Structural Basis for the Unusual Q_y Red-Shift and Enhanced Thermostability of the LH1 Complex from <i>Thermochromatium tepidum</i>

While the majority of the core light-harvesting complexes (LH1) in purple photosynthetic bacteria exhibit a Q_y absorption band in the range of 870–890 nm, LH1 from the thermophilic bacterium <i>Thermochromatium tepidum</i> displays the Q_y band at 915 nm with an enhanced thermostability. These properties are regulated by Ca²⁺ ions. Substitution of the Ca²⁺ with other divalent metal ions results in a complex with the Q_y band blue-shifted to 880–890 nm and a reduced thermostability. Following the recent publication of the structure of the Ca-bound LH1-reaction center (RC) complex [Niwa, S., et al. (2014) <i>Nature</i> <i>508</i>, 228], we have determined the crystal structures of the Sr- and Ba-substituted LH1-RC complexes with the LH1 Q_y band at 888 nm. Sixteen Sr²⁺ and Ba²⁺ ions are identified in the LH1 complexes. Both Sr²⁺ and Ba²⁺ are located at the same positions, and these are clearly different from, though close to, the Ca²⁺-binding sites. Conformational rearrangement induced by the substitution is limited to the metal-binding sites. Unlike the Ca-LH1-RC complex, only the α-polypeptides are involved in the Sr and Ba coordinations in LH1. The difference in the thermostability between these complexes can be attributed to the different patterns of the network formed by metal binding. The Sr- and Ba-LH1-RC complexes form a single-ring network by the LH1 α-polypeptides only, in contrast to the double-ring network composed of both α- and β-polypeptides in the Ca-LH1-RC complex. On the basis of the structural information, a combined effect of hydrogen bonding, structural integrity, and charge distribution is considered to influence the spectral properties of the core antenna complex

Excitonic and Vibrational Coherence in the Excitation Relaxation Process of Two LH1 Complexes as Revealed by Two-Dimensional Electronic Spectroscopy

Ultrafast excitation relaxation within a manifold exciton state and long-lived vibrational coherence are two universal characteristics of photosynthetic antenna complexes. In this work, we studied the two-dimensional electronic spectra of two core light-harvesting (LH1) complexes of <i>Thermochromatium</i> (<i>Tch.</i>) <i>tepidum</i>, native Ca²⁺-LH1 and modified Ba²⁺-LH1. The role of the vibrational coherence in the exciton relaxation was revealed by comparing the two LH1 with similar structures but different electronic properties and by the evolution of the exciton and vibrational coherence as a function of temperature

Direct Observation of Energy Detrapping in LH1-RC Complex by Two-Dimensional Electronic Spectroscopy

The purple bacterial core light harvesting antenna-reaction center (LH1-RC) complex is the simplest system able to achieve the entire primary function of photosynthesis. During the past decade, a variety of photosynthetic proteins were studied by a powerful technique, two-dimensional electronic spectroscopy (2DES). However, little attention has been paid to LH1-RC, although its reversible uphill energy transfer, trapping, and backward detrapping processes, represent a crucial step in the early photosynthetic reaction dynamics. Thus, in this work, we employed 2DES to study two LH1-RC complexes of Thermochromatium (Tch.) tepidum. By direct observation of detrapping, the complex reversible process was clearly identified and an overall scheme of the excitation evolution in LH1-RC was obtained

Bacterial Light-Harvesting Complexes Showing Giant Second-Order Nonlinear Optical Response as Revealed by Hyper-Rayleigh Light Scattering

The second-order nonlinear optical (NLO) properties of light-harvesting complexes (LHs) from the purple photosynthetic bacteria Thermochromatium (Tch.) tepidum were investigated for the first time by means of hyper-Rayleigh scattering (HRS). The carotenoid (Car) molecules bound to the isolated LH1 and LH2 proteins gave rise to second-harmonic scattering; however, they showed an opposite effect of the collective contribution from Car, that is, the first hyperpolarizability (β) reduced substantially from (10 510 ± 370) × 10^–30 esu for LH1 to (360 ± 120) × 10^–30 esu for LH2. Chromatophores of Tch. tepidum also showed a giant hyperpolarizability of (11 640 ± 630) × 10^–30 esu. On the basis of the structural information on bacterial LHs, it is found that the effective β of an LH is governed by the microenvironment and orientational correlation among the Car chromophores, which is concluded to be coherently enhanced for LH1. For LH2, however, additional destructive effects between different Car molecules may account for the small β value. This work demonstrates that LH1 and native membranes of purple bacteria can be potent NLO materials and that HRS is a promising spectroscopic means for investigating structural information of pigment–protein supramolecules

Carotenoid Singlet Fission Reactions in Bacterial Light Harvesting Complexes As Revealed by Triplet Excitation Profiles

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 <i>Tch. tepidum</i> and <i>Rba. sphaeroides</i> 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 <i>Rba. sphaeroides</i> 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

Spectroscopic and Thermodynamic Characterization of the Metal-Binding Sites in the LH1–RC Complex from Thermophilic Photosynthetic Bacterium Thermochromatium tepidum

The light-harvesting 1 reaction center (LH1–RC) complex from thermophilic photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits enhanced thermostability and an unusual LH1 <i>Q_y</i> transition, both induced by Ca²⁺ binding. In this study, metal-binding sites and metal–protein interactions in the LH1–RC complexes from wild-type (B915) and biosynthetically Sr²⁺-substituted (B888) Tch. tepidum were investigated by isothermal titration calorimetry (ITC), atomic absorption (AA), and attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopies. The ITC measurements revealed stoichiometric ratios of approximately 1:1 for binding of Ca²⁺, Sr²⁺, or Ba²⁺ to the LH1 αβ-subunit, indicating the presence of 16 binding sites in both B915 and B888. The AA analysis provided direct evidence for Ca²⁺ and Sr²⁺ binding to B915 and B888, respectively, in their purified states. Metal-binding experiments supported that Ca²⁺ and Sr²⁺ (or Ba²⁺) competitively associate with the binding sites in both species. The ATR-FTIR difference spectra upon Ca²⁺ depletion and Sr²⁺ substitution demonstrated that dissociation and binding of Ca²⁺ are predominantly responsible for metal-dependent conformational changes of B915 and B888. The present results are largely compatible with the recent structural evidence that another binding site for Sr²⁺ (or Ba²⁺) exists in the vicinity of the Ca²⁺-binding site, a part of which is shared in both metal-binding sites

Thermal Adaptability of the Light-Harvesting Complex 2 from <i>Thermochromatium tepidum</i>: Temperature-Dependent Excitation Transfer Dynamics

The photosynthetic purple bacterium <i>Thermochromatium (Tch.) tepidum</i> is a thermophile that grows at an optimal temperature of ∼50 °C. We have investigated, by means of steady-state and time-resolved optical spectroscopies, the effects of temperature on the near-infrared light absorption and the excitation energy transfer (EET) dynamics of its light-harvesting complex 2 (LH2), for which the mesophilic counterpart of <i>Rhodobacter</i> (<i>Rba.</i>) <i>sphaeroides</i> 2.4.1 (∼30 °C) was examined in comparison. In a limited range around the physiological temperature (10–55 °C), the B800-to-B850 EET process of the <i>Tch. tepidum</i> LH2, but not the <i>Rba. sphaeroides</i> LH2, was found to be characteristically temperature-dependent, mainly because of a temperature-tunable spectral overlap. At 55 °C, the LH2 complex from <i>Tch. tepidum</i> maintained efficient near-infrared light harvesting and B800-to-B850 EET dynamics, whereas this EET process was disrupted in the case of <i>Rba. sphaeroides</i> 2.4.1 owing to the structural distortion of the LH2 complex. Our results reveal a remarkable thermal adaptability of the light-harvesting function of <i>Tch. tepidum</i>, which could enhance our understanding of the survival strategy of this thermophile in response to environmental challenges

Proteomic and Comparative Genomic Analysis of Two <i>Brassica napus</i> Lines Differing in Oil Content

Ultrastructural observations, combined with proteomic and comparative genomic analyses, were applied to interpret the differences in protein composition and oil-body characteristics of mature seed of two <i>Brassica napus</i> lines with high and low oil contents of 55.19% and 36.49%, respectively. The results showed that oil bodies were arranged much closer in the high than in the low oil content line, and differences in cell size and thickness of cell walls were also observed. There were 119 and 32 differentially expressed proteins (DEPs) of total and oil-body proteins identified. The 119 DEPs of total protein were mainly involved in the oil-related, dehydration-related, storage and defense/disease, and some of these may be related to oil formation. The DEPs involved with dehydration-related were both detected in total and oil-body proteins for high and low oil lines and may be correlated with the number and size of oil bodies in the different lines. Some genes that corresponded to DEPs were confirmed by quantitative trait loci (QTL) mapping analysis for oil content. The results revealed that some candidate genes deduced from DEPs were located in the confidence intervals of QTL for oil content. Finally, the function of one gene that coded storage protein was verified by using a collection of <i>Arabidopsis</i> lines that can conditionally express the full length cDNA from developing seeds of <i>B. napus</i>