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Bacterial Light-Harvesting Complexes Showing Giant Second-Order Nonlinear Optical Response as Revealed by Hyper-Rayleigh Light Scattering Fei Ma, Long-Jiang Yu, Xiao-hua Ma, Peng Wang, Zheng-Yu Wang-Otomo, and Jian-Ping Zhang J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b07461 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016
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Bacterial Light-Harvesting Complexes Showing Giant Second-Order Nonlinear Optical Response as Revealed by Hyper-Rayleigh Light Scattering
Fei Ma, 1* Long-Jiang Yu, 2 Xiao-Hua Ma, 1 Peng Wang,1 Zheng-Yu Wang-Otomo,2 Jian-Ping Zhang1*
1
Department of Chemistry, Renmin University of China, Beijing 1000872, P. R. China 2
Faculty of Science, Ibaraki University, Mito, Ibaraki 310-8512, Japan
*To whom correspondence should be addressed. Tel: +86-10-62516604; Fax: +86-10-62516444 E-mail:
[email protected] (F. Ma);
[email protected] (J.-P. Zhang)
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Abstract The second-order nonlinear optical properties of light-harvesting complexes (LHs) from purple photosynthetic bacteria Thermochromatium (Tch.) tepidum was investigated for the first time by means of hyper-Rayleigh scattering (HRS). The carotenoid molecules bound in the isolated LH1 and LH2 proteins both gave rise to second-harmonic scattering, however, with an opposite effect of collective contribution from carotenoid, i.e. the first hyperpolarizability (β) reduced substantially from (10510±370)×10−30 esu for LH1 to (360±120)×10−30 esu for LH2. Chromatophores of Tch. tepidum were also found a giant hyperpolarizability of (11640±630)×10−30 esu. Based on the structural information of bacterial LHs, it is found that the effective β of a LH is governed by the micro-environment and the orientational correlation among the carotenoid chromophores, which is concluded to be coherently enhancement for LH1. For LH2, however, additional destructive effects between different Car molecules may account for the small β value. This work demonstrates that the LH1 complexes and native membranes of purple bacteria can be potent nonlinear optical materials, and that HRS is a promising spectroscopic means for investigating structural information of the pigment-protein supramolecules.
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Introduction Photosynthetic pigment-protein complexes have been proposed as potential materials for nonlinear optical (NLO) applications, such as electro-optic modulators and frequency-doublers, owing to the bulky π-electron conjugation characteristics of pigment cofactors and the noncentrosymmetric protein environment which may enable high first hyperpolarizability (β) and hence strong second-order NLO responses.1–5 For the characterization of β, hyper-Rayleigh scattering (HRS), an optical means based on two-photon absorptivity and second harmonic light detection, has been shown to be both powerful and convenient,6–9 and this technique has been applied to a variety of chemical, material or chromophore-containing protein systems in solution since its early development in 1990’s. 1,2,8,10–19 In the present work, the second-order NLO phenomena of purple bacteria were investigated by means of HRS. Purple bacteria are perhaps the simplest and most widely studied photosynthetic organisms, whose photosynthetic membranes contain peripheral light-harvesting complexes, denoted as LH2, and reaction centers (RC) encircled by core light-harvesting complexes (LH1), referred to as LH1-RC core complexes.20–26 These pigment-protein complexes are nanoscale trans-membrane assemblies embedded in lipid-bilayer vesicles called chromatophores (see Figure 3E-G). We have quantitatively investigated the hyperpolarizability response of the LH1-RC and LH2 proteins from Thermochromatium (Tch.) tepidum in their respective isolated forms or integrated natively in chromatophores, and have explored the structural origins of the second-order NLO responses. It is shown that the spatial orientation of the conjugated carotenoid molecules plays an essential role in the macroscopic second-order NLO response of these pigment-protein complexes.
Experimental section Sample preparation. All-trans-Spx and β-Car were purchased from Sigma, and was recrystallized before using. Spectral grade tetrahydrofuran and chloroform were purchased from Chemical Reagent Ltd. Spx and β-Car solutions were prepared in a concentration range of 10−6 to 10−5 M. The chromatophore, LH1-RC and LH2 complexes of Tch. tepidum were prepared and purified following previously reported procedures.24,27,28 The complexes were suspended in 20 mM Tris-HCl buffers (pH 7.5) ACS Paragon Plus Environment
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containing
0.08%
(w/v)
n-octyl-β-D-glucopyranoside
(β-OG)
for
LH1-RC
or
0.1%
(w/v)
n-dodecyl-β-D-maltoside (DDM) for LH2. The RC of Tch. tepidum was purified according to previously reported procedures29 and suspended in 20 mM Tris-HCl buffer (pH 7.5) containing 0.05% (w/v) DDM. The concentration of LH1-RC and LH2 suspensions were 10−6 to 10−5 M, determined with the extinction coefficient of 4.32×106 L·mol−1·cm−1 at 915 nm for LH1-RC and 1.57×106 L·mol−1·cm−1 at 850 nm for LH2. The concentration of RC suspensions were 10−5 to 10−4 M, with the extinction coefficient of 2.88×105 L·mol−1·cm−1 at 802 nm. The Rs. rubrum LH1-RC complex was prepared by solubilizing the chromatophores with 1.0 % w/v DDM in 20 mM Tris-HCl (pH 8.5) buffer for 60 min at room temperature, followed by differential centrifugation. The supernatant was loaded onto a DEAE column (Toyopearl 650S, TOSOH) equilibrated at 4 ˚C with 20 mM Tris-HCl buffer (pH 7.5) containing 0.1 % w/v of DDM. The LH1-RC fraction was eluted by a linear gradient of NaCl from 0 mM to 250 mM, and the peak fractions with A878/A280 > 2.2 were collected for subsequent measurement. The Rs. rubrum LH1-only complex was prepared by a procedure reported previously30. The Rs. rubrum Car-free LH1-RC complex was prepared by selectively extracting the Car from the purified LH1-RC complex with benzene. The extracting process was repeated three times, and the benzene-treated LH1-RC was dried by vacuum and then dissolved in 20 mM Tris-HCl (pH 7.5) buffer containing 0.05% w/v DDM. For the three LH1-RC complexes of Rs. rubrum, the extinction coefficient 3.8×106 L·mol−1·cm−1 at Qy maxima was used. HRS measurement. Near-infrared HRS excitation laser pulses (880−1200 nm, ∼7 ns, 10 Hz) were supplied by an optical parametric oscillator (MOPO-SL, Spectra-Physics Laser Inc., Mountain View, California, USA) driven by a Q-switched Nd3+:YAG laser (Pro-230, Spectra Physics Laser Inc., Mountain View, California, USA). The excitation beam (3 mJ/pulse) was focused with a lens (focal length 250 mm) into the sample cell. The HRS signal was collected with a camera lens (f/1.4), and passed through a quarttz depolarizer before focusing onto the entrance slit of a triplet spectrograph (Tri-Vista, Princeton Instruments/Acton, Trenton, NJ, USA), and was detected with an ICCD detector (Intensifire: UNIGENTM; CCD: II PI-MAX, Princeton Instruments/Acton, Trenton, NJ, USA). The ICCD detector was electrically gated (10 ns) and synchronized to the laser flash with an electronic delay (DG 535). Spectra resolved HRS ACS Paragon Plus Environment
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signal was obtained by averaging 2500 acquisitions. All of the measurments were performed at room temperature (298 K). For each β measurement, three independent measurements were employed to obtain the error bar. After each measurement, the degree of the solution degradation during experiment was monitored by UV-Vis spectra. Typically, less than 2% degradation of the starting solution was observed. Calculation of β. The HRS signal collected usually superposes on a two-photon luminescence background, as the black line in Figure 1A. After subtracting this background (red line), the obtained spectrum (blue line) was fitted with a Gaussian function (orange line), with the amplitude as the HRS intensity I(2ω). For high concentration samples, the self-absorption of the HRS signal is not negligible, so I(2ω)
(pink
circles
in
Figure
1B)
was
calibrated
with
Beer-Lambert
Law,
using
I cali ( 2ω ) = I ( 2ω ) / exp ( −ε 2ω cl ) (black circles), where ε 2ω is the extinction coefficient at second harmonic wavelength. For each β, six samples with a serial of concentrations were measured independently. The slope of the linear fitting line (black line) of I cali ( 2ω ) against concentration, Slopes, is used to calculate βs, with
β s2 =
Slopes 2 β r , where Sloper is for the external standard, p-Nitroaniline in Sloper
dimethylsulfoxide, obtained with the same way as Slopes. βr at 1000 nm used was 34.5×10−30 esu.31
Figure 1. Procedure for HRS signal progress (A) and calculation of β (B). ACS Paragon Plus Environment
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Results and discussion
Figure 2. Normalized absorption spectra (black, left vertical axis) and experimental β values (blue circle with error bar, right vertical axis) for all-trans-Spx in (A) tetrahydrofuran and (B) chloroform. Blue lines are fitting curves of the data points with Gaussian functions.
The main pigments in purple bacteria are bacteriochlorophyll (BChl) a and carotenoid (Car). At the HRS excitation wavelengths of 900−1200 nm, the β values of BChl a free in chloroform solution are rather small, ˂4×10−30 esu. Car exhibits a much larger first hyperpolarizability (Figures 2 and S1, Table 1), which is mainly due to the two-photon resonance enhancement with the optically active singlet-excited state S2 (1Bu+). Various factors contribute to the electronic hyperpolarizability of Car, such as the number of conjugated C=C bonds (N), the electron-withdrawing side or end substituents, and the polarity of solvents or protein surroundings.1,3,32,33 Comparing spirilloxanthin (Spx, N=13) and β-carotene (β-Car, N=11) in different solvents, we see that the structural and environmental factors are substantially influencing on the hyperpolarizability of Car (Figures 2 and S1). As seen in Figure 2 for Spx in tetrahydrofuran (chloroform), the HRS excitation profile, i.e. the dependence of β on the excitation wavelength, can be described by a Gaussian model function, revealing a 8 nm (15 nm) redshift of the peak response wavelength with respect to the 0→0 vibronic absorption maximum of the UV-visible spectra. Such redshift is due to the discrepancy in the origins of one- and two-photon absorptive transition energies.34
Table 1. The β values (×10−30 esu) of complexes and chromatophore from different purple bacteria. HRS excitation ACS Paragon Plus Environment
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wavelength was 1000 nm. RC Tch. tepidum Rs. rubrum Rs. rubrum (RC free) Rs. rubrum (Car-less) BChl a in chloroform Spx in THF
LH2
LH1-RC
Chromatophore
390 ± 60 360 ± 120 10900 ± 370 11640 ± 630 10070 ± 390 9240 ± 500 170 ± 50 ˂4 630 ± 60
Figure 3. Normalized absorption spectra (black, left vertical axis) and experimental β values (blue circle with error bar, right vertical axis) for the RC (A), LH1-RC (B), LH2 complexes (C) and chromatophore (D) of Tch. tepidum. Blue lines are fitting curves of the data points with Gaussian functions. The arrangements of carotenoids (Spx) in the Tch. tepidum LH1-RC (E, 3wmm24) and Rps. acidophila LH2 (F, 2fkw22) complexes are shown. (G) is the model structure of the native photosynthetic membrane of Rb. sphaeroides.25
Figure 3A-D show the HRS excitation profiles for the RC, LH1-RC, LH2 complexes and the chromatophores of Tch. tepidum. The aromatic amino acids of these membrane proteins contribute little to the overall first hyperpolarizability because of their rather small β values (
