Fluorescence and Absorption Detected Magnetic Resonance of Chlorosomes from Green Bacteria...

Fluorescence and Absorption Detected Magnetic Resonance of Chlorosomes from GreenBacteria Chlorobium tepidumand Chloroflexus aurantiacus. A Comparative Study†

Donatella Carbonera,* Enrica Bordignon, and Giovanni GiacomettiDepartment of Physical Chemistry, UniVersity of PadoVa, Via Loredan 2, 35131 PadoVa, Italy

Giancarlo AgostiniCentro Studi Stati Molecolari Radicalici ed Eccitati CNR,Via Loredan 2, 35131 PadoVa, Italy

Alberto Vianelli and Candida VanniniDepartment of Structural and Functional Biology, UniVersity of Insubria,Via J.H. Dunant 3,21100 Varese, Italy

ReceiVed: May 12, 2000; In Final Form: October 4, 2000

A comparative study on the isolated chlorosomes fromChloroflexus aurantiacus, a green filamentousphotosynthetic bacterium andChlorobium tepidum, a green sulfur photosynthetic bacterium, was done byODMR (optically detected magnetic resonance). Correlation between the results obtained by fluorescenceand absorption detection is shown to be a source of information about the functional interactions amongpigments. Analogies and differences are pointed out between the light-harvesting systems of the two species.Triplet states are easily detected in both bacteria at 1.8 K under steady-state illumination and are assigned toBChl c, BChl a, and carotenoid molecules. Carotenoids are found to be able to quench BChla triplet states,but no evidence of BChlc triplet states quenching by this triplet-triplet transfer mechanism is found in bothsystems. Then from the data it appears that some carotenoids are in close contact with BChla molecules.The relevance of this finding to the localization of carotenoids in the chlorosomes is discussed. InCb. tepidumthree different pools of BChlc oligomers connected to BChla were found by detection of their triplet state,while only one pool of BChlc was evidenced inCf. aurantiacus. The latter appears to be unconnected, atleast at 1.8 K, to BChla. On the other hand, heterogeneity in the BChla triplet population was detected inCf. aurantiacus. Even though the two bacteria show common features in the way the light excitation inducestriplet formation at low temperature, the detected triplet states show spectroscopic properties that stronglydepend on the system. The results clearly indicate that differences in pigment organization exist both in thecore and in the baseplate of the chlorosomes from the two different bacteria.

Introduction

From a phylogenetic point of view the two families of greenbacteriaChlorobiaceaeandChloroflexaceaeare considered twoseparate phyla.1 However, unexpectedly, they contain verysimilar light harvesting entities, the so-called chlorosomes. Thecomparative study on the structure-function relationships ofthe isolated chlorosomes ofChloroflexus aurantiacusandChlorobium tepidum, as representative of the two families, isthen of some interest.

Chlorosomes are organelles consisting of a core and of anenvelope. The core contains preponderantly bacteriochlorophyll(BChl) c, d, or e, depending on the bacterium, assembled intorodlike elements2-5 as oligomers. The pigment-pigment inter-actions are important in determining the supramolecular orga-nization in the rods.6,7 Proteins are present in the lipid envelope,

which is formed by a monolayer of galacto-lipids, but possiblynot in the rod elements.5 A baseplate in the envelope connectsthe chlorosomes to the cytoplasmic membrane. Chlorosomesfrom green sulfur bacteria are found to be larger compared tothe chlorosomes from green filamentous bacteria.5

A small number of BChla molecules (up to 1% inCb.tepidumand up to 5% inCf. aurantiacuswith respect to thetotal amount of BChlc) are complexed with proteins8 and likelylocated in the baseplate9,10 assembly, although the finding thatBChl a is correlated to the CsmA protein raises some doubtsabout the location of BChla in the baseplate.8 BChl a is alsofound in a membrane-bound antenna complex (B808-866) ingreen filamentous bacteria and in the well-known FMO-proteinin green sulfur bacteria. These latter two antenna complexesare thought to mediate the energy transfer from the chlorosomesto the reaction centers.5 The main structural difference thereforeis found in the contact region between the chlorosomes and thecytoplasmic membrane. InCf. aurantiacusthe baseplate is indirect contact with the membrane, while inCb. tepidumtheFMO-protein is assumed to mechanically couple the chlorosomeand the membrane.

Carotenoids also contribute to the whole pigment compositionof chlorosomes. They represent 30% of pigment content inCf.

† Abbreviations: ODMR, optically detected magnetic resonance; ZF-FDMR, zero field fluorescence detected magnetic resonance; ZF-ADMR,zero field absorption detected magnetic resonance; MIA, microwavesinduced absorption; T-S, triplet-minus-singlet; ZFS, zero field splitting;BChl, bacteriochlorophyll;Cb., Chlorobium; Cf., Chloroflexus; RC, reactioncenter: ISC, Inter System Crossing.

* Corresponding author. Fax:+39(49)8275135. E-mail: [email protected].

246 J. Phys. Chem. B2001,105,246-255

10.1021/jp001778+ CCC: $20.00 © 2001 American Chemical SocietyPublished on Web 11/30/2000

aurantiacusand 10% inCb. tepidum. Chlorobactene is theprevailing species ofCb. tepidum, followed by 1′-2′-dihydro-chlorobactene whileâ-carotene andγ-carotene are mainly foundin Cf. aurantiacus. A 20% of the total carotenoid content inCf. aurantiacusis represented by glucoside and OH- caro-tenoids, while only a few percent of this B-type carotenoidsare found inCb. tepidum. The localization of carotenoids inthe chlorosomes is still uncertain. The role of carotenoids inchlorosomes is probably of the same kind as that alreadyrecognized in other photosynthetic antenna systems, where theyserve as light-harvesting pigments and as photoprotectors fromsinglet oxygen, although this latter function should not be sorelevant in anaerobic organisms. Quinones are also found inchlorosomes and seem to play an important role in affectingthe fluorescence and the energy transfer under oxic conditionsin green sulfur bacteria11 while their role in green filamentousbacteria is not clear.12

The organization of pigments in the chlorosomes has beenunder discussion during the last 10 years and is still controver-sial. Due to molecular aggregation, excitonic states seem todominate the absorption characteristic of BChlc molecules inthe chlorosomes; however, the presence of different BChlcpools has been suggested by several authors.10,13Analogies anddifferences between the two families of green bacteria have alsobeen pointed out.5,14

We use both ZF-FDMR and ZF-ADMR to detect tripletformation at low temperature, under broad band photoexcitation,in isolated chlorosomes fromCb. tepidum and from Cf.aurantiacuswith the aim of throwing light on energy transferprocesses and pigment interactions in these two species of greenbacteria. Previous work on other photosynthetic systems hasshown that these techniques are helpful for structure and funcionanalyses on both antenna and reaction center complexes.

FDMR of whole cells and isolated chlorosomes ofCb.phaeobacteroidesandCb. limicola has been already reportedby Psencik et al.15 Indeed, several triplet species have beendetected and assigned to BChla, carotenoids, and BChle (orc) molecules of the chlorosomes. In this study we performODMR for the first time in the isolated chlorosomes of a greenfilamentous bacterium and extend the previous work on thechlorosomes of green sulfur bacteria. FDMR is done bydetecting the fluorescence changes selectively on the BChlcand BChl a emission bands at different wavelengths, uponmodulation of triplet population by resonant amplitude modu-lated microwaves. In this way, energy transfer pathways canbe proven and the presence of different pigment pools can alsobe investigated.

Microwave-induced absorption gives the triplet minus singlet(T-S) spectra of the different triplet states observed by FDMRand allows the assignment to specific pigment in the chlorosomeabsorption spectrum. Since ADMR monitors the bulk populationdirectly, it is possible to evaluate the relative triplet yield ofdifferent pigments in a more reliable way as compared to theFDMR. ADMR in isolated chlorosomes from the two familiesof green bacteria is here used for the first time and the resultsare compared.

Materials and Methods

Chlorosomes fractions ofCb. tepidumwere prepared accord-ing to the procedure of Gerola et al.16 (omitting NaSCN) to afinal concentration of 1 mg BChl/mL. The absorption spectrumwas the same as those reported in the literature for the samekind of preparation. The samples were poised at pH 7.4 withpotassium phosphate buffer (10 mM) containing 25% (w/w)

sucrose and diluted to 200µg BChl/mL before the addition of20 mM dithionite under nitrogen. This reduction procedureimproves significantly the signal-to-noise ratio of the tripletspectra, as expected on the basis of the redox potential controlof fluorescence and energy transfer yields already reported.11,17

Cells ofCf. aurantiacus, strain J-10-fl, were grown at 55°C,under anaerobic conditions and under illumination (9µE m-2

s-1). Chlorosomes were prepared using NaSCN throughout thepurification as described by Gerola and Olson,16 except that Tris10 mM pH 8 buffer18 was used. The samples were poised atpH 7.8 with Tris buffer (10 mM) to a final concentration of200µg of BChl/mL. The absorption spectrum was the same asthose previously reported for the same kind of preparation.19

Glycerol was added at 66% vol/vol just before freezing ofthe samples to avoid matrix cracking, and oxygen was removedusing a glucose/glucose oxidase system as described.20

The home-built FDMR apparatus was previously described.21

The detection wavelength was selected by cutoff and band-passfilters in the range 770-850 nm (bandwidths 10 nm). TheADMR apparatus, together with the experimental setup for thedetection of triplet minus singlet (T-S) spectra, was alsopreviously described.22 Different modulation frequencies of theamplitude modulated microwaves have been used dependingon the triplet lifetime: 325 Hz for carotenoids and BChla tripletstates and 33 Hz for BChlc triplet states.

Results

The absorption spectra of isolated chlorosomes are dominatedby the bands of BChlc in oligomeric form. At room temperaturethe Qy band is found at 753 nm inCb. tepidum, while in Cf.aurantiacusit is found at 741 nm, both red-shifted with respectto the monomer absorption (670 nm). The BChla molecules,which are present in a small amount, absorb in the longwavelength tail of the Qy band of BChlc, at about 795 nm. Ashoulder at 510 nm is indicative of the presence of carotenoidsin both systems. The relative amount of BChla and carotenoidsis higher in Cf. aurantiacusand is clearly observed in theabsorption spectra (not shown).

Figure 1 shows the emission spectra of isolated chlorosomesof Cb. tepidumand Cf. aurantiacus, taken at 1.8 K. Thefluorescence spectra show two main bands. The first, centeredat 783 nm inCb. tepidumand at 764 inCf. aurantiacus, is dueto the emission from BChlc; the other, centered at 827 nm inCb. tepidumand at 819 nm inCf. aurantiacus, is due to BChla. The emission band due to BChla is the most intense inCf.aurantiacuswhile the emission band due to BChlc dominatesthe fluorescence spectrum ofCb. tepidum. In the figure thetransmittance of the band-pass filters used for the FDMRdetection are also shown.

The FDMR results are presented in Figures 2-6. With thefluorescence detection set at different wavelengths, several tripletstates have been detected, which can be assigned to BChlc,BChl a, and carotenoid molecules. The assignments can be doneon the basis of the ZFS parameters and, in some cases, on thebasis of the microwave-induced triplet-minus-singlet (T-S)spectra (see below).

BChl c Triplet States. The changes in the maximum of theBChl c emission band due to resonant microwave fields revealthe presence of triplet states under illumination at 1.8 K, in bothsamples (see Figure 2). InCf. aurantiacustwo transitionscentered at 648 and 875 MHz have been detected correspondingto the|D| - |E| and|D| + |E| transitions of an oligomeric BChlc triplet state23 having |D| ) 0.0254 cm-1 and |E| ) 0.0038cm-1. Similarly, in Cb. tepidumtwo transitions centered at 641

ODMR of Chlorosomes J. Phys. Chem. B, Vol. 105, No. 1, 2001247

and about 900 MHz have been found, corresponding to a tripletstate with|D| ) 0.0257 cm-1 |E| ) 0.0043 cm-1, as alreadyfound in ref 23. BChlc triplet states can be populated in bothsystems by low-temperature illumination, but comparison of thetwo spectra shows that several differences are present.

The signs of FDMR signals are opposite in the two bacteria.Moreover the|D| + |E| transition splits into two components(at about 856 and 901 MHz) indicated by the arrows in Figure2, only in Cb. tepidum. There are thus two slightly differentBChl c triplet states in this system. It is interesting to note thatthe relative intensity of the FDMR signals due to these twodifferent triplet states depends on the growing conditions: atlow light intensity the 856 MHz species dominates, while athigh light the 901 MHz component prevails (data not shown).The T-S spectra taken at the resonance frequencies of thesetwo triplet states in the Qy absorption region of BChlc, confirmthat the triplet states are due to two BChlc species in oligomericform. Indeed, the main singlet-singlet bleachings are found at740 and 760 nm (Figure 2). The intensities of the T-S signalsare quite low, indicating a very small triplet yield of BChlcunder steady-state illumination at low temperature. The evenlower intensity of the signal inCf. aurantiacusprevents thedetection of the T-S spectrum of the BChlc triplet statedetected by FDMR. As shown in Figure 3 for the most intensetransitions (|D| - |E|), the dependence of the FDMR signalson the detection wavelength is also different in the chlorosomesof the two bacteria. The signals due to BChlc triplet states arewell detectable up to 850 nm in the case ofCb. tepidum, while

they disappear at about 800 nm inCf. aurantiacus. The sign ofBChl c FDMR signals does not change when passing from theemission band of BChlc itself to the emission band of BChlawhile the signal intensity is maintained. Apparently, inCf.aurantiacusthe molecules carrying the triplet states belong toa BChl c pool that is unconnected to BChla fluorescentmolecules, and for this reason the FDMR disappears as soonas the BChlc fluorescence component vanishes.

When the |D| + |E| transitions are detected at differentwavelengths (range 800-900 MHz), a new transition centeredat 828 MHz, with a maximum of intensity at 800 nm, appearsonly in the spectra ofCb. tepidum(Figure 4b); this transitionoverlaps with the|D| + |E| transition of the BChla triplet state(see below). However, it is distinguishable at detection wave-lengths (790-810 nm) at whichTBChl a does not contribute.We assign this triplet state to a third different BChlc tripletstate on the basis of double resonance experiments (data notshown), which indicate a|D| - |E| transition at about 630 MHz.Interestingly, this third pool of BChlc (oligomer) shows adifferent polarization and a selection for long emitting BChlcmolecules.

BChl a Triplet States.By monitoring the microwave-inducedfluorescence changes in the emission band of BChla, togetherwith the above-discussed FDMR signals due to BChlc in Cb.tepidum, new FDMR signals, in a different microwave range,have been detected in both bacteria. The new transitions are

Figure 1. Fluorescence spectra of isolated chlorosomes taken at 1.8K: (a) Cf. aurantiacus; (b) Cb. tepidum. Excitation: white light from250 W tungsten lamp, filtered by CuSO4 solution (1 M, 5 cm). 1.5 nmresolution. Band-pass filters used in the FDMR experiments to selectthe detection wavelength are also shown (dashed lines).

Figure 2. FDMR spectra of isolated chlorosomes: (a)Cf. aurantiacus(detection wavelength: 770 nm); (b)Cb. tepidum(detection wave-length: 780 nm). Experimental conditions:T ) 1.8 K; modulationfrequency, 33 Hz; microwave power, 500 mW; scan rate, 5 MHz/s.Inset: T-S spectra taken at 901 MHz (solid line) and 856 MHz (dashedline) indicated by arrows in the FDMR spectrum ofCb. tepidum.Experimental conditions:T ) 1.8 K; modulation frequency, 33 Hz;microwave power, 500 mW; scan rate, 0.1 nm/s.

248 J. Phys. Chem. B, Vol. 105, No. 1, 2001 Carbonera et al.

shown in Figure 4 and can be assigned to BChla triplet statesby comparing the ZFS parameters of the observed triplet stateswith the in vivo and in vitro values reported for BChla tripletstates.24

Also in the case of BChla triplet population, analogies anddifferences characterize the two type of chlorosomes. InCb.tepidumthe resonance frequencies are centered at 457 and 820MHz, the third transition being too weak to be detected. TheFDMR transitions correspond, in this case, to a decrease ofemission and can be assigned to BChla present in the baseplate,as previously suggested9,10 on the basis of the ZFS:|D| )0.02213 cm-1, |E| ) 0.0061 cm-1. It is worth noting that theseBChl a transitions are not observed when the BChlc emissionis detected at 780 nm, possibly due to the lack of back energytransfer at 1.8 K, if this is the main factor influencing the steady-state populations of the BChlc emitters when the tripletpopulation of BChla is modulated. Another possibility is thatthe BChla molecules giving the triplet states are unconnectedfrom the BChl c molecules and absorb light only directly. InCf. aurantiacusthe signals have opposite sign and the 2Etransition is detectable at about 400 MHz. The|D| + |E|transition on the other hand is expected to be found at 875 MHz;however, the overlap with the stronger transitions due tocarotenoids is large in this microwave region (see the followingsection).

These are not the only differences that can be found bycomparing the two systems. In fact, changing the detectionwavelength in the BChla emission band from 810 to 850 nm,we found that inCb. tepidumthe signal shapes and the resonancefrequencies do not change, while inCf. aurantiacus thetransitions change dramatically (Figure 4). The behavior of the

line shape suggests the presence of two different BChla tripletstates, correlated to different optical properties. In the figure afitting with two Gaussian components for each transition isshown. From the fitting we may see that one triplet state showsthe maximum of intensity at 820 nm and the other at 840 nm.These components have different polarization, but both show adetectable 2E transition.

Carotenoid Triplet States. Together with the signal due toBChl a triplet states, other FDMR transitions have been detectedin both systems when monitoring the BChla emission, but notwhen monitoring the BChlc emission. These transitions, shownin Figure 5a,b, are easily assigned to carotenoid triplet stateson the basis of the ZFS values, which correspond roughly to anumber of 9-10 conjugated double bonds25 (|D| )0.0334 cm-1;|E| ) 0.00347 cm-1), and on the basis of the characteristicpattern of the T-S spectra, reported in Figure 5a,b, which havebeen detected in correspondence with the maximum of the low-frequency FDMR transitions (2E). The T-S spectra in bothsamples show positive bands at 475, 510, and 555 nm andnegative bands at about 485 and 525 nm, assigned respectivelyto triplet-triplet and singlet-singlet absorption. Very similarT-S spectra of carotenoids in photosynthetic antenna complexeshave been found previously both in higher plants,26-28 and inpurple bacteria.29 The intensity of the T-S signal is low,indicating small carotenoid triplet population. For this reasonthe T-S spectra were not detected in the broader|D| + |E|transitions. As already found for all the carotenoid triplet states

Figure 3. FDMR spectra of the BChlc, corresponding to the|D| -|E| transitions, taken at different detection wavelengths, as indicatedin the figures: (a)Cf. aurantiacus; (b) Cb. tepidum.

Figure 4. FDMR spectra of isolated chlorosomes taken at differentdetection wavelengths, as indicated in the figures. Experimentalconditions: T ) 1.8 K, modulation frequency, 320 Hz; microwavepower, 500 mW; scan rate, 5 MHz/s. (a)Cf. aurantiacus(detectionwavelength: from 810 to 850 nm). Dashed lines correspond tocomponents of a fitting function which is the sum of Gaussian curves.In the fitting of the signals at different detection wavelengths, thefrequencies, reported in Table 1, of the Gaussian conponents are keptfixed and only their relative contributions are changed. (b)Cb. tepidum(detection wavelength: from 780 to 850 nm).


reported in photosynthetic systems, special features are foundin the BChls absorption regions of the T-S spectra, due to thechange in the electronic state of the nearby carotenoidmolecules.26-29

The dependence of the FDMR signals (|D| + |E| transitions)on the detection wavelength is shown in Figure 6. An analysisof the line shape shows that several components contribute tothe signals. InCf. aurantiacusthe relative contribution of thedifferent components changes with the detection wavelength,while no changes, except for the intensity of the resonance, arefound in Cb. tepidum. The heterogeneity is better seen in the|D| + |E| transitions, which are more structure sensitive thanthe stronger 2E transitions.

Again, together with some analogies, differences between thetwo bacteria are found. Actually, no|D| - |E| transitions havebeen detected inCb. tepidumwhile in Cf. aurantiacusthe |D|- |E| transition is clearly seen and is inhomogeneouslybroadened in a similar way with respect to the|D| + |E|transition. In the chlorosomes ofCb. tepidumthe |D| - |E|transition is expected at about 900 MHz. In this microwaverange the transitions due to BChlc triplet states are also found.However, we can exclude the presence of an overlapping|D|- |E| transition due to carotenoids since, when the frequencymodulation of the amplitude of the microwaves is changed from33 to 325 Hz, the signal disappears completely (not shown)while the |D| + |E| and the 2E transitions of carotenoids areusually found to show a maximum of intensity at the latterfrequency, due to the lifetime of carotenoids triplet state, whichis close to 10µs. On the contrary, BChlc triplet states are long-lived (milliseconds) and for this reason, the intensity of thesignal decreases when the modulation frequency is increased.

Discussion

The energy transfer processes which occur in the chlorosomesare aimed to funnel the energy toward the reaction centers boundto the cytoplasmic membrane. Energy can be transferred in afew tens of picoseconds from BChlc to BChla pigments, whichare thought to be coordinated to protein in the baseplate.8,14,16

The efficiency of this process ranges from 30 to 90%,6,14,30-32

depending on the system and on the experimental conditions.In green sulfur bacteria, quenching of BChlc excited states anddecrease of energy transfer to BChla is observed underoxidizing conditions.11,16 This redox-depending quenching,which is not found inCf. aurantiacus, appears to involve somecomponents that are inactive in their reduced state, likelyquinone molecules.12,33

At physiological temperatures the excitation can move withinlarge domains of the chlorosomes,34-36 and the lifetimes of BChlc excited states are found to be longer5 in green sulfur bacteriathan inCf. aurantiacus. The kinetics of energy transfer withinthe BChlc antenna molecules have remained unclear, but theyare assumed to occur in less than 10 ps.13 It has been suggested14

that spectral heterogeneity plays a larger role in chlorosomesfrom Cb. tepidumthan in chlorosomes fromCf. aurantiacus.Moreover, the BChlc transition moments seem to be morecollinear inCf. aurantiacusthan inCb. tepidum,14 as deducedby the anisotropy of fluorescence decay.

Hole burning experiments at 1.8 K allowed to recognize thelowest excited singlet state of BChlc aggregates in the twobacteria at 774 nm inCb. tepidumand at 752 nm inCf.aurantiacus.37

Carotenoids are assumed to transfer excitation energy to BChlc with a transfer efficiency that inCf. aurantiacuswas calculated

Figure 5. FDMR and T-S spectra of carotenoid triplet states of isolated chlorosomes. Experimental conditions:T ) 1.8 K; modulation frequency,320 Hz; microwave power, 500 mW. (a)Cf. aurantiacus: (upper traces) FDMR taken at 830 nm, scan rate 5 MHz/s; (bottom trace) T-S spectrumtaken at 198 MHz (2E transition), resolution 3 nm, scan rate 0.08 nm/s. (b)Cb. tepidum: (upper traces) FDMR taken at 830 nm, scan rate 5 MHz/s;(bottom trace) T-S spectrum taken at 208 MHz (2E transition), resolution 3 nm, scan rate 0.08 nm/s.


to be 65% at 4 K,32 and probably most of the energy transferfrom the carotenoids to BChla proceeds via BChlc. Recently,Psencik et al.15 have shown that the carotenoid triplet state canbe formed under illumination of cells and of isolated chloro-somes suggesting a possible photoprotective role for thesepigments, similar to the one they play in purple bacteria andhigher plants.38,39

In this general description of the excited states and of theenergy transfer within chlorosomes, the formation of tripletstates under steady-state illumination seems to be a process withvery low probability. It is well-known, however, that energeticdisorder leads to localization at very low temperature. If thedifference in the energy among different oligomer excited statesis below thekT value at room temperature, it may well becomecritical at l.8 K, the temperature of ODMR experiments. In sucha way localization of triplet state can be possible even in theBChl c population and the triplet states can be used as probesof the antenna structure and of the energy transfer processes.

The first result of our experiments is that in isolatedchlorosomes, from the two green bacteria studied, triplet statesare populated by low-temperature illumination, in all kinds ofpigments present: BChlc, BChl a, and carotenoids. This factsuggests that the general pigment distribution and the generalfeatures of energy transfer processes and decay mechanisms arequite similar in the two species. However, despite thesesimilarities, the details of triplet formation and their spectralcharacteristics point to a different “local” organization withinthe chlorosomes.

As expected, the triplet yields of all the observed species arevery low, as indicated by the microwave induced relativechanges of fluorescence (∆I/I values of about 10-6 in the FDMRspectra) and as indicated by the microwave induced∆A valuesdetectable in the T-S spectra (10-7-10-6).

BChl c. The formation of BChl c triplet states underillumination is larger inCb. tepidum, and three different BChlc pools are observed where the triplet states become localized.As seen in the T-S spectra taken at 856 and 901 MHz, theBChl c oligomers that undergo the change in the electronic stateabsorb respectively at 767 and 747 nm. These values are veryclose to the absorption wavelengths of the BChlc mainpopulations found by optical experiments.10,13,40

The third component (828 MHz) gives a very weak T-Sspectrum (not shown) with a main bleaching at about 765 nmand seems to be associated with long emitting BChlc molecules(790-800 nm). This finding points to a higher heterogeneityof BChl c oligomers in green sulfur bacteria compared to greenfilamentous bacteria. All these pools are, directly and/orindirectly, connected to BChla via energy transfer, as provedby the fact that FDMR of BChlc triplet states may be performednot only by detection of BChlc emission but also by detectionof BChl a fluorescence. The sign of FDMR signals is conservedgoing from 770 to 850 nm detection wavelength, as expectedfor a triplet state localized in a molecule that is a donor of singletexcitation, as BChlc is assumed to be with respect to BChla.A different situation is found forCf. aurantiacus. In this caseBChl c triplet states become populated at low temperature undercontinuous illumination, but the triplet state can only be detectedwhen monitoring the BChlc emission. This suggests that thetriplet trap is not connected, at least at the very low temperatureof the experiments, to BChla molecules and that the back-transfer to other BChlc molecules is also poor. It can be seenthat the FDMR signal has a maximum at 780 nm, while theBChl c emission maximum is centered at 765 nm, indicatingthat the triplet state is likely to be localized in some BChlcmolecules absorbing at the red tail of the Qy absorption band.In principle, it is also possible that the observed triplet state islocalized in a population that has been damaged during thesample preparation. The different sign of FDMR transitionsfound for the two triplet states with respect to the third one inCb. tepidumand with respect to the observed transition inCf.aurantiacusis surprising. However, the polarization pattern ofthe signals is sensitive to the environment and to the pigmentorganization. The differences among different pools may thenbe explained on this basis. In any case the chance to populatethe triplet state in BChlc oligomers that are functional to theenergy transfer process is higher in the green sulfur bacteriumthan in the gliding bacterium. The formation of BChlc tripletstates under illumination could in principle represent a dangerfor the photosystems in the presence of oxygen. The highertriplet yield in Cb. tepidumwould make this species moresusceptible to damage. However, it is well-known that in aerobicconditions, quenchers of BChlc excited states reduce theirlifetimes. Under these conditions even the formation of tripletstates becomes unlikely. The quenching mechanism is notpresent inCf. aurantiacus; however, in this case the triplet yieldis much lower and it does not represent a serious problem forthe system.

The ZFS parameters of the BChlc triplet states are shown inTable 1. The values are in good agreement with those alreadyreported, obtained in whole cells ofCb. tepidumand assignedto BChl c in oligomeric form.23 Our T-S results, at least in

Figure 6. FDMR spectra of carotenoid triplet states of isolatedchlorosomes taken at different detection wavelengths, as indicated inthe figures, in the microwave range corresponding to the|D| - |E|and|D| + |E| transitions: (a)Cf. aurantiacus; (b) Cb. tepidum. Otherexperimental conditions as in Figure 5.


Cb. tepidum, correlate the triplet formation with the bleachingsof oligomers of BChlc singlet states.

BChl a. BChl a molecules are found in the chlorosomes ofgreen bacteria. The organization of these molecules is stilluncertain and their location in the baseplate has been proposedon the basis of several experimental evidence. Taking accountof the average dimensions of chlorosomes, and the averagenumber of BChls per chlorosome (103), van Noort and col-leagues41 calculated, inChlorobium Vibrioforme, that therewould be about 90 BChla in the envelope, their average distancebeing 65 Å. This distance is too high to explain the experimentalfinding by the same group that randomization of the excitationamong BChla molecules, measured by time-resolved anisotropydecay, has a time constant of about 20 ps. This would suggeststhat BChla is organized in clusters.

The role of the proteins present in the envelope is also unclear.Evidence of the role of the CsmA protein in the ligation of BChla has been obtained inCf. aurantiacuson the basis of the effectinduced by SDS, 1-hexanol, and Triton on the composition oftreated chlorosomes;8 in view of the sequence homology ofCsmA betweenCf. aurantiacusand Cb. tepidum, this mighthold true for the latter species as well. The relative amount ofBChl a with respect to the BChlc is higher in the chlorosomesof Cf. aurantiacus, 4% vs 1%. In Cf. aurantiacus twospectroscopically different BChla species are suggested toexist42-45 based on the time-resolved fluorescence resultsobtained in whole cells and to the experiments done by Mimuroet al.45 on the effects induced by 1-hexanol treatment in thebaseplate. The conclusion of that work was that there is a BChla pool, absorbing at 793 nm and fluorescing at 813 nm at roomtemperature, which is a functional baseplate component for theenergy transfer to the B808-866 complex, and a second BChla pool (absorption maximum at 813 nm and fluorescencemaximum at 826 nm), which is an additional baseplate formnot involved in the energy transfer to the membrane. Differently,in green sulfur bacteria the energy transfer is thought to occurbetween the BChla molecules in the baseplate and the BChlamolecules in the FMO protein before reaching the reactioncenters.

The FDMR spectra of BChla triplet states in the chlorosomesclearly show that the pigment organization differs in the twospecies studied. The ZFS parameters are reported in Table 1.In both systems BChla triplet states can be detected underillumination at very low temperature; however, inCf. auran-tiacustwo types of BChla pool have been detected correspond-ing to different optical and magnetic properties, as suggested

by the fact that the intensity of different FDMR signals has themaximum at different wavelengths of detection.

These two pools are probably related to those previouslyreported.45 In Cb. tepidumonly one type of BChla triplet statehas been detected. A marked difference is also found whenconsidering the polarization of the BChla FDMR signals inthe two bacteria. If the assignment is correct, as we believe onthe basis of the behavior of the signal intensity with the detectionwavelength and on the basis of the deduced ZFS values, theBChl a triplet states inCf. aurantiacusshow an unusualpolarization. Indeed, the 2E transitions are well detectable, whilein the light harvesting complexes from purple bacteria and insolvent,46 BChl a triplet states have a polarization that resemblesthe one found inCb. tepidum: two transitions (|D| - |E| and|D| + |E|) comparable in intensity and with the same sign. Sincethe polarization, besides the molecular structure, can beinfluenced by several factors such as pigment-pitment interac-tions and/or exposure to lipid or protein environment, theODMR data strongly suggest a different organization of thesemolecules in the two types of chlorosomes.Cf. aurantiacus,which lacks the FMO proteins, seems to have a more complexdistribution of BChla pigments likely in the contact region withthe cytoplasmic membrane.

Carotenoids. Carotenoids are nonfluorescent species andhave a very low ISC probability, so that their triplet states arenot easily populated by photoexcitation from the excited singletstates.

The common function of carotenoids in photosyntheticsystems is 2-fold.39,47-52 First they act as efficient antennapigments and transfer excitation to (B)Chl molecules, in thepigment-protein light-harvesting complexes. Second, theyfunction as quenchers of (B)Chl triplet states to avoid singletoxygen formation, both in the antenna complexes and in somereaction centers. Since green sulfur bacteria andCf. aurantiacusare anaerobes (although the latter is a facultative aerobe), itseems surprising to find that, in both bacteria, some carotenoidsare able in principle to perform the photoprotective role, assuggested by our finding that carotenoid triplet states arepopulated. Actually, green sulfur bacteria may encounter oxicconditions in their environment due to vertical mixing of zonesin the chemocline in stratified lakes. In factCb. tepidum12 underoxidizing conditions activates a mechanism in the chlorosomesthat quenches excited BChlc singlet states, decreasing theenergy transfer to the BChla in the baseplate. This uncouplingmechanism is reversible and prevents the formations of toxicreactive oxygen species from photosynthetically producedreactants in the reaction centers.Cf. aurantiacus, on the otherhand, does not possess such a quenching mechanism, probablybecause its PSII-type reaction center does not readily formreactive oxygen. However,Cf. aurantiacusis a facultativeaerobe and can be harmfully exposed to oxygen during thetransition from photosynthetic to respiratory metabolism inaerobic environments. Both systems, then, can be exposed tooxygen and the possibility to form singlet oxygen in thechlorosomes cannot be ruled out if the BChl triplet state can bepopulated under particular conditions.

By ODMR we found that both BChlc and BChla tripletstates become populated in isolated chlorosomes at least at lowtemperature, but the main triplet species present in the chloro-somes under illumination at low temperature are carotenoidtriplet states. FDMR of these triplet states, in both bacteria, canonly be done by detection of BChla fluorescence.

We found that the carotenoid triplet states are not detectedby BChl c fluorescence at 780 nm, even though, as is the case

TABLE 1: ZFS Parameters of All Assigned Triplet States inIsolated Chlorosomes ofCf. aurantiacusand of Cb. tepiduma

pigment2|E|

(MHz)|D| - |E|(MHz)

|D| + |E|(MHz)

|D|(cm-1)

|E|(cm-1)

Chloroflexus aurantiacusBChl c n.d. (-) 648 (-) 875 0.0254 0.0038BChl a (I) (+) 395 (+) 468 n.d. 0.0222 0.0066BChl a (II) (+) 385 (-) 470 n.d. 0.0221 0.0064carotenoids (+) 198 (+) ∼830 (+) ∼1050 0.0314 0.0033

Chlorobium tepidumBChl c (I) n.d. (+) 640 (+) 868 0.0252 0.0038BChl c (II) n.d. (+) 640 (+) 905 0.0258 0.0044BChl c (III) n.d. (*) 634 (+) 829 0.0246 0.0033BChl a n.d. (-) 456 (-) 818 0.0213 0.0060carotenoids (+) 206 n.d. (+) ∼1100 0.0333 0.0034

a (+): microwaves induced fluorescence increase. (-): microwavesinduced fluorescence decrease. (*): double resonance data. n.d.: notdetected.


of Cb. tepidum, the fluorescence yield is higher at 780 nm thanat 820 nm. This suggests a closer contact of carotenoids to BChla species rather than to BChlc and then, as a consequence, alocation of carotenoids in the region where BChlsa are present,likely the baseplate of the chlorosomes. The finding seems tobe in contrast with the conclusions drawn by Freese et al.53 basedon the Stark effect, LD, and CD in normal and carotenoid-deficient chlorosomes ofCf. aurantiacus. In that case, thespectral changes in the Qy transition of BChlc observed in theabsence of carotenoids seem to suggest the vicinity of caro-tenoids and BChlc in the chlorosomes, even though theobserved effect is quite small. However, with the ODMRtechnique we select only the carotenoids that are able to quenchBChl triplet states. Triplet-triplet transfer requires closedistances between the pigments involved in the energy transfersince the Dexter exchange mechanism depends on the overlapof the excited-state molecular orbitals. Singlet-singlet energytransfer may be possible at larger distances since it may proceedvia the Forster mechanism, which depends on the strength ofthe transition dipoles between ground and excited states. Thenwe may not exclude that some carotenoid population is able totransfer singlet excitation to BChlc molecules in the core ofthe chlorosomes, as is indeed suggested by Takaichi,54,55 whofound the core of chlorosomes, in bothCb. tepidum and Cf.aurantiacus, enriched inâ andγ carotene.

In several antenna complexes of higher plants and purplebacteria, T-S spectra taken at the resonance frequencies ofcarotenoid triplet states show characteristic features in the Qy

absorption region of (B)Chls.26-29 These spectral features havebeen interpreted as due to electronic interactions between thecarotenoid molecule carrying the triplet state and Chl moleculesclose to it and likely involved in the triplet-triplet transferprocess that populates the carotenoid triplet state. At wave-lengths corresponding to the Qy absorption region of BChla(800-830 nm), small negative and positive bands seem indeedto be present in the T-S spectrum of the chlorosomes, recordedat the resonance frequency of carotenoid triplet state.

It has been noted29 that the intensity of these interaction bandsis directly correlated to the rate of triplet transfer. We may thensay that in the chlorosomes the efficiency of triplet-triplettransfer is not very high, since the interaction bands are veryweak. This is also in agreement with the presence of detectabletriplet states of BChlc and BChla molecules, which indicatethat the carotenoids present in the chlorosomes are not able toquench the BChls with high efficiency. It is possible that thecarotenoids which are able to quench BChla triplet states arelocated in the baseplate where they may have a main structuralrole in anchoring the chlorosomes to the membrane, as alreadysuggested,56 and the fact of being able to play a photoprotectivefunction may have been only fortuitous at the beginning of theexistence of these ancient photosystems, when the atmospherecontained no oxygen.

The line shape of the|D| + |E| transitions in the FDMRspectra shows that in both samples several components con-tribute to the whole spectra. InCf. aurantiacusthis heterogeneitycan also be found in the spread of the|D| + |E| transition, whichis also detectable. The strong 2E transitions are less broad dueto a small spread in theE parameter for the observed tripletstates. TheD values are determined by the effective distancebetween the two unpaired electrons along the long polyene chainaxis (z axis). TheE values depend on the asymmetry of thedipolar tensor perpendicular to the long polyene chain axis, andthe variation of the electron distribution in thexy plane isprobably less influenced than the variation along the chain axis.

According to the empirical relationship given by Ros et al.,57

the D and E values depend on the inverse of the number ofconjugated double bonds. If we consider the pigment composi-tion55,58 in the chlorosomes, the carotenoids which are presentin larger amount are chlorobactene and 1,2-dihydrochlorobactene(10 conjugated double bonds) plus a small amount ofγ-carotene(10 conjugated double bonds) and glucoside-γ-chlorobactenein Cb. tepidum; γ-carotene,â-carotene (9 conjugated doublebonds) and a 20% amount of glucoside-γ-carotene inCf.aurantiacus. On the basis of the number of conjugate doublebonds and on the basis of the carotenoids abundance, we wouldexpect to find the same transition frequencies in the two samplesexcept for theâ-carotene, which should have somewhat largerZFS values. On the contrary inCf. aurantiacusthe resonant|D| + |E| transitions occur at lower frequencies with respect tothe observed transitions inCb. tepidum. Since by ODMR weare selecting only the carotenoid populations that are able topopulate the triplet state, these could differ substantially withrespect to the chemical composition. Moreover, the environmentand the molecular conformation may influence the resonancefrequencies as well. Since the polarization pattern of carotenoidtriplet states inCf. aurantiacusis different compared to thatobserved inCb. tepidum, and at least for some components,the resonance frequencies are also different, the exposure ofthe involved carotenoids to a different environment as well asa difference in their conformation, may play an important rolein determining the magnetic characteristic of the triplet states.We then suggest that if BChlsa are localized in the baseplate,then the latter also contains carotenoids molecules, but theorganization, and likely the amount of these pigments in thetwo bacteria, is different. Possibly, the glucoside-carotenoidsin Cf. aurantiacusare more involved in the structure of thebaseplate. Actually, they are found to be present in the baseplatewith a 50% contribution.55

Finally, a further difference found between the two systemsis that the carotenoid triplet states are formed with some siteselection by quenching of BChla molecules: we found thatthe line shape of the transitions depends on the detectionwavelength in the 800-850 nm range only inCf. aurantiacus.This fact, together with the observed heterogeneity in thedetected BChla triplet states, points toward an higher complex-ity of the contact region between the chlorosomes and themembrane, likely due to the absence of the FMO protein.

The results concerning triplet formation in prereduced chlo-rosomes fromCb. tepidumare very similar to the results fromCb. limicola reported by Psencik etal.;15 however, we foundthat the detection of FDMR signals in the emission band ofBChl c gives prevalent contributions of BChlc triplet states.No |D| + |E| FDMR transitions to be assigned to carotenoidshave been detected. Very weak signals in the 2E microwaveregion have been detected but the overlap with 2E transitionsdue to BChlc triplet states does not allow us to assign them tocarotenoids with certainty. InCf. aurantiacusthe signals in thisregion are even less intense. Following these experimentalresults we may assume that the carotenoid populations that areable to populate the triplet state are mainly interacting with BChla molecules and are then localized close to them.

Conclusions

The comparison of ODMR results obtained from the analysisof the spectra of isolated chlorosomes in the two bacteria chosenas representative of the two phylogenetically distinct speciesof green bacteria, revealed important analogies and differencesbetween the two systems.


The general energy transfer processes and the formation oftriplet states under illumination at low temperature follow thesame line. We found that BChlc, BChl a, and carotenoid tripletstates are populated in low yield in the chlorosomes in bothsystems. The carotenoid triplet states seem to be formed byquenching of BChla triplet states, and a weak interactionbetween carotenoids and BChla molecules is a common featurein Cf. aurantiacusand inCb. tepidum. These results stronglysuggest that some carotenoid molecules are present in closecontact with BChla molecules, likely in the baseplate, where,in principle, they are able to perform also a photoprotectiverole.

Several differences, however, appear when considering thecharacteristics of these triplet states, which reveal as, either inthe core or in the baseplate, the local organization of thepigments is characteristic of the system. The chlorosomes ofCb. tepidumpresent distinct BChlc-oligomers easily distin-guished because of the associated triplet states and of their T-Sspectra. The origin of these different forms, which are connectedto BChl a via energy transfer also at the very low temperatureof the ODMR experiments, is not clear; it may, however,depends on the degree of alkylation of the homologues. On thecontrary, in the chlorosomes ofCf. aurantiacus, we foundspectrally distinct forms of BChla, instead of BChlc. Ahomogeneous BChlc population inCf. aurantiacushas beenalso suggested59 by pump-probe experiments and may berelated to the lack of different triplet traps at very lowtemperature.

The possible role of different pools of BChla in the energytransfer process to the RCs remains uncertain.

Carotenoids quench selectively different BChla forms inCf.aurantiacus, while in Cb. tepidumno site selection has beenfound. Again this can be taken as an indication of the higherlevel of complexity in the baseplate ofCf. aurantiacuswhichlacks the FMO proteins.

We are planning to perform ODMR experiments on wholecells of the two green bacteria strains, under different redoxconditions, to correlate the information we already gained inisolated chlorosomes to the interactions of these antenna systemswith the reaction centers.

Acknowledgment. This investigation was supported in partby MURST BIOSTRUTT program and by EC contract NO.ERB FMRX- 0214; both agencies are gratefully acknowledged.We also thank Prof. Shinichi Takaichi (Nippon Medical School,Kawasaki), Prof. Shigeru Itoh (Nagoya University), and Prof.Katsumi Matsuura (Tokyo Metropolitan University) for usefuldiscussions.

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