S CIS (15,25) method are shown in Fig. 10. The esti-mated Qy band position of BChl a–water monomer is at 773.5nm and the corresponding spectrum is shown in Fig. 10a. Themajor Qy band components of the dimer (Fig. 10b), trimer(Fig. 10c), and tetramer (Fig. 10d) are red-shifted by 575,1041, and 1350 cm�1, respectively, as compared to the mono-mer band. This is a clear demonstration of orbital overlapeffects between the chromophores at close distances. The pre-dicted shift for the tetramer is very close to the experimentallyobserved shift. Such strong interactions are present also in theB850 ring of LH2 and in the B875 ring of LH1 light harvestingantenna of purple bacteria where they determine the positionof the B850 and B875 bands in the spectrum. Recently we havebeen able to simulate experimental absorption and CD spectraof these complexes correctly by using the CIEM approach.31

As the quantum chemical computations become insurmounta-ble for aggregates larger than tetramers of Chl’s and BChl’s itis obvious that the CIEM method is a lucrative option to beused for the study of aggregates of larger size in various envir-onments, in solution or in protein.

Conclusions

The purpose of the present work was to study spectroscopicproperties (including transient behaviour) of aggregated bac-teriochlorophylls in non-polar solution and to simulate therecorded spectra. In the simulations molecular mechanicsmethod was used to create model stuctures. By using modelstructures, absorption and CD spectra of the aggregates werecalculated with three different approaches: (1) exciton modelbased on dipole–dipole interaction (DDEM); (2) excitonmodel based on nearest neighbour interaction energiesobtained from semi-empirical calculations (CIEM); (3) semi-empirical ZINDO/S CI calculations up to tetramers.The structure calculations suggest linear H-type binding for

the BChl a–dioxane aggregate with periodic repetition of twointernal tetramers. The result is in agreement with experimen-tal findings of needle-like structures for dioxane chlorophylland bacteriochlorophyll aggregates.4 Both DDEM and CIEMapproaches predicted blue shifted Qy transition energies of theaggregates contradicting the experimental findings. The shapeof the calculated CD spectrum dioxane aggregate was approxi-mately the same as the experimental band profile and a shift ofsome 800 cm�1 of the calculated spectrum produced almostperfect match of the two spectra. The three-dimensional modelstructure containing also J-interactions was not able to explainthe experimental shift either. It is concluded that in the BChla–dioxane system a considerable environmental shift is pre-sent, not accounted for in the calculations, which probablybecomes of importance in the three dimensions. For the one-dimensional BChl a–water aggregate, a helical J-type structurewas predicted. The calculated diameter of the helical stringaggregate of 22 nm is very close to the experimentally deter-mined value of 21 nm8 of the real micelles in solution. Thedipole–dipole approximation of interaction failed in predictingthe experimentally observed spectrum also in this aggregate.The CIEM method predicted a shift of 1288 cm�1 not far fromthe experimental shift of 1436 cm�1. Even better agreementwith the experiment was obtained when the spectroscopic Qy

transition energies were obtained from ZINOD/S CIS

(15,15) calculation by considering a Bchl a–water tetramer asa single supermolecule. It is concluded then that quantum che-mical interactions become important at close distancesbetween large interacting chromophores like Chl’s and BChl’s.

Acknowledgement

The authors acknowledge the financial support from the Acad-emy of Finland (Contracts No 34192 and 44546) and comput-ing resources from the Finnish National Centre of ScientificComputing (CSC), Espoo, Finland.

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