Carotenoid Singlet Fission Reactions in Bacterial Light Harvesting

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Cite This: J. Am. Chem. Soc. 2017, 139, 15984-15993

Carotenoid Singlet Fission Reactions in Bacterial Light Harvesting Complexes As Revealed by Triplet Excitation Profiles Jie Yu,† Li-Min Fu,† Long-Jiang Yu,‡,§ Ying Shi,† Peng Wang,† Zheng-Yu Wang-Otomo,‡ and Jian-Ping Zhang*,† †

Department of Chemistry, Renmin University of China, Beijing 100872, P. R. China Faculty of Science, Ibaraki University, Mito 310-8512, Japan § Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, Japan ‡

S Supporting Information *

ABSTRACT: Carotenoids (Cars) in bacterial photosynthesis are known as accessory light harvesters and photoprotectors. Recently, the singlet fission (SF) reaction initiated by Car photoabsorption has been recognized to be an effective excitation deactivation channel disfavoring the light harvesting function. Since the SF reaction and the triplet sensitization reaction underlying photoprotection both yield triplet excited state Cars (3Car*), their contribution to the overall 3Car* photoproduction are difficult to disentangle. To tackle this problem, we resorted to the triplet excitation profiles (TEPs), i.e., the actinic spectra of the overall 3Car* photoproduction. The TEPs combined with the conventional fluorescence excitation spectra allowed us to extract the neat SF contribution, which can serve as a spectroscopic measure for the SF reactivity. This novel spectroscopic strategy was applied to analyze the light harvesting complexes (LHs) from Tch. tepidum and Rba. sphaeroides 2.4.1. The results unambiguously showed that the SF reaction of Cars proceeds with an intramolecular scheme, even in the case of LH1-RC from Rba. sphaeroides 2.4.1 likely binding a secondary pool of Cars. Regarding the SF-reactivity, the geometric distortion in the conjugated backbone of Cars was shown to be the structural determinant, while the length of the Car conjugation was suggested to be relevant to the effective localization of the geminate triplets to avoid being annihilated. The SF reaction scheme and structure−activity relationship revealed herein will be useful not only in deepening our understanding of the roles of Cars in photosynthesis, but also in enlightening the applications of Cars in artificial light conversion systems. present between the S2(1B+u ) and S1(2A−g ) states, however, its identity and physiological roles remain unclear.12−20 For light harvesting, the overall efficiency of Car-to-BChl singlet EET varies from ∼30% to ∼90% depending on the antenna complexes, and a longer conjugation of Car (NCC ≥ 11) tends to exhibit a lower efficiency.1−6 The detailed light harvesting and photoprotection mechanisms of Car have been extensively investigated on the basis of the excited-state properties of Cars12−17,21 as well as the crystallographic structures of photochemical reaction centers (RCs),22,23 peripheral light-harvesting complexes 2 (LH 2s),24,25 and light-harvesting complexes 1 (LH 1s) associated with RCs (referred to as core complexes).26 Car photoprotection is governed by the BChl-to-Car triplet EET reaction proceeding in a time scale of ∼20 ns. This together with a 3 BChl* lifetime of ∼80 μs, as determined in absence of Cars, ensures nearly 100% quenching of 3BChl*.16,27−29 Despite the high quenching efficiency, bacterial photosynthetic organisms

1. INTRODUCTION Carotenoids (Cars) in bacterial photosynthesis absorb light in the blue-green spectral region, where bacteriochlorophylls (BChl) do weakly, and pass the photoexcitation to BChls via singlet excitation energy transfer (EET). They can also quench the triplet excitation of BChls (3BChl*) that otherwise would sensitize the formation of harmful 1O2*, or directly scavenge 1 O2* in contingency. Besides the light harvesting and photoprotection roles, Cars help to stabilize the supramolecular pigment−protein assemblies.1−8 Naturally occurring photosynthetic Cars are C40-hydrocarbons with 9−13 conjugated CC double bonds (NCC; see Scheme 1 for the major Cars to be discussed). In analogy to the electronic structures of linearly polyenes with a C2h symmetry,9,10 the lowest-lying singlet-excited state S1(2A−g ) of Cars is optically dark, while the second lowest-lying singlet-excited state S2(1B+u ) is dipoleallowed from the ground state S0(1A−g ), which is responsible for the intense visible light absorption.11 Both S1(2Ag−) and S2(1B+u ) states are capable of conducting the Car-to-BChl singlet EET. An additional low-lying singlet excited state, referred to as S* and/or 1B−u in the literature, may energetically © 2017 American Chemical Society

Received: September 14, 2017 Published: October 20, 2017 15984

DOI: 10.1021/jacs.7b09809 J. Am. Chem. Soc. 2017, 139, 15984−15993

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Journal of the American Chemical Society

containing LH 1s revealed a 3Car* yield of ∼30% originated from the singlet fission (SF) reaction of Spx, which was considered to be partially responsible for the low lightharvesting efficiency of Spx (25% at 77 K).33 In photosynthesis, the Car SF reaction, a 1Car* splitting into a pair of 3Car*, was first observed for the cells of Rhodospirillum rubrum,34,35 and later found for isolated LHs with ultrafast spectroscopies12,33 as well as with laser flash photolysis in conjunction with magnetic-field effects.36,37 Recent studies on the Spe- or Spx-reconstituted B850-LHs from Rhodobacter (Rba.) sphaeroides R-26.1 have revealed the dependence of Car SF reactivity on the molecular configuration,38 which was consolidated with the LH 2s containing different Cars (NCC = 9−11) from Rba. sphaeroides G1C and Rba. sphaeroides 2.4.1.39 In ref 33, the Car SF reaction was suggested to be an intramolecular process, i.e., a pair of SF-yielded 3Spx* reside in the two halves of a Spx molecule. This scheme, however, has been challenged by the time-resolved electron paramagnetic resonance (EPR) studies on isolated LH 2s.40,41 The results favor an intermolecular SF scheme, i.e., the SF reaction takes place between a pair of neighboring Cars, which is likely mediated by the proximate BChls. The intermolecular scheme also draws support from early studies on the effects of magnetic-field on 3Car* photogeneration in LH 2s.37 Beyond photosynthesis, the Car SF reactions have been attracting considerable interests from the field of organic photoelectronics such as photovoltaics. This phenomenon, originally observed in the molecular crystals of aromatic compounds,13,42 is potent to break the Shockley-Queisser limit of light-to-electricity conversion efficiency (33%), because it theoretically supports a 200% quantum efficiency of photonto-electron conversion.42−46 Recently, SF reactions have been

Scheme 1. Molecular Structures of the Major Carotenoids Discussed in This Work

generally show a low 3Car* yield, e.g., 2% for chromatophores as measured under weak photoexcitation30 and 2−15% for isolated LH 2s containing neurosporene or spheroidene.31 The low 3Car* photoproduction is due to the low 3BChl* yield, which resulted from the kinetic competition of the slow 1BChl* → 3BChl* intersystem crossing (ISC), ∼7 ns, with the fast 1 BChl* → BChl internal conversion (IC), 1.4 ns.16,29,32 In chromatophores or LH1-RCs, the 1BChl* lifetimes are shortened to ten to a few tens of picoseconds, thus the 3 BChl* yields via ISC, and hence the 3Car* production via the triplet EET, decline with respect to the isolated LHs. A recent ultrafast spectroscopic work on the RC-devoid and Spx-

Figure 1. Steady-state electronic absorption and carotenoid resonance Raman spectra of the LH1-RC and LH2 complexes from (A, B) Tch. tepidum and (C, D) Rba. sphaeroides 2.4.1. In panels A and C, the absorption features of Car (Car) and BChl (Soret, Qx, Qy) are indicated, and the spectra are normalized at the Qy absorption maxima. The Raman excitation wavelength was 532 nm. 15985


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Figure 2. Kinetics traces probed at the maximal ESA wavelengths of 3Car* (λPr) for the LHs from (A, B) Tch. tepidum and (C, D) Rba. sphaeroides 2.4.1 under different excitation wavelengths (λEx). The excitation pulse energy was kept constant upon varying the excitation wavelength. The steady state absorption spectrum (dashed curve) is shown in each panel for reference.

demonstrated for covalently linked chromophore dimers47 and collision formed excimers48 in solutions, as well as for tetracene or pentacene crystals49 and Car aggregates.50−52 Meanwhile, SF reaction mechanisms have been attracting considerable theoretical interests.53−55 The natural Car assemblies differ from the solution-phase Car aggregates in molecular separation and packing circumstance: The nearest Car-Car distance in LHs (>1 nm) is substantially larger than that in the artificial aggregates (van der Waals sphere). In addition, Cars in LHs stay in specific molecular geometries owing to protein binding and BChl interactions. Therefore, it is intriguing to uncover the structure−activity relationship of Car SF in natural photosynthetic systems. However, despite the existing research efforts, issues of the intermoleular or intramolecular reactions, the influence from Car conformation and pigment−protein surroundings, and the identity of the precursory singlet state remain to be further investigated. Herein, we report a spectroscopic protocol to derive the neat SF contribution to 3 Car* photoproduction, which can serve as a measure of the SF reactivity. This was established on the basis of the triplet excitation profiles (TEPs) of Cars, reflecting the overall 3Car* photoproduction as a function of the actinic light wavelength. This novel strategy allows us to disentangle the relative yields of 3 Car* originated from the triplet sensitization (TS) and the SF reactions. This together with the bacterial LHs binding a variety of Cars provide us with a unique opportunity to gain deeper insights into some of the aforementioned unsettled issues. Our results strongly support the intramolecular reaction scheme of Car SF in bacterial LHs. In addition, both conformational twist and conjugation size are shown to be crucial in boosting the SF reactivity of Cars.

LH1-RC-Rba, and LH2-Rba, were prepared following the procedures described in refs 56−59. The electronic absorption spectra of these LH preparations, as depicted in Figure 1, are typical, like those reported in literatures.12−15,56,58,60 The LH1-RC preparations in 20 mM Tris·HCl buffer (pH 7.5) with 0.05% (w/v) n-dodecyl-β-D-maltoside (DDM), and the LH2 preparations in 20 mM Tris·HCl buffer (pH 8) with 0.05% (w/v) DDM were used for spectroscopic measurements. 2.2. Steady State Absorption, Fluorescence, and Resonance Raman Spectroscopies. Absorption spectra were recorded on a Cary 50 spectrophotometer (Varian). Fluorescence spectra and fluorescence excitation spectra (FESs) were measured with a FLS 980 fluorescence spectrometer (Edinburgh Instrument, U.K.) for which the LHs were adjusted to ODQy = 0.02 to minimize the effect of self-absorption. To calibrate the FESs in 675−920 nm covering the Qy bands of BChls, we measured the relative fluorescence quantum yields of the LHs with a home-built apparatus appended to the FLS 980 spectrometer, and the data were used as the reference for spectral calibration. See Supporting Information, SI, S1 for details. Car resonance Raman spectra of the LHs (ODQy = 0.5) were recorded on a XploRA PLUS spectrometer (Horiba, Paris, France) under an excitation wavelength of 532 nm. These measurements were performed at room temperature (298 K). 2.3. Transient Absorption and Triplet Excitation Profiles (TEPs). The TEP measurements were based on a transient absorption (TA) apparatus described in detail elsewhere.67,71 Briefly, the excitation laser pulses (7 ns, 10 Hz) were supplied by an optical parametric oscillator (OPO) driven by an Nd:YAG laser (Quanta-Ray Pro-Series, Spectra Physics Lasers Inc.). A white light source (LDLSEQ-1500, Energetiq) provided the visible to near-infrared probing light, which was sent to a monochromator (SP2500i, Princeton Instruments, U.S.A.) after interrogation with the photoexcited volume. The probe light was detected with an Si-PIN photodiode (model S3071, Hamamatsu Photonics) attached to the monochromator, and the electrical signals were fed to a digital storage oscilloscope (LeCroy WaveSurfer HDO-4054, Chestnut Ridge). The time resolution of the TA apparatus was 50 ns as the full-width at half-maximum of the instrumental response function. The LHs (OD480 nm = 0.5) subjected to the TA measurements were circulated with a peristaltic pump between a reservoir and a quartz cell

2. MATERIALS AND METHODS 2.1. Sample Preparation. The LHs from Thermochromatium (Tch.) tepidum and Rba. sphaeroides 2.4.1, LH1-RC-Tch, LH2-Tch, 15986


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Journal of the American Chemical Society of 0.5 cm optical path length. A sample volume of 18 mL was kept in a reservoir, which was bubbled with high purity nitrogen for oxygen removal. Three independent measurements with renewed samples were performed for each LH sample so as to derive the data statistics. To avoid excitation saturation and other unwanted nonlinear effects, the pulse energy was kept as low as 0.2 mJ (Figure S2). Each kinetics trace was averaged for 300 laser shots to reach a detection sensitivity of ∼10−5. All of the measurements were done at room temperature (298 K). As shown in Figure 2, for each LH preparation, the excitation wavelength was tuned to cover the Car and the BChl-Qy absorption bands (cf. Figure 1), and the Tn ← T1 kinetics of 3Car* were recorded at the wavelength of maximal excited state absorption (ESA). The TEP was then constructed by plotting the maximal ESA amplitudes emerged in the kinetics traces at the delay time of 0.15 μs as a function of the excitation wavelength. Since the excitation pulse energy were kept constant upon varying the excitation wavelength, a TEP can be regarded as an actinic spectrum of the relative yield of 3Car*.

C stretching (ν1 mode) and the symmetric C−C stretching coupled with C−H bending (ν2 mode), respectively. Those at ∼1000 cm−1 stem from the in-plane methyl rocking (ν3 mode). Importantly, the Raman lines at ∼970 cm−1, originating from the out-of-plane C−H wagging (ν4 mode), are characteristic to the twist of the conjugated backbone, because this mode would be otherwise inactive owing to its decoupling to the backbone conjugation.38 Thus, the distinct ν4 lines of LH1-RC-Tch, LH2Tch, and LH1-RC-Rba indicate the geometric distortion of Cars in these LHs. To the contrary, LH2-Rba does not show the ν4 signature, indicating a planar Car geometry in this case. 3.2. Carotenoid Triplet Excitation Profiles of the LH Complexes. Triplet excited-state properties of Cars both free in solutions and bound to bacterial LHs are well documented, and the Tn ← T1 transition energy of 3Car* is known to be proportional to 1/NCC.30,60,65,66 The characteristic ESA spectra of 3Car* observed in this work are in accord with those reported in the literature. (See SI S4 for typical TA spectra.) Irrespective to the excitation wavelengths, both LH2Rba and LH1-RC-Rba showed ESAs peaking at 530 nm, which are ascribed to the triplet excited states of Spe and its derivatives with NCC = 10. The ESAs of LH1-RC-Tch peaking at 580 nm can be attributed to the Tn ← T1 absorption of 3 Spx*. In the case of LH2-Tch, we assign the ESA peaking at 565 nm to the major Car composition with NCC = 11 (rhodopin with minor lycopene, 68.7%) rather than to Spx, which is plausible in view of the lower Spx content (spirilloxanthin with minor OH-spirilloxanthin, 22.6%). As supporting evidence, Spx in LH1-RC-Tch shows a 15 nm redshifted ESA and a 10 cm−1 red-shifted ν1 Raman line (Table S1). In the TEP measurements, we selectively excite the Car and the BChl components by tuning the photoexcitation wavelength while keeping a constant excitation pulse energy. At the delay time of 0.15 μs when the kinetics traces reached maxima (Figure 2), all of the singlet EET reactions and the excitation conversion/relaxation processes in the LHs are completed. The TEPs obtained by plotting the maximal ESA amplitude against the excitation wavelength are presented in Figure 3. Note that the TEPs are normalized to the (1−T) spectra at the Qy bands of B915 (3A), B850 (3B, 3D), and B875 (3C) for the following considerations: Upon selective excitation to the red-most Qy bands, the low-energy BChls are raised directly into the S1states (1BChl*). In this case, the 3Car* photoproduction must be formed via the BChl-to-Car triplet EET reactions, because the possibility of SF-yielding 3Car* can be excluded for energetic reasons. However, photoexcitations to the blue side of the red-most Qy bands may bring about SF-yielded 3Car* in addition to the triplet-EET-resulted 3Car*. Thus, the spectral normalization allows the intuitive recognition of the SF contribution by inspecting the TEPs (vide infra). The normalized presentation of TEPs may be analogous to that of FESs for evaluating the efficiency of singlet EET. The aforementioned mechanisms of 3Car* photogeneration can be expressed as 3BChl* + Car → BChl + 3Car* for the triplet sensitization (hereafter denoted TS), and 1Car* ⇄ 3 Car*⊗3Car* for SF. In terms of the 3Car* photoproduction, TS and SF are linear and nonlinear processes, respectively. For the TS mechanism, it is noteworthy that the BChls involved are the red-most absorbing BChls, because this pool of low-energy BChls serve as the excitation trap of an LH complex. For the SF mechanism, the backward arrow is a prompt for the possible annihilation of the triplet pair.

3. RESULTS 3.1. Spectroscopic Characterization of the LH Preparations. As seen in Figure 1, both Car and Qy absorption features are distinctly different between the two bacteria: The vibronic features of Car absorption (450−550 nm) are less clear in the cases of Tch. tepidum, which is partially due to the multicompositional Cars varying in NCC for both LH2-Tch and LH1-RC-Tch (Table 1). In comparison, the Car vibronic Table 1. Carotenoid Compositions, Partial Contents and Number of Conjugated CC bonds (NCC) for the LHs from Tch. tepidum and Rba. sphaeroides 2.4.1 complex Tch. tepiduma LH1-RC

composition

rhodopin OH-spirilloxanthin spirilloxanthin LH2 rhodopin lycopene anhydrorhodovibrin OH-spirilloxanthin spirilloxanthin Rba. sphaseroides 2.4.1b LH1-RC hydroxy-spheroidene demethylspheroidene spheroidenone spheroidene spirilloxanthin LH2 spheroidene a

NCC

content (%)

11 13 13 11 11 12 13 13

3.3 4.4 92.3 66.5 2.2 8.7 2.2 20.4

9 10 10 10 13 10

23.8 9.9 3.0 62.4 0.9 >90

Ref 58. bRef 70.

features are much more distinct in the case of Rba. sphaeroides 2.4.1. For this species, the major Car composition (>90%) are Spe and Spe-derivatives in both types of LHs. In addition, LH2Tch exhibits broader B800 and B850 bands, while LH1-RC-Tch shows a ∼ 40 nm red shift of the Qy band with reference to LH1-RC-Rba. See SI S3 for more detailed spectral characterization. It is to be noted that, compared to LH2-Rba, LH1-RCRba shows a nearly doubled absorption amplitude of Car, which is indicative of the presence of two Car molecules in a α,β-subunit of this type of complex.43,70 Resonance Raman spectroscopy was employed to identify the molecular configurations of Cars bound in the LHs,38,61−64 and the results are presented in Figure 1. The key Raman lines at ∼1510 cm−1 and ∼1150 cm−1 arise from the symmetric C 15987


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Figure 3. (A−D) Triplet excitation profiles (TEPs, red), 1-T spectra (black), and fluorescence excitation spectra (FESs, blue) for the LHs form Tch. tepidum and Rba. sphaeroides 2.4.1. In each panel, the spectra are normalized to the red-most Qy band. For comparison, both upper and lower limits of coordination are set as the same for each panel. Error bars were obtained on the basis of three independent measurements.

The TEPs in Figure 3 provide us with some new insights into the mechanisms of 3Car* photogeneration. Except for the SF reaction, the singlet excitation transfer and relaxation processes prior to the TS reactions result in a decline of the 3Car* photoproduction, because these cascading kinetic processes will certainly decrease the overall yield of 3BChl*. Therefore, for TS-dominated 3Car* photogeneration, the (1−T) spectrum serves as an upper limit of the TEP spectrum. This is indeed the case for LH2-Rba as demonstrated in Figure 3D. In the other cases (Figure 3A−C), the TEPs are substantially higher than the (1−T) spectra in the Car absorption regions, which clearly prove the involvement of Car SF reactions. The following points can be drawn from the TEPs: (i) The TS mechanisms are in operation for all of the LHs in question. (ii) Tch. tepidum. The SF-production of 3Car* are observed for both types of LHs upon Car excitation. (iii) Rba. sphaeroides 2.4.1. Upon Car excitation, the SF contribution to 3Car* photoproduction is confirmed for the LH1-RCs, but not the LH 2s. These results will be discussed in terms of reaction scheme and the structure−activity relationship of Car SF (see the Discussion). 3.3. Disentangle the SF and TS Contribution to the 3 Car* Photoproduction. We first examine the relationship between the TS-induced TA spectra of 3Car* and the steadystate fluorescence excitation spectra (FESs). For this purpose, we will consider the timing of 0.15 μs when 3Car* ESA reached the maxima, rather than the temporal evolution of 3Car*. Assuming an excitation photon quantity ϕ(λEx) (in mol·L−1) of an actinic laser pulse at the excitation wavelength λEx, we can write the 3Car* population density (in mol·L−1) as follows: Δn T(λEx ) = ϕ(λEx ) × [1 − T(λEx )] × ηS(λEx ) × ηISC × ηT

According to Beer−Lambert’s law, the TS-induced TA spectra of 3Car* is as follows: ΔODTTS(λEx ) = Δn T(λEx ) × ε T(λPr) × l

(2)

where ε (λPr) represents the extinction coefficient of the Tn ← T1 absorption of 3Car* at the probing wavelength of λPr, and l is the optical path length of the sample. On the other hand, we can write the FES spectra as follows: T

FES(λEx ) ≡ ϕ(λEx ) × [1 − T (λEx )] × ηs(λEx ) × ϕF

(3)

where ΦF stands for the fluorescence quantum yield of lowenergy BChls in the S1 state. Here, we assume that the quantities ϕ(λEx), T(λEx), and ηS(λEx) are the same as those in the TA measurements. Taken eqs 1, 2, and 3 together, we reach the following relation: ΔODTTS(λEx ) = K × FES(λEx )

(4)

where K = ηISC × ε (λPr) × l/ΦF is a constant for a given T measurement. Here, ΔODTS (λEx) represents the actinic spectrum of TS-yielded 3Car*, which can be regarded as the TEP lacking of the SF contribution. Equation 4 proves that ΔODTTS(λEx) shares the same spectral line-shape of FES(λEx), and, therefore, they can be normalized to each other as done in Figure 3. In general, a TEP in Figure 3 may constitute both TS and SF contribution. By subtracting a normalized ΔFES(λEx) spectrum from a TEP(λEx) spectrum, we can readily derive the neat SF contribution to the 3Car* photoproduction (ΔODTSF(λEx)). The results thus obtained are shown in Figure 4. It is seen from Figure 4 that, except for LH2-Rba, the SF contribution is rather distinct in the spectral regime of Car excitation (λEx, 450−550 nm). In the BChl excitation regime, however, the SF contribution is about 10-fold weaker. Therefore, it is the Car excitation, rather than the BChl excitation, that effectively induces the Car SF reaction. We will T

(1)

where ηS(λEx) stands for the efficiency of singlet EET from the selectively excited pigments to the low-energy BChls, and ηISC represents the ISC efficiency of these BChls. For bacterial LHs, the efficiency of BChl-to-Car triplet EET, ηT, is known to be unity.16,27−29 15988


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both limited and scattered, which together with the considerable BLC-ESA spectral overlap prevent us from making any reliable assessment. As proven briefly below (for more details, see SI S5), such circumstances can be averted by comparing the TS-resultant ESA-to-BLC ratio under the Qyexcitation of low-energy BChls (denoted as RTS) to the SFresultant ESA-to-BLC ratio under the Car excitation (denoted as RSF). Upon the Qy-excitation of low-energy BChls (B850, B875, or B915), the 3Car* population yielded via TS is responsible for both ESA and BLC of an ΔOD spectrum. According to Beer− Lambert’s law, the ESA-to-BLC ratio in this case is RTS = εT/εG. Upon the Car excitation, both SF and TS mechanisms contribute to the 3Car* photoproduction. The protocols for deriving the SF-induced ΔODTSF can be applied to obtain the SF-induced BLC amplitude of Car (ΔODGSF, SI S5), which allow us to obtain the ratio, RSF = ΔnTSFεT/ΔmGSFεG. Here, ΔnTSF G and ΔmSF represent the SF-yielded 3Car* populations responsible for the ESA and the BLC, respectively, which can be different depending on the SF reaction schemes. Taken together, the following relation holds, T ΔnSF G ΔmSF

=

R SF RTS

(5)

Importantly, eq 5 does not involve the extinction coefficients. It turns out that the 3Car* photoproduction via SF would undergo an intermolecular scheme (ΔnTSF = ΔmGSF) in the case of RSF/RTS = 1, or an intramolecular scheme (ΔnTSF = 2ΔmGSF) in the case of RSF/RTS = 2. Thus, the spectroscopic parameter, RSF/RTS, can serve as a criterion for the SF reaction scheme. On the basis of the formulas described in Section 3.3 for deriving the neat SF contribution, we could determine the values of RTS and RSF from the respective experimental data of TPEs and ΔODSF (see SI S5 for details). Figure 5 presents the RSF/RTS with referring to RTS for the LHs. Herein, the RSF/RTS values of ∼2 for the LH1-RCs from both Tch. tepidum and Rba. sphaeroides 2.4.1 prove the intramolecular SF scheme. However, an RSF/RTS value of ∼1.4 for LH2-Tch suggests that not all of the Car compositions are involved in the SF reactions, and/or that the annihilation of triplet pairs may take place before the delay time of 0.15 μs. In the case of LH2-Rba, where TS predominates the 3Car* photoproduction, an RSF/RTS value (∼0.92) slightly smaller than 1 can be due to the incomplete Car-to-BChl singlet EET (∼90%). Nevertheless, our results unambiguously show that, upon Car excitation, SF reactions in bacterial LHs proceed with an intramolecular scheme. Here, we note that, despite a high stoichiometric composition of Car (vide supra), the SF reactions in the LH1-RC-Rba complexes also proceed in an intramolecular scheme, because the RSF/RTS value of 2, by definition, precludes the intermolecular scheme. Since the SF mechanism is suggested to be not advantageous to the light harvesting function of Cars,20,32,33,39 it may be intriguing to correlate the SF reactivity to the Car-to-BChl singlet EET efficiency of Cars. Assume a yield of 10% for 3Car* photogenerated via TS under the Qy excitation,31 the SFresultant 3Car* yields, as estimated from the TEPs in Figure 3, are estimated to be ∼25% and ∼12% for LH1-RC-Tch and LH2-Tch, respectively. These SF-resultant triplet yields show a reverse trend to the variation of Car-to-BChl singlet EET efficiency, which are ∼20% and ∼30% for the above LHs in the same order. As an extreme, Spe molecules in LH2-Rba exhibit negligible SF reactivity, correlating reversely to the rather high

Figure 4. Neat SF contribution: 3Car* photoproduction via the SF mechanism for the LH1-RCs (red) and LH 2s (blue) from (A) Tch. tepidum and (B) Rba. sphaeroides 2.4.1. (See text for details.).

consider the possible origins of the weak background in Discussion.

4. DISCUSSION We have measured the TEPs that reflect the overall 3Car* photoproduction via both TS and SF reactions in bacterial LHs, and derived the neat SF contribution to the 3Car* photoproduction. These results provide us with a unique opportunity to look deeper into the SF mechanisms of Cars in the LHs. Hereafter, we will examine the issue of intermolecular or intramolecular schemes of the SF reaction, as well as the influence of Car molecular structures and surroundings on the SF reactivity. 4.1. Intermolecular or Intramolecular SF Reaction Schemes. For the SF reactions under Car photoexcitation, it is understandable that an intermolecular and an intramolecular reaction will result in different numbers of photobleached ground-state Car molecules (cf. Scheme 2). Ideally, the depletion numbers will be 2 and 1 for an intermolecular and an intramolecular reaction, respectively. Given the extinction coefficients of the Tn ← T1 absorption (εT) and the S2 ← S0 absorption (εG) of Cars, the depletion number can in principle be assessed based on the ESA-to-BLC amplitude ratio and the Beer−Lambert law. Unfortunately, the documented εT data are 15989


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Scheme 2. Side Views (Upper) and Bottom Views (Lower) of the Pigment Orientation of (A) LH2 and (B) LH1 Complexesa

a

Numerals represent the edge-to-edge molecular separation in Å. A pair of neighboring Cars and their vicinal BChls are displayed. The intramolecular singlet fission (SF) is illustrated schematically with the triplet pair ↑↑···↑↑. These diagrams are based on PDB 1LGH for the LH2 of Rs. molischianum25 and PBD 3WMM for the LH1 of Tch. tepidum.26

4.2. Effects of Conformation and Conjugation Length on the SF Reactivity of Car. As discussed above, in LH2-Rba adopting the major Car composition of Spe (>90%), Cars exhibit no obvious SF reactivity. This can be recognized intuitively by inspecting the coincidence of the TEP and FES spectra in Figure 3D or the nearly vanishing SF-yielded 3Car* in Figure 4. Apparently, 3Car* photogeneration in LH2-Rba is predominated by the TS mechanism irrespective of the excitation wavelengths. In LH1-RC-Rba where Spe and Spederivatives constitute a major Car composition (>75%), Car excitation results in substantial 3Car* production via the SF mechanism. Since Spe and its derivatives have the same conjugation length, NCC = 10, and thereby similar electronic structures, the drastic difference in the SF activities between LH2-Rba and LH1-RC-Rba can be ascribed to the difference in Spe molecular conformations. Specifically, the geometric distortion of the conjugated backbone, as revealed by the resonance Raman for LH1-RC-Rba, seems vital for the SF reactivity. LH2-Tch contains a major pool of Cars with NCC = 11 (68.7%) and a minor fraction of longer conjugated Cars, whereas LH1-RC-Tch adopts Spx with NCC = 13 as the major Car composition (>90%). Car molecules in these LHs take twisted conformations, and hence the SF mechanism is active. By comparing the actinic spectra of the SF-yielded 3Car* in Figure 4, e.g., LH2-Tch vs LH1-RC-Tch and LH1-RC-Rba vs LH1-RC-Tch, we see a prominent conjugation-length effect, i.e., a longer conjugation is propitious to a higher 3Car* yield.

Figure 5. Comparison of the SF-resultant ESA-to-BLC ratio under Car photoexcitation (RSF) to the TS-resultant ESA-to-BLC ratio under Qy excitation of the low-energy BChls (RTS). To avoid the complication from the extinction coefficients of 3Car*, RSF/RTS instead of RSF is used for the comparison (see text for details). Error bars were obtained on the basis of three independent measurements.

Car-to-BChl singlet EET efficiency of ∼90%. It is surprising to see that, for LH1-RC-Rba, both the 3Car* yield via SF (∼30%) and the Car-to-BChl singlet EET efficiency (∼50%) are moderately high, which deviates from the aforementioned general trend of efficiency correlation. This may be relevant to the 2 pools of Cars, i.e., they play different roles in this type of core complex. 15990


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Journal of the American Chemical Society 3

The structure−activity relationship of SF revealed in the present work agrees well with the literature reports. Spx has the longest conjugation (NCC = 13) among the naturally occurring photosynthetic Cars, however, it is almost SF inactive with an all-trans and planar conformation in solutions33 or reconstituted in the B850-LH complexes from Rba. sphaeroides R-26.1.38 In contrast, with a geometric distortion in bacterial LHs, the short conjugated neurosporene (NCC = 9) exhibits significant SF reactivity.18,39 Taken together, a twist of the conjugated backbone appears as the structural determinant for the SF reactivity, while a long conjugation facilitates the 3Car* yield of SF reaction. Our conclusion on the intramolecular SF reaction scheme of Cars is in accord with that reached by ultrafast TA studies on bacterial LHs,32,33 but is opposite to the intermolecular scheme proposed by Klenina et al. based on the EPR properties of 3 Car*.40,41 In refs 40 and 41, the authors investigated, by the use of time-resolved EPR spectroscopy with a time resolution of 150 ns, the mechanism of 3Car* formation via SF reaction in the isolated LH 2s from Allochromatium (Alc.) minutissimum containing multicompositional Cars (Rdp, An-Rdv, and Spx. Scheme 1). The transient EPR spectra of 3Car*, recorded at a few microseconds after the nanosecond laser flash at 470 nm, agreed well with those documented for the TS-yielded 3Car*, and simulation of the observed spectra yielded the zero-fieldsplitting (ZFS) parameters. On the basis of the ZFS parameters and the patterns of the EPR spectra, the authors exclude the intramolecular scheme of Car SF reaction by arguing that significant distortion of the EPR spectral shape would otherwise be observed owing to the strong magnetic dipole−dipole and exchange interactions between a pair of triplets in the same Car molecule. The proposed intermolecular scheme was supported by the close similarity between an EPR spectrum of the LH2 preparation and that of the solid film casted with the total Car extract from Alc. minutissimum chromatophores.40 In ref 41, the authors further demonstrated a decrease in the EPR-signal amplitude of 3Car* with increasing the extent of B850 photobleaching, which allowed them to suggest the involvment of BChls (B850 and B800) in mediating the intermolecular SF reaction between a pair of Car molecules. Here, we argue that the EPR properties of 3Car* as revealed in refs 40 and 41 can also be accounted for by the intramolecular scheme based on the effects of molecular and electronic structures of Cars on the SF reactivity as summarized below. The structural effects on the SF reactivity of Cars can be integrated into the following hypothetical schemes.18,33,38 A geometric distortions of Car conjugated backbones, such as the molecular twist as identified by resonance Raman spectroscopy, can effectively decouple a SF-yielded triplet pair. On the other hand, a long enough linear conjugation can support the spatial localization of the decoupled triplet excitations and in effect prevent them from annihilation. It is noteworthy that the intramolecular SF scheme, a pair of geminate triplets residing in two halves of a Car molecule, is restricted to the photosynthetic Car assemblies. In artificial Car aggregates, on the other hand, the intermolecular SF reactions may also take place owing to the tightly packed Car molecules.50,52 4.3. Environmental Effects on the SF Reactivity of Car. As pointed out in refs 4, 33, 38, and 68, in bacterial LHs, protein binding and pigment packing are crucial for the conformational distortion and hence the SF reactivity of Cars. In the present work, the weak background signals beyond the Car excitation regimes in the actinic spectra of SF-yielded

Car*, as shown in Figure 4, are indicative of the electronic contribution from the surroundings of Car. In the case of LH1RC-Tch, the actinic spectrum of SF-yielded 3Car* shows vanishingly small background in the Qy excitation region. However, in the other cases the background signals are nonnegligible although rather weak. Because these backgrounds are the residues left by subtracting the FESs from the TEPs as normalized in Figure 3, they represent the 3Car* production via any mechanisms other than TS as discussed below. In LH1-RCs, Cars adopted in RCs may contribute to the 3 Car* production, especially when charge recombination occurs in the RCs.34,37 However, the weak backgrounds were observed also for LH 2s without RCs being involved. As shown in Scheme 2, Cars in LH 2s or LH 1s orient parallel to each other, and they keep van der Waals contact with the macrocycles of B850s or B915s. In LH 2s, the nearest Car-B800 edge distance falls into the van der Waals sphere (∼3 Å) despite a nearly perpendicular orientation. In fact, spectral evidence for the 3 Car*-BChl orbital interaction have recently been reported for LH2-Tch.60,67 Such kind of electronic coupling was previously considered as the basis for BChls as mediators of intermolecular Car SF reactions.40,41 However, under the Qy excitation, the S1state energy of B850 and B875 are definitely not high enough to drive any SF reactions involving Cars. Here, we tentatively ascribe the weak background in Figure 4 to the excitation fusion reactions,69 i.e., annihilation of low-energy BChl excitations to form higher-lying singlet excited BChls, which eventually convert into 3BChl* via IC and ISC and subsequently into 3 Car* via TS. In view of the closely packed circular aggregates of BChls, it may not be surprising to see the effects of triplet− triplet or singlet−triplet annihilation reactions despite the rather weak laser excitation. Finally, we comment on the implication of Car SF reactions in bacterial photosynthesis. Our results, i.e., the reverse correlation of SF efficiency with the Car-to-BChl singlet EET efficiency and the geometric and conjugation-length effects on the SF reactivity, are consistent with previous ultrafast TA studies on bacterial LHs,20,32,33,39 where the SF reaction from a precursory S* state of Car was shown to proceed in a time scale of ∼10 ps, which is competitive with the Car-to-BChl singlet EET reaction from the S* state. In bacterial photosynthesis, a relatively low Car-to-BChl singlet EET efficiency (∼30%) is generic for Cars of longer conjugation (NCC ≥ 11), which is ascribed to the inefficient and even closed Car(S1(2A−g )) → BChl(Qy) EET pathway for energetic reasons.39,59 In this relation, the rapid SF deactivation of S* excitation may further contribute to the low efficiency of Car energy transfer to BChl. However, such an adverse effect in the light-harvesting function of Car seems dependent on the electronic structures and protein interactions of Cars in LHs, an issue which deserves further investigation.32,33 Nevertheless, the SF reactivity is relatively high in the LHs binding longer conjugated Cars (NCC ≥ 11) of poor light-harvesting function, and Cars in these LHs are suggested to play the major roles of photoprotection and structural stabilization. In addition, since the geometric distortion of Cars, a structural determinant for effective SF, is induced by interacting with protein surroundings, photosynthetic bacteria may adopt such a regulating mechanism to enhance their photoprotective function in response to environmental stress. 15991


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(3) Ritz, T.; Damjanovic, A.; Schulten, K.; Zhang, J.-P.; Koyama, Y. Photosynth. Res. 2000, 66, 125−144. (4) Polívka, T.; Sundström, V. Chem. Rev. 2004, 104, 2021−2071. (5) Polívka, T.; Frank, H. A. Acc. Chem. Res. 2010, 43, 1125−1134. (6) Dilbeck, P. L.; Tang, Q.; Mothersole, D. J.; Martin, E. C.; Hunter, C. N.; Bocian, D. F.; Holten, D.; Niedzwiedzki, D. M. J. Phys. Chem. B 2016, 120, 5429−5443. (7) Lang, H. P.; Hunter, C. N. Biochem. J. 1994, 298, 197−205. (8) Plumley, F. G.; Schmidt, G. W. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 146−150. (9) Tavan, P.; Schulten, K. J. Chem. Phys. 1986, 85, 6602−6609. (10) Tavan, P.; Schulten, K. Phys. Rev. B: Condens. Matter Mater. Phys. 1987, 36, 4337. (11) Hudson, B.; Kohler, B. Annu. Rev. Phys. Chem. 1974, 25, 437− 460. (12) Zhang, J.-P.; Inaba, T.; Koyama, Y. J. Mol. Struct. 2001, 598, 65− 78. (13) Balevičius, V.; Pour, A. G.; Savolainen, J.; Lincoln, C. N.; Lukeš, V.; Riedle, E.; Valkunas, L.; Abramavicius, D.; Hauer, J. Phys. Chem. Chem. Phys. 2015, 17, 19491−19499. (14) Balevičius, V., Jr; Abramavicius, D.; Polívka, T.; Galestian Pour, A.; Hauer, J. J. Phys. Chem. Lett. 2016, 7, 3347−3352. (15) Kloz, M.; Weißenborn, J.; Polívka, T.; Frank, H. A.; Kennis, J. T. Phys. Chem. Chem. Phys. 2016, 18, 14619−14628. (16) Niedzwiedzki, D. M.; Hunter, C. N.; Blankenship, R. E. J. Phys. Chem. B 2016, 120, 11123−11131. (17) Feng, J.; Tseng, C. W.; Chen, T.; Leng, X.; Yin, H.; Cheng, Y. C.; Rohlfing, M.; Ma, Y. Nat. Commun. 2017, 8, 71. (18) Niedzwiedzki, D. M.; Swainsbury, D.; Martin, E. C.; Hunter, C. N.; Blankenship, R. E. J. Phys. Chem. B 2017, 121, 7571−7585. (19) Wohlleben, W.; Buckup, T.; Hashimoto, H.; Cogdell, R. J.; Herek, J. L.; Motzkus, M. J. Phys. Chem. B 2004, 108, 3320−3325. (20) Kosumi, D.; Maruta, S.; Horibe, T.; Nagaoka, Y.; Fujii, R.; Sugisaki, M.; Cogdell, R. J.; Hashimoto, H. J. Chem. Phys. 2012, 137, 064505. (21) Mendes-Pinto, M. M.; Sansiaume, E.; Hashimoto, H.; Pascal, A. A.; Gall, A.; Robert, B. J. Phys. Chem. B 2013, 117, 11015−11021. (22) Deisenhofer, J.; Epp, O.; Miki, K.; Huber, R.; Michel, H. Nature 1985, 318, 618−624. (23) Nogi, T.; Fathir, I.; Kobayashi, M.; Nozawa, T.; Miki, K. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 13561−13566. (24) McDermott, G. M.; Prince, S. M.; Freer, A. A.; Hawthornthwaite-Lawless, A. M.; Papiz, M. Z.; Cogdell, R. J.; Isaacs, N. W. Nature 1995, 374, 517−521. (25) Koepke, J.; Hu, X.; Muenke, C.; Schulten, K.; Michel, H. Structure 1996, 4, 581−597. (26) Niwa, S.; Yu, L. J.; Takeda, K.; Hirano, Y.; Kawakami, T.; WangOtomo, Z. Y.; Miki, K. Nature 2014, 508, 228. (27) Nagae, H.; Kakitani, T.; Katoh, T.; Mimuro, M. J. Chem. Phys. 1993, 98, 8012−8023. (28) Farhoosh, R.; Chynwat, V.; Gebhard, R.; Lugtenburg, J.; Frank, H. A. Photosynth. Res. 1994, 42, 157−166. (29) Kosumi, D.; Horibe, T.; Sugisaki, M.; Cogdell, R. J.; Hashimoto, H. J. Phys. Chem. B 2016, 120, 951−956. (30) Monger, T. G.; Cogdell, R. J.; Parson, W. W. Biochim. Biophys. Acta, Bioenerg. 1976, 449, 136−153. (31) Cogdell, R. J.; Hipkins, M. F.; MacDonald, W.; Truscott, T. G. Biochim. Biophys. Acta, Bioenerg. 1981, 634, 191−202. (32) Papagiannakis, E.; Kennis, J. T.; van Stokkum, I. H.; Cogdell, R. J.; van Grondelle, R. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 6017− 6022. (33) Gradinaru, C. C.; Kennis, J. T.; Papagiannakis, E.; van Stokkum, I. H.; Cogdell, R. J.; Fleming, G. R.; Niederman, R. A.; van Grondelle, R. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 2364−2369. (34) Rademaker, H.; Hoff, A. J.; Van Grondelle, R.; Duysens, L. N. Biochim. Biophys. Acta, Bioenerg. 1980, 592, 240−257. (35) Cogdell, R. J.; Land, E. J.; Truscott, T. G. Photochem. Photobiol. 1983, 38, 723−725.

5. CONCLUSIONS We have devised TEP spectroscopy to investigate the scheme and structure−activity relationship of Car SF reactions in bacterial LHs. The TEPs combined with the conventional FESs, the actinic spectra of Car triplet yield and BChl fluorescence yield, respectively, allowed us to extract the neat SF contribution to the 3Car* photoproduction, which can be used as a spectroscopic measure of the SF reactivity. We further compared the SF-resultant ESA-to-BLC ratio under the Car excitation (RSF) to the TS-resultant ratio under the Qyexcitation of low-energy BChls (RTS), which can be taken as a criterion, without arousing the equivocal excited-state extinction coefficients, for the intermolecular or intermolecular schemes of Car SF reactions. These novel analytical methods were applied to the LHs from Tch. tepidum and Rba. sphaeroides 2.4.1, and the results unambiguously showed that the Car SF reactions take an intramolecular scheme, even in the case of LH1-RC-Rba likely binding a secondary pool of Cars. Regarding the SF-reactivity, the geometric distortion in the conjugated backbone of Cars, as induced by protein binding, was shown to be the structural determinant, while the length of Car linear conjugation was suggested to be relevant to the effective localization of the geminate triplets to avoid being annihilated. The Car assemblies in the natural photosynthetic LHs differ from the artificial Car aggregates or crystallites in the molecular packing orders. Therefore, the present work will be useful not only in deepening our understanding of the structure−activity relationship of Cars in photosynthesis, but also in enlightening the application of Cars in artificial light conversion systems.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b09809. Calibration of near-infrared fluorescence excitation spectra, linear response regime of photoexcitation, spectral features of steady-state absorption and resonance Raman scattering, representative TA spectra, and calculation of ESA-to-BLC ratios (PDF)

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Jian-Ping Zhang: 0000-0002-9216-2386 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The project was supported by the Natural Science Foundation of China (21673288, 21173265; 21411130185). We are indebted to Prof. A. V. Nemukhin at the Research Computing Center of the M.V. Lomonosov Moscow State University for stimulating discussion.

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REFERENCES

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Carotenoid Singlet Fission Reactions in Bacterial Light Harvesting

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