Cooperative Photoprotection by Multi-Compositional Carotenoids in

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Cooperative Photoprotection by Multi-Compositional Carotenoids in the LH1 Antenna from a Mutant Strain of Rhodobacter sphaeroides Jie Yu, Liming Tan, Tomoaki Kawakami, Peng Wang, Li-Min Fu, Zheng-Yu Wang-Otomo, and Jian-Ping Zhang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b06080 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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The Journal of Physical Chemistry

Cooperative Photoprotection by Multi-Compositional Carotenoids in the LH1 Antenna from a Mutant Strain of Rhodobacter sphaeroides

Jie Yu,†,a Li-Ming Tan,† Tomoaki Kawakami,‡ Peng Wang,† Li-Min Fu,† Zheng-Yu Wang-Otomo,‡ and Jian-Ping Zhang†,*

†

Department of Chemistry, Renmin University of China, Beijing 1000872, P. R. China ‡

Faculty of Science, Ibaraki University, Mito 310-8512, Japan

a

Present address: Department of Pharmacy, Weifang Medical University, Weifang, Shandong 261053, P. R. China

E-mail addresses of authors J. Yu: [email protected] L.-M. Tan: [email protected] T. Kawakami: [email protected] P. Wang: [email protected] L.-M. Fu: [email protected] Z.-Y. Wang-Otomo: [email protected]

*To whom correspondence should be addressed Tel: +86-10-62516604; Fax: +86-10-62516444 E-mail: [email protected] (J.-P. Zhang)

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Abstract To explore the photoprotection role of multi-compositional carotenoid (Car) in photosynthetic purple bacteria, we investigated, by means of triplet excitation profile (TEP) combined with steady state optical spectroscopies, the core light-harvesting complex-reaction center of a mutant strain of Rhodobacter (Rba.) sphaeroides (m-LH1-RC) at room temperature. TEP spectra revealed that spheroidene and derivative (Spe) preferentially protect bacteriochlorophylls (BChls) of relatively lower site energy by quenching the triplet excitation (3BChl*), however, spirilloxanthin (Spx) does so irrespective to the site energy of BChls. Triplet excitation results showed the triplet excitation energy transfer (EET) reaction in a timescale of ∼0.5 µs from Spe and derivatives as a major component (∼85%) to Spx as a minor component (∼8%), suggesting the coexistence of different kinds of Cars in individual LH1 complex. The nonequivalent quenching potency and the triplet EET reaction between Cars constitute the cooperative photoprotection by multi-compositional Cars in bacterial photosynthesis.


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1. Introduction In bacterial photosynthesis, carotenoid (Car) plays important roles in light harvesting and photoprotection, as well as in assembling of functional pigment-protein complexes. Car absorbs sun light in the visible spectral region where bacteriochlorophyll (BChl) does weakly and transfers the singlet excitation to BChl, and quenches BChl triplet excitation (3BChl*) that would otherwise sensitize the formation of detrimental singlet oxygen (1O2). In this way, Car carries out light harvesting and photoprotection, respectively.1-6 In photosynthetic pigment-protein complexes, the physiological functions of Car rely on its unique molecular and electronic structures, which are analogous to the conjugated linear polyene,7,8 as well as on its close proximity to BChl molecules.9-13 Accordingly, the mechanisms of Car in light harvesting and photoprotection have been extensively investigated on the basis of Car excited state property14-16 and the high-resolution structures of various pigment-protein complexes, e. g., photochemical reaction center (RC),17,18 light harvesting complex 2 (LH2)9,10 and light harvesting complex 1 (LH1) encircling RC (referred to as LH1-RC)11-13. Regarding photoprotection, as established by both experimental19-24 and theoretical25-28 investigations, the BChl-to-Car triplet excitation energy transfer (EET) reaction proceeding in a ∼10 ns timescale can ensure a nearly unitary quenching efficiency of 3BChl*, because the 3BChl* lifetime is more than 3 orders of magnitude longer (∼80 µs) in the absence of Car quenchers. For bacterial LHs, recent ultrafast spectroscopic studies have revealed the singlet fission (SF) reaction of Car proceeding in a 100 fs timescale, which yields a pair of triplet excitation from a singlet excitation of Car.29-32 Interestingly, the SF reaction in bacterial LHs has recently been suggested to be an intramolecular process correlating with a decreased efficiency of Car-to-BChl singlet EET.29,33 The LH complexes of photosynthetic purple bacteria with known structures seem to be dominated by a single Car composition. For instances, the LH2s from Rs. molischianum and Rps. acidophila, 3 ACS Paragon Plus Environment


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respectively, mainly incorporate lycopene and rhodopin glucoside. The LH1s from Thermochromatium (Tch.) tepidum and Blastochloris (Blc.) viridis mainly adopt spirilloxanthin and dihydroneurosporene, respectively. In fact, the majority of bacterial LHs are equipped with multi-compositions of Car,34-39 and it is therefore interesting to explore the implication of presence of multiple Car components. Besides, it is also intriguing to understand whether the Cars coexist in individual LH complex. We have tried to investigate these issues, by means of time-resolved absorption spectroscopy, for the LH2s from Rhodopseudomonas (Rps.) palustris and Tch. tepidum, both contain at least 5 different kinds of Car with various number of conjugated double bonds (nc=c). For Rps. palustris LH2 we observed Car-to-Car triplet EET processes at cryogenic temperature (77 K),37 while for Tch. tepidum LH2 we showed the importance of a minor Car composition, anhydrorhodovbrin (8.7 %), in trapping the singlet excitation of Car.38 However, the Car-to-Car triplet EET reaction was not observed for Tch. tepidum LH2.39 Nevertheless, in view of the recently unraveled high-resolution structures and the new excitation dynamics results of bacterial LHs, it is timely to explore the interplay of different Car compositions in conducting the accessary light harvesting and the photoprotection functions.

Hydroxyspheroidene (OH-Spe, nc=c=9)

Demethylspheroidene (DM-Spe, nc=c=10)

Spheroidenone (Spo, nc=c=10)

Spheroidene (Spe, nc=c=10)

Spirilloxanthin (Spx, nc=c=13)

Scheme 1. Molecular structures of the carotenoids (Cars) discussed in this work.

In the present work, we target on a unique core complex from a mutant strain of Rhodobacter (Rba.)


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sphaeroides consisting of chimeric Rba. sphaeroides RC and Tch. tepidum LH1.40 This type of m-LH1RC complex contains multi-compositional Cars with nc=c varying from 9 to 13 (Scheme 1). Spheroidene (Spe) and its derivatives with nc=c=10 constitute the major Car component, whereas spirilloxanthin (Spx) with nc=c=13 presents as a minor component (Table 1). The m-LH1-RC protein was subjected to submicrosecond time-resolved absorption (TA) spectroscopy to derive the triplet excitation profile (TEP), a spectroscopic technique capable of differentiating the origin of 3Car*, i.e. the formation of 3Car* via SF or triplet sensitization.33 To avoid the complication from Car SF reaction, the m-LH1-RC preparation was photoexcited into the LH1-Qy band, i.e. BChls are excited into the lowest lying singlet excited state. Therein, the size of excitation energy is unfavorable for any SF reactions among Car and BChl molecules. Based on the TEP spectra and the TA kinetics, we propose the cooperative photoprotection of different kinds of Car, which is discussed in light of the recently disclosed high-resolution LH1-RC structure of Tch. tepidum.11,12

TABLE 1. Carotenoid Composition (in %) of the m-LH1-RC Complex from a Mutant Strain of Rba. sphaeroides, and the LH1-RC Complexes from Tch. tepidum and Rba. sphaeroides DP2. Carotenoid (Abbreviation) Hydroxyspheroidene (OH-Spe) Demethylspheroidene (DM-Spe) Spheroidenone (Spo) Spheroidene (Spe) Rhodopin (Rhd) OH-spirilloxanthin (OH-Spx) Spirilloxanthin (Spx)

nc=c 9 10 10 10 11 13 13

m-LH1-RC40 6.9 9.4 1.8 74.2   7.7

Rba-LH1-RC40 23.8 9.9 3.0 62.4   0.9

Tch-LH1-RC34     3.3 4.4 92.3

2. Materials and Methods 2.1. Sample Preparation. The m-LH1-RC complex was prepared from a mutant strain of Rba. sphaeroides, which genetically incorporated the full-length LH1 α,β-polypeptides of Tch. tepidum, while all the other components remained the same as those from a LH2-deficient Rba. sphaeroides, 5 ACS Paragon Plus Environment


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strain DP2.40 The LH1-RC complexes from Tch. tepidum and Rba. sphaeroides DP2, denoted as TchLH1-RC and Rba-LH1-RC, were purified following the procedures described in ref 41 and ref 40, respectively. The LH1-RC preparations in 20 mM Tris-HCl buffer (pH 8) with 0.05% (w/v) n-dodecyl-

β-D-maltoside (DDM) were used for spectroscopic measurements. 2.2. Steady State Spectroscopies. Absorption spectra were recorded on a Cary 50 spectrophotometer (Varian). Fluorescence spectra were measured with a FLS 980 fluorescence spectrometer (Edinburgh Instrument, UK), for which the LHs were adjusted to ODQy = 0.02 to minimize the effect of selfabsorption. Instrumental response of fluorescence excitation spectra in 675-920 nm covering the Qy bands of LH1-BChl were calibrated as described elsewhere.33 Car resonance Raman spectra of the complexes (ODQy = 0.5) were recorded on a XploRA PLUS spectrometer (Horiba, Paris, France) under laser excitation at 532 nm. 2.3. TA and TEP Spectroscopies. The TEP measurements were based on a nanosecond TA apparatus.42,43 Briefly, an optical parametric oscillator (OPO) driven by a Nd:YAG laser (Quanta-Ray Pro-Series, Spectra Physics Lasers Inc.) delivered the excitation laser pulses (7 ns, 10 Hz). A continuous-wave white light source (LDLS-EQ-1500, Energetiq) provided the visible-to-near infrared probing light, which was sent to a monochromator (SP2500i, Princeton Instruments, USA) after interrogating with the photoexcited volume. The probe light was detected with a Si-PIN photodiode (model S3071, Hamamatsu Photonics) attached to the monochromator, and the electrical signal was fed to a digital storage oscilloscope (LeCroy WaveSurfer HDO-4054, Chestnut Ridge). The time resolution of was 50 ns as the full-width at half maximum (FWHM) of the instrumental response function (IRF). The samples (OD480nm = 0.5) subjected to TA measurements were circulated with a peristaltic pump between a reservoir and a quartz cell of 1 cm optical path length. A sample volume of 18 mL was kept in the reservoir, which was bubbled with high purity nitrogen for oxygen removal. Three independent 6 ACS Paragon Plus Environment

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measurements with renewed samples were performed for each LH1-RC preparation so as to derive the data statistics. To avoid excitation saturation and other unwanted nonlinear effects, the pulse energy was kept at low as 0.2 mJ. Each kinetics trace was averaged for 300 laser shots to reach a detection sensitivity better than 10−5. All of the measurements were done at room temperature (298 K). For each pigment-protein preparation, the excitation wavelength was tuned to cover the Qy band (cf. Figure 1), and the TA kinetics of 3Car* were recorded at the Car(Tn←T1) absorption maxima. The TEP spectra was then constructed by plotting the maximal TA amplitudes against the excitation wavelength. Since the excitation pulse energy was kept constant on varying the excitation wavelength, a TEP can be regarded as an actinic spectrum of relative 3Car* yield, which is analogous to a fluorescence excitation spectrum.

m-LH1-RC (Mutant strain) Rba-LH1-RC (Rba. sphaerides) Tch-LH1-RC (Tch. tepidum)

875 915

505

Absorbence

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


550

Soret

300

400

Car

Qx

500

600

LH1-Qy

700

800

900

1000

Wavelength (nm)

Figure 1. Steady-state absorption spectra of m-LH1-RC, Rba-LH1-RC and Tch-LH1-RC normalized at the Qy absorption maxima. Major bands of Car and BChl (Soret, Qx and LH1-Qy) are indicated.

3. Results 3.1. Spectroscopic Characterization of m-LH-RC. Figure 1 compares the steady-state absorption spectrum of m-LH1-RC to those of the other pair of LH1-RCs, which display the following differences. (i) Car absorption. Compared to Rba-LH1-RC, m-LH1-RC shows similar spectral pattern and ∼30% reduction in relative absorbance. The stronger Car absorption of Rba-LH1-RC is likely due to the 7 ACS Paragon Plus Environment


presence of a secondary Car molecule in an α,β-subunit.35,40,44 The Car absorption exhibit distinct vibronic features, which are attributed to Spe and derivatives with a composition exceeding ∼90% (Scheme 1, Table 1). On the other hand, the Car absorption of Tch-LH1-RC is dominated by Spx (>92.3%), giving rise to the red-shifted and blurred vibronic features. (ii) LH1-Qy absorption. The mLH1-RC and Tch-LH1-RC complexes show intimately similar LH1-Qy bands, indicating that in these proteins the associated circular LH1-BChl aggregates are organized in a similar manner. Besides, the LH1-Qy bands of m-LH1-RC and Tch-LH1-RC are ∼40 nm red shifted with respect to Rba-LH1-RC.

ν2

Raman intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1120

1140

1160

ν1

1480

1180

1500

1520

1540

ν3

940

960

900

980

ν4

1000

1100

1200

1300

1400

1500

1600

−1

Raman shift (cm )

Figure 2. Car resonance Raman spectra of m-LH1-RC (red), Rba-LH1-RC (grey) and Tch-LH1-RC (black). Laser excitation wavelength was 532 nm. Insets show the details of the ν1, ν2 and ν4 spectral lineshapes.

Resonance Raman spectroscopy was employed to identify the molecular configuration of Cars in the LH1 complexes (Figure 2). The key Raman lines at ∼1510 cm−1 and ∼1150 cm−1, respectively, arise from the symmetric C=C stretching (ν1 mode) and the symmetric C-C stretching coupled with C-H bending (ν2 mode). Those at ∼1000 cm−1 stem from the in-plane methyl rocking (ν3 mode). The particular Raman lines at ∼970 cm−1, originating from the out-of-plane C-H wagging (ν4 mode), are characteristic to the twist of Car conjugated backbone, as in the case of planar geometry this mode would be Raman inactive owing to the lack of coupling to the conjugated backbone.45-49 Both ν1 and ν2 bands of m-LH1-RC split into two sub-bands, and each of them can be decomposed into the corresponding bands of Rba-LH1-RC and Tch-LH1-RC (Figure S1). However, the ν4 band of m8 ACS Paragon Plus Environment

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LH1-RC showing more distinct split cannot be simply approximated by a linear combination of the ν4 bands of the other LH1-RCs. These results confirm that m-LH1-RC binds multi-compositions of Car. Most importantly, the occurrence of ν4 band indicates the geometric distortion of Car conjugated backbones in different types of LH1-RCs.

Spx (T n←T 1 )

(A)

1.0

∆t=0.15 µs

(II)

1

(B)

(I)

(II)

m-LH1-RC λe x= 895 nm

0.5

Tch-LH1-RC (λ ex = 910 nm)

(I)

0 Spe (T n←T 1 ) m-LH1-RC ( λe x= 930 nm)

∆mOD

2

∆ mOD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.0 ∆t 0.15 µs

-0.5

-1

0.5 µs 1 µs

Rba-LH1-RC (λ ex = 880 nm)

-2 475

500

525

550

575

Wavelength (nm)

600

1.5 µs

-1.0

2 µs

475

500

525

550

575

600

Wavelength (nm)

Figure 3. Car(Tn←T1) spectra for different types of LH1-RCs recorded at the indicated excitation wavelengths (λex) and delay times (∆t). (A) Comparison of the transient spectra at ∆t=0.15 µs. (B) Car(Tn←T1) spectra of m-LH1-RC at early delay times.

3.2. Car(Tn←T1) Spectra of m-LH-RC. Figure 3A compares the characteristic Car(Tn←T1) spectrum of m-LH1-RC to those of the other LH1-RCs. The m-LH1-RC complex exhibits two distinct bands at ∼580 nm (band-I) and ∼530 nm (band-II). The former and the latter, respectively, appear at similar transition wavelengths of the major TA bands of Tch-LH1-RC and Rba-LH1-RC. Figure 3B shows the time resolved TA spectra for m-LH1-RC photoexcited at 895 nm. It is seen that, except a relatively stronger band-I, the TA spectra are similar to that of m-LH1-RC under photoexcitation at 930 nm (Figure 3A). In addition, band-II decays monotonically whereas band-I shows a rise phase prior to decay, which is more clearly demonstrated by comparing the time evolution behavior in Figure 4A (and in Figure S2 for that at different λex). From band-I to band-II, the variation of Tn←T1 transition energy follows the reciprocal relation of the transition energy with the conjugation length (nc=c) for LH-bound Cars.19,37,50,51 9 ACS Paragon Plus Environment


Specifically, the transition energy, 17,240 cm−1 and 18,780 cm−1 for band-I and band-II, respectively, agrees with those of 3Spx* (17,361 cm−1) and 3Spe* (18,975 cm−1) reported for the LH1-RCs of Rhodospirillum (Rsp.) rubrum S1 and Rba. sphaeroides 2.4.1, respectively.50

(B)

(A) ∆mOD (normalized)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Kinetics Model & Fitting Results

0.6 3Spe*

0.4

τEET=0.51 (0.56) EET

0.0 0.5 1.0 1.5 2.0

3Spx*

band-I @ 580 nm

0.2

τ1=1.59 (1.29) 0.0

isc

band-II @ 530 nm 0

4

8

12

τ2=2.79 (2.79)

isc

16

Delay time (µs)

Figure 4. (A) TA kinetics probed at 580 nm (band-I, red) and 530 nm (band-II, black) for m-LH1-RC photoexcited at 895 nm (cf. Figure 3B). Inset shows the kinetics details at early delay times. (B) Kinetics model for global fitting the kinetics curves in panel A. Time constants (τ, µs) are obtained by setting N1(0) = N2(0) = 1 as initial condition, and those in parenthesis are obtained with initial condition N1(0) = 1 and N2(0) = 0. Solid lines in panel A are fitting curves, which are indistinguishable with different initial conditions.

In the case of m-LH1-RC, the trailing edges of the kinetics curves in Figure 4A could be fitted to a mono-exponential function, and the decay time constants thus derived are 2.8 µs and 1.6 µs, respectively, for the kinetics curves at 580nm (band-I) and 530 nm (band-II). The different kinetics behavior of band-I and band-II implies that they are originated from different Car components. As shown in Figure S3, in the cases of Tch-LH1-RC or Rba-LH1-RC, the pair of kinetics curves at 530/580 nm decayed in phase, sharing a same decay time constant of 2.5 µs or 3.6 µs, respectively. For Tch-LH1RC or Rba-LH1-RC, the identical kinetics behavior of band-I and band-II indicates that the TA spectra are originated from the same Car with a predominant composition, i.e. Spx (92.3%) for Tch-LH1-RC


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and Spe and derivatives (99%) for Rba-LH1-RC. In addition, the shorter and the longer time constants, respectively, are in good agreement with those of 3Spx* (2.9 µs) and 3Spe* (4.6∼5.6 µs) of various bacterial LH1s.50 Based on the kinetics behavior and the aforementioned characteristic Tn←T1 transition energy, we ascribe band-I and band-II observed for m-LH1-RC to 3Spx* and 3Spe*, respectively, which is to be consolidated by the kinetics correlation of band-I with band-II (vide infra). Further inspecting the kinetics traces in Figure 4A, we see that the decay of band-II correlated with the rise of band-I. We therefore simultaneously fit the pair of kinetics traces to the kinetics model depicted in Figure 4B. In this model, the Spe-to-Spx triplet EET reaction is considered, which is energetically favorable in view of the triplet excited state energy of Spe (7,100 cm−1) and Spx (5,950 cm−1). Similar decay-to-rise correlation holds for m-LH1-RC photoexcited at Car band and at the red edge of LH1-Qy band (cf. Figure S2). Table 2 summarizes the time constants obtained by simultaneously fitting the kinetics data to the kinetics model. It is seen that, owing to the triplet EET reaction, the 3Spe* lifetime (1.13∼1.62 µs) is considerably shortened with reference to the documented values (4.6∼5.6 µs). The average triplet EET time constant is 0.5 µs, and the time constants exhibit weak dependence on λex. Here, it is important to note that, in contrast to the case of m-LH1-RC, the referencing LH1-RCs did not show any decay-to-rise correlation between the kinetics pairs at 530/580 nm (cf. Figure S3), which is understandable in view of the same origin of band-I and band-II for Tch-LH1-RC or Rba-LH1-RC. TABLE 2. Decay time constants (τi, i = 1,2) and triplet EET time constant (τEET) for m-LH1-RC photoexcited at different wavelengths (λex).a λex (nm) τ1 (µs) τ2 (µs) τEET (µs) 495 1.13 (0.91) 2.71 (2.71) 0.35 (0.38) 895 1.59 (1.29) 2.79 (2.79) 0.51 (0.56) 930 1.62 (1.51) 2.88 (2.88) 0.69 (0.71) a Time constants were derived by fitting the kinetics traces at 530 nm and 580 nm to the model in Figure 4B by setting N1(0) = N2(0) = 1 as initial condition. Those in parenthesis are obtained with initial condition N1(0) = 1 and N2(0) = 0.



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3.3. Carotenoid Triplet Excitation Profiles of m-LH-RC. Figure 5 shows the TEPs probed at the absorption maxima of 3Spx* (band-I) and 3Spe* (band-II) for m-LH1-RC, which can be regarded as the actinic spectra of 3Spx* and 3Spe*. The TEPs in the visible spectral region are presented in Figure S4. In the spectral region of 725-825 nm, we see weak TEPs for both 3Spx* and 3Spe* (Figure 5), which is likely contributed by the RC-associated Cars upon photoexcitation of the accessary BChls of RC (absorbing around 800 nm). However, in the LH1-Qy region (850-950 nm), the contribution from RCCars, if any, can be even smaller, because the stoichiometric ratio of RC-Car to LH1-Car is as low as 1:16, and the steady-state absorption of the special pair BChl2 of RC (absorbing around 865 nm) is much weaker than that of LH1-BChls. Here, it is worthy of noting that, upon LH1-Qy excitation, the possibility of SF reactions is excluded for energetics reason, i.e. EQy < ET + E'T. In addition, all of the intra-complex singlet EET and excitation conversion/relaxation processes complete in a timescale comparable to an IRF (∼50 ns). Therefore, the 3Spx* or 3Spe* population at ∆t = 0.15 µs must be originated from triplet EET reactions. As such, the TEPs in the LH1-Qy spectral region should be consistent with the (1−T) spectrum or with the fluorescence excitation spectrum as previously observed for a few native bacterial LHs.33 However, as seen in Figure 5, the TEP of 3Spe*, but not 3Spx*, differs distinctly from the (1−T) or the fluorescence excitation spectrum. To be specific, the TEP of 3Spe* deviates from the blue edge of the (1−T) spectrum, whereas the TEP of 3Spx* almost fully coincides with the LH1-Qy band. The difference in the TEPs of 3Spe* and 3Spx* is to be ascribed to selective quenching of 3BChl* by Spe.


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0.006

λpr = 580 nm (TEP of 3Spx*)

λpr = 530 nm (TEP of 3Spe*)

∆ OD (normalized)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.004

0.002

0.000

LH1-Qy

800

850

900

950

Actinic wavelength (nm)

Figure 5. The (1-T) spectrum (solid grey), the fluorescence excitation spectrum (dotted blue) and the triplet excitation profile (TPE, symbols) probed at 530 nm (black) and 580 nm (red) for m-LH1-RC. Error bars were based on three independent measurements. Spectra are normalized at the LH1-Qy maxima. Shaded area emphasizes the difference in TEPs.

4. Discussions We have investigated the triplet excitation dynamics of m-LH1-RC with submicrosecond TA spectroscopy by tuning the actinic wavelength over the LH1-Qy band. The major observations include (i) the sizable TA amplitude of 3Spx* despite a minor composition (7.7%), (ii) the sub-microsecond decay-to-rise correlation of 3Spe* with 3Spx*, as well as (iii) the difference in the TEPs probed at the maxima of Spe(Tn←T1) and Spx (Tn←T1) absorption. These experimental phenomena are to be discussed on the basis of the available Tch-LH1-RC structure, which allows us to propose the mechanism of corporative photoprotection by multi-Car compositions. 4.1. Triplet EET Paths in Native Tch-LH1-RC. According to Dexter’s theory, for effective triplet EET, a donor (D) and an acceptor (A) have to be in close proximity for a significant strength of electronexchange interaction.22,26,53 Such a structural prerequisite, for the BChl-to-Car triplet EET in bacterial LHs, is satisfied by the short edge-to-edge distances between BChl and Car cofactors (see Scheme 2A for Tch-LH1-RC). Recent time-resolved spectroscopic studies have shown that in LH2s this triplet EET proceeds at a timescale of ∼10 ns.22-25,52 On the other hand, the direct Car-to-Car triplet EET reaction



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seems unlikely because of the large separation (∼14 Å) as in the case of Tch-LH1-RC. The rate of triplet EET reaction (k) drops rapidly with increasing the D-A separation (R) following the relation,53,54 k=

2π KJ DA e − ( 2 R / L ) , h

(1)

where K is a parameter relevant to the strength of D-A electron-exchange interaction, JDA is the spectral overlap integral between D emission and A absorption, and L is an average of the van der Waals radius of the initial and the final states of the D-A system. Scheme 2A displays the mutual orientation of BChl and Car cofactors in an α/β-subunit (1) and its neighboring subunits (0) and (2) of Tch-LH1. As measured from the C35 of the π-conjugated chromophore of Spx(1) to the nearest edge of BChls, Spx(1) keeps the closest contact with BChl(0)β of the neighboring subunit (0) (R(0)β−(1) = 3.66 Å), rather than with the BChl pair of the same subunit (Rα(1)−(1) = 5.38 Å, R(1)β−(1) = 11.84 Å). Assuming an L of 3 Å for convenience and using the R values between Spx(1) and BChl(0)β or BChl(1)α as depicted in Scheme 2A, the k(1)α→(1)/k(0)β→(1) and k(1)β→(1)/k(0)β→(1) ratios are estimated to be 32% and 0.4%, respectively, meaning that the rates of triplet transfer from BChl(1)α and BChl(1)β to Spx(1) decrease drastically with reference to that from BChl(0)β. However, a ∼90% reduction in the rate would not cause considerable drop of 3BChl* quenching efficiency because of the rather long intrinsic lifetime of 3BChl* (∼80 µs).


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Scheme 2. Schematic orientation of pigment cofactors (A) in the native Tch-LH1-RC complex (PBD 5Y5S),12 where Spx(1) BChl(1)α and BChl(1)β belong to the same α/β-subunit, and (B) hypothesized for the m-LH1-RC complex containing Spe and Spx. For clarity, the bacteriopheophytins of BChls were truncated.

Regarding the BChl-BChl separation of Tch-LH1-RC, the averaged inter-subunit Mg-Mg distance (8.72 Å) is shorter than the intra-subunit one (8.88 Å),12 a circumstance which is similar to LH2s.9,10 Note that the BChl-BChl separations in Tch-LH1 are considerably shorter than those in LH2s. As seen in Scheme 2A, the nearest edge-to-edge BChl-BChl distances are R(0)β-(1)α = 3.37 Å and R(1)α-(1)β =3.49 Å, both of which are shorter than the closest Spx-BChl separation (R(0)β-(1) = 3.66 Å). Therefore, β-BChl transfers triplet excitations to α-BChl more efficiently than to Spx, i.e. both inter- and intra-subunit BChl-to-BChl triplet EET reactions must be taken into account in discussing the photoprotection mechanism of Car as previously suggested for LH2s21,26. In this context, a recent theoretical work has shown that, for Tch-LH1-RC, the site energy of α-BChls are relatively lower than that of β-BChls.55 Taken together, the 3BChl* excitation is prone to reside on α-BChls, from where the triplet excitation is eventually directed to Spx.

4.2. Hypothetical Pigment Orientation and Triplet EET Paths in m-LH1-RC. As seen in Scheme 1, Spe, the major Car component of m-LH1-RC, differs from Spx both in the methoxyl substituents and in



Page 16 of 26

the geometric location of the conjugated π-electron system. It is important to consider the mutual orientation of the chromophores of BChl and Spe, because this will substantially affect the efficiency of triplet excitation transfer. Unfortunately, such information on the pigment orientation of m-LH1-RC is unavailable. It seems reasonable to assume that the methoxyl terminal of Spe is oriented similarly as Spx in native Tch-LH1-RC, i.e. the methoxyl of Spe approaches LH1-BChls, because the LH1-Qy absorption bands of m-LH1-RC and Tch-LH1-RC, rather sensitive to both the aggregation form and the microenvironment of LH1-BChls, are nearly identical. This vision, however, conflicts with the spectral observation: As highlighted in Figure 5, the TEP spectrum of 3Spe* deviates distinctly from the fluorescence excitation or (1-T) spectrum at the blue edge of LH1-Qy band, indicating that BChls of relative higher site energy, i.e. β-BChls, are rather inefficient in transferring triplet excitation to Spe molecules. If the methoxyl terminal of Spe in m-LH1-RC is oriented similarly as Spx in Tch-LH1-RC, Spe would receive triplet excitation from β-BChls similarly as Spx does and, accordingly, the TEP of 3

Spe* would coincide with the blue edge of fluorescence excitation or (1-T) spectrum. However, this is

apparently not the case. To explain the aforementioned spectral inconsistency, we herein propose the pigment orientation of m-LH1-RC. As shown schematically in Scheme 2B, the hydrophobic end of Spe approaches the BChl aggregate, whereas the methoxyl end extends towards the N-terminals of α/β-polypeptides (cytoplasmic side). For Spe, the nearest distances from the C28 of π-conjugation to the neighboring BChls are R(0)β-(1) = 5.44 Å and R(1)α-(1) = 5.96 Å, which are 1.78 Å and 0.58 Å larger than those of Tch-LH1-RC, respectively. Such significant increase in R has to be taken into account, because this may substantially retard the BChl-to-Car triplet EET process.26 Nevertheless, in our hypothetical scheme (Scheme 2B), β3

BChl* can be more efficiently quenched by α-BChl rather than by Spe owing to the crucial R

dependence of the triplet EET rate. This explains the discrepancy between the TEP of 3Spe* and the (1–


Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


T) spectrum (Figure 5). On the other hand, the agreement of the TEP of 3Spx* with the (1–T) spectrum over the Qy band proves that Spx is able to protect both α-BChl and β-BChl irrespective to the difference in static energy, which is understandable in view of the close Spx-BChl contact.

4.3. Car-to-Car Triplet EET and Corporative photoprotection in m-LH1-RC. As demonstrated for Tch. tepidum LH2,39,42 Spx in triplet excited state interacts tightly with the ring aggregate of BChl in ground state to form a triplet excimer-like species, which gives rise to a characteristic spectral signature to the longer-wavelength side of the LH2-Qy band. Such kind of ‘interactive’ spectral features were also reported for the LHC II complexes of higher plant.56 In the case of m-LH1-RC complex, Spe keeps van der Waals contact with BChls, and the LH1-BChls are tightly packed as in native Tch-LH1-RC (Scheme 2). In addition, the hybrid m-LH1-RC binds Ca2+ similarly as the native Tch-LH1-RC does,40 and thus reduces the static disorder of BChls by enhancing the protein rigidity. In view of the structural peculiarities of m-LH1-RC, we propose that the LH1-BChls serve as a bridge mediating the transfer of 3

Spe* excitation to its neighboring Spx in a sub-microsecond timescale (∼500 ns). This triplet EET

process is rather slow compared to others, which can be due to the weak BChl-mediated 3Spe*-Spx coupling. It is known that photosynthetic Cars exhibit an extremely low quantum yield of intersystem crossing (∼10−6).57 In addition, the photoexcitation in the Qy spectral region, rather than direct Car excitation, avoids the complication by Car SF reaction. Therefore, the TEP spectra in the Qy region are merely originated from the triplet sensitization reactions. This together with the spectrally and kinetically distinguishable 3Spe* and 3Spx* species allows us to identify the triplet EET reaction between different kinds of Car. The hypothetical Spe-to-Spx triplet EET mechanism readily accounts for the sizable Spx(Tn←T1) absorption despite a low fractional content (7.7%), i.e., this can be explained by the ∼70% efficiency of triplet transfer from Spe to Spx as estimated by using the kinetics parameters in Table 2. In addition, this



Page 18 of 26

mechanism implicitly means the coexistence of different kinds of Cars in an m-LH1-RC complex, because an efficient triplet transfer reaction is feasible only at a van der waals D-A contact. As such, the multi-compositions of Car are in a position to quench 3BChl* excitation in a cooperative manner. Besides the Spe-to-Spx triplet EET herein observed for m-LH1-RC at room temperature, this process of triplet transfer was reported for multi-Car containing Rps. palustris LH2 at 77 K.37 However, for Tch. tepidum LH2 containing multi-compositions of Car, the triplet transfer reaction was not observed either at 77 K or at room temperature.39 Most likely, in the case of m-LH1-RC, the intrinsically higher rigidity and hence the less static disorder of the LH1-BChl aggregate is crucial for BChls to act as a mediator of the Spe-to-Spx triplet transfer. However, whether the cooperative photoprotection holds for other types of LHs containing multi-compositions of Car remained to be further explored. Finally, we look into the efficiency of Car-to-BChl singlet EET in the m-LH1-RC complex. In spite of a high composition of Spe and derivatives, the Spe-to-BChl singlet EET efficiency is as low as 40% as determined from the (1–T) and the fluorescence excitation spectrum in the visible spectral region (Figure S4). In contrast, Spe in LH2 gives rise to a much higher singlet EET efficiency, e.g., >90 % in the case of Rba. sphaeroides 2.4.1.31,33 In general, Cars with nc=c ≤ 10 favor the accessory light harvesting, whereas those with nc=c≥11 exhibits a relatively low light-harvesting efficiency.31,39 The relatively low singlet EET efficiency of Spe and derivatives in m-LH1-RC is partially due to the SF deactivation of 1

Spe*, which becomes active in case the molecular geometry distorts in the π-conjugated region as

indicated by the appearance of the ν4 band in the resonance Raman spectrum (Figure 2). The


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involvement of SF reaction upon direct photoexcitation of Car is demonstrated by the substantially stronger TEPs of 3Spe* and 3Spx* compared to the fluorescence excitation spectrum (Figure S4).31

5. Conclusions In summary, we have investigated the triplet excited state dynamics of the m-LH1-RC complex from a mutant strain of Rba. sphaeroides at room temperature. The maximal Tn←T1 absorption amplitude of Spx as a minor Car component (90%). The TA kinetics of 3Spe* and 3Spx* exhibited a tight decay-to-rise correlation at a sub-microsecond timescale, indicating the involving of Spe-to-Spx triplet excitation transfer. This together with a detailed analysis of the TEPs of 3Spe* and 3Spx* in the LH1-Qy spectral region allow us to propose the selective photoprotection function of Spe and derivatives, i.e. they mainly quench the triplet excitation of BChls bearing relatively low static energy (responsible for the red portion of the LH1-Qy absorption). All of the observations are well explained by a hypothetical pigment orientation scheme for the m-LH1-RC complex, which is analogous to the crystallographic structure of the native LH1-RC complex of Tch. tepidum.11,12 In this scheme, the methoxyl-less end of a Spe molecule is suggested to approach the LH1-BChls, and the rigid BChl aggregate is proposed to mediate the Spe-toSpx triplet EET process at a sub-microsecond timescale (∼500 ns). In view of the generically higher site



Page 20 of 26

energy of β-BChl than α-BChl in the native Tch-LH1-RC complex,55 we propose for m-LH1-RC that the triplet transfer from β-BChl to α-BChl has to be taken into account in the mechanism of photoprotection as previously suggested for LH2s.21,26 It is concluded that the triplet excitation transfer among different types of Cars and BChls in m-LH1-RC constitute a cooperative photoprotection mechanism for the pigment-protein complex. It is interesting to further examine m-LH1-RC and other types of bacterial LHs equipped with multi-compositional Cars by the use of TA spectroscopy covering subpicosecond-tomicrosecond window of delay time. This present work provides a deeper insight into the photoprotection roles of multi-compositional Cars in bacterial photosynthesis.

Acknowledgments The project has been supported by the Natural Science Foundation of China (NSFC, No. 21673288); by JSPS KAKENHI, JP16H04174 (Z.-Y.W.-O.); and by a JSPS Research Fellowship (T.K.). We thank M. T. Madigan for providing the Tch. tepidum strain MC.

Supporting Information Available: Raman line deconvolution, additional kinetics data, TEPs in visible region. This material is available free of charge via the Internet at http://pubs.acs.org.

References 20 ACS Paragon Plus Environment

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Table of Contents

3Spx* 3Spe* Triplet excitation profiles of m-LH1-RC complex

80

60

0.004

40

(1− T)

0.006

∆ OD

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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0.002 20

LH1-Qy

0.000 800

850

900

0 950

Actinic Wavelength (nm)


Cooperative Photoprotection by Multi-Compositional Carotenoids in

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