LH1-RC Complex Research Articles

High-Performance UV Enhancer Molecules Coupled with Photosynthetic Proteins for Ultra-Low-Intensity UV Detection

Summary Dual attributes of UV-photo-responsive organic-ionic conductors are exploited in bio-photoelectrochemical cells based on photosynthetic RC-LH1 proteins from Rhodobacter sphaeroides. These UV enhancer molecules (UVEM) can generate small photocurrents in the absence of protein and are also effective electrolytes for photocurrent generation by RC-LH1 complexes in response to near-infrared excitation. Mixing RC-LH1 and UVEM components strongly enhanced UV photocurrents relative to those obtained with protein or UVEM alone, an effect that is attributed to energy transfer from the hetero-anthracene chromophore of the UVEM to the carotenoids of the RC-LH1 complex. RC-LH1/UVEM bio-photoelectrochemical cells were superior to conventional RC-LH1 cells in terms of UV external quantum efficiency, photo-response sensitivity, and photocurrent rise-decay times. These bio-photodetectors could detect weak UV radiation with intensities as low as 2 μW/cm2. This combination of photosynthetic proteins with dual-function electrolytes is the first attempt to construct fully functional bio-photoelectrochemical UV photodetector based on natural components.

Orientation assignment of LH2 and LH1-RC complexes from Thermochromatium tepidum reconstituted in PC liposome and their ultrafast excitation dynamics comparison between in artificial and in natural chromatophores

In this study, the artificial chromatophore was constructed by reconstitution of light-harvesting complex 2 (LH2) and core-complex (LH1-RC) from Thermochromatium tepidum in egg phosphatidylcholine (PC) liposome. By calcium ion dependent absorption changes, the orientations of protein complexes in liposomes solely were assigned. Ultrafast EET dynamics were studied by fs time-resolved absorption spectroscopy on both the artificial and natural chromatophores at their RC ‘open’ and ‘close’ states, respectively. The results indicated that these two membrane systems have similar EET dynamic profiles, and the orientations of LH2 and LH1-RC complexes in artificial chromatophores were opposite to those in natural chromatophores.

Structure of photosynthetic LH1-RC supercomplex at 1.9 Å resolution.

Light-harvesting complex 1 (LH1) and the reaction centre (RC) form a membrane-protein supercomplex that performs the primary reactions of photosynthesis in purple photosynthetic bacteria. The structure of the LH1-RC complex can provide information on the arrangement of protein subunits and cofactors; however, so far it has been resolved only at a relatively low resolution. Here we report the crystal structure of the calcium-ion-bound LH1-RC supercomplex of Thermochromatium tepidum at a resolution of 1.9 Å. This atomic-resolution structure revealed several new features about the organization of protein subunits and cofactors. We describe the loop regions of RC in their intact states, the interaction of these loop regions with the LH1 subunits, the exchange route for the bound quinone QB with free quinone molecules, the transport of free quinones between the inside and outside of the LH1 ring structure, and the detailed calcium-ion-binding environment. This structure provides a solid basis for the detailed examination of the light reactions that occur during bacterial photosynthesis.

Towards first-principles calculation of electronic excitations in the ring of the protein-bound bacteriochlorophylls

Modeling electronic excitation of bacteriochlorophyll (BChl) molecules in light-harvesting (LH) antennae from photosynthetic centers presents a challenge for the quantum theory. We report on a quantum chemical study of the ring of 32 BChl molecules from the bacterial core complex LH1-RC. Diagonal and off-diagonal elements of the excitonic Hamiltonian matrices are estimated in quantum chemical calculations of relevant fragments using the TD-DFT and CIS approaches. The deviation of the computed excitation energy of this BChl system from the experimental data related to the Qy band maximum of this LH1-RC complex is about 0.2 eV. We demonstrate that corrections due to improvement in modeling of an individual BChl molecule and due to contributions from the protein environment are in the range of the obtained discrepancy between theory and experiment. Differences between results of the excitonic model and direct quantum chemical calculations of BChl aggregates fall in the same range.

Proposal for verifying dipole properties of light-harvesting antennas

For light harvesters with a reaction center complex (LH1-RC complex) of three types, we propose an experiment to verify our analysis based upon antenna theories that automatically include the required structural information. Our analysis conforms to the current understanding of light-harvesting antennas in that we can explain known properties of these complexes. We provide an explanation for the functional roles of the notch at the light harvester, a functional role of the polypeptide called PufX or W at the opening, a functional role of the special pair, a reason that the cross section of the light harvester must not be circular, a reason that the light harvester must not be spherical, reasons for the use of dielectric bacteriochlorophylls instead of conductors to make the light harvester, a mechanism to prevent damage from excess sunlight, an advantage of the dimeric form, and reasons for the modular design of nature. Based upon our analysis we provide a mechanism for dimerization. We predict that the dimeric form of light-harvesting complexes is favored under intense sunlight. We further comment upon the classification of the dimeric or S-shape complexes. The S-shape complexes should not be considered as the third type of light harvester but simply as a composite form.

Engineered photoproteins that give rise to photosynthetically-incompetent bacteria are effective as photovoltaic materials for biohybrid photoelectrochemical cells.

Reaction centre/light harvesting proteins such as the RCLH1X complex from Rhodobacter sphaeroides carry out highly quantum-efficient conversion of solar energy through ultrafast energy transfer and charge separation, and these pigment-proteins have been incorporated into biohybrid photoelectrochemical cells for a variety of applications. In this work we demonstrate that, despite not being able to support normal photosynthetic growth of Rhodobacter sphaeroides, an engineered variant of this RCLH1X complex lacking the PufX protein and with an enlarged light harvesting antenna is unimpaired in its capacity for photocurrent generation in two types of bio-photoelectrochemical cells. Removal of PufX also did not impair the ability of the RCLH1 complex to act as an acceptor of energy from synthetic light harvesting quantum dots. Unexpectedly, the removal of PufX led to a marked improvement in the overall stability of the RCLH1 complex under heat stress. We conclude that PufX-deficient RCLH1 complexes are fully functional in solar energy conversion in a device setting and that their enhanced structural stability could make them a preferred choice over their native PufX-containing counterpart. Our findings on the competence of RCLH1 complexes for light energy conversion in vitro are discussed with reference to the reason why these PufX-deficient proteins are not capable of light energy conversion in vivo.

Probing structure–function relationships in early events in photosynthesis using a chimeric photocomplex

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium Thermochromatium tepidum requires Ca2+ for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the Tch. tepidum LH1 complex in an engineered Rhodobacter sphaeroides mutant strain. This system contained a chimeric pufBALM gene cluster (pufBA from Tch. tepidum and pufLM from Rba. sphaeroides) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed Tch. tepidum wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in Rba. sphaeroides) was incorporated into the Tch. tepidum LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in Rba. sphaeroides were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the Tch. tepidum LH1 complex; in contrast, α-N50 does not participate in Ca2+ coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated Tch. tepidum LH1-RC complex and have unambiguously identified the location of Ca2+ within this key antenna complex.

Spirilloxanthin incorporation into the LH2 and LH1-RC pigment-protein complexes from a purple sulfur bacterium Allochromatium minutissimum

Incorporation of spirilloxanthin into carotenoidless LH2 and LH1-RC complexes from a purple sulfur bacterium Allochromatium (Alc.) minutissimum was studied. Carotenoidless cells of Alc. minutissimum were obtained using diphenylamine, a carotenoid biosynthesis inhibitor. In the course of incorporation of the carotenoid mixture, the composition of which corresponded to that of Alc. minutissimum control photosynthetic membranes, no selective incorporation of spirilloxanthin into the LH1-RC complex was detected. It is assumed that in vivo carotenoids are not incorporated into the LH2 and LH1-RC complexes from a common pool. Pure spirilloxanthin destroys both the LH2 and LH1-RC complexes. Within the concentration range of spirilloxanthin in the incorporated mixture from 27% to 52%, it was found to be incorporated into the LH2 and LH1-RC complexes with the efficiency of 13% and 33%, respectively. The possible existence of different sites of assembly for the LH2 and LH1-RC complexes is discussed, as well as of two fractions of LH2 complexes, in one of which rhodopin may be integrated, and in the other (minor) one, spirilloxanthin.

Origin of the S* Excited State Feature of Carotenoids in Light-Harvesting Complex 1 from Purple Photosynthetic Bacteria.

This spectroscopic study investigates the origin of the transient feature of the S* excited state of carotenoids bound in LH1 complexes from purple bacteria. The studies were performed on two RC-LH1 complexes from Rba. sphaeroides strains that bound carotenoids with different carbon-carbon double bond conjugation N, neurosporene (N = 9) and spirilloxanthin (N = 13). The S* transient spectral feature, originally associated with an elusive and optically silent excited state of spirilloxanthin in the LH1 complex, may be successfully explained and mimicked without involving any unknown electronic state. The spectral and temporal characteristics of the S* feature suggest that it is associated with triplet-triplet annihilation of carotenoid triplets formed after direct excitation of the molecule via a singlet fission mechanism. Depending on pigment homogeneity and carotenoid assembly in the LH1 complex, the spectro-temporal component associated with triplet-triplet annihilation may simply resolve a pure T-S spectrum of a carotenoid. In some cases (like spirilloxanthin), the T-S feature will also be accompanied by a carotenoid Stark spectrum and/or residual transient absorption of minor carotenoid species bound into LH1 antenna complex.

Effects of Calcium Ions on the Thermostability and Spectroscopic Properties of the LH1-RC Complex from a New Thermophilic Purple Bacterium Allochromatium tepidum.

The light harvesting-reaction center (LH1-RC) complex from a new thermophilic purple sulfur bacterium Allochromatium (Alc.) tepidum was isolated and characterized by spectroscopic and thermodynamic analyses. The purified Alc. tepidum LH1-RC complex showed a high thermostability comparable to that of another thermophilic purple sulfur bacterium Thermochromatium tepidum, and spectroscopic characteristics similar to those of a mesophilic bacterium Alc. vinosum. Approximately 4-5 Ca2+ per LH1-RC were detected by inductively coupled plasma atomic emission spectroscopy and isothermal titration calorimetry. Upon removal of Ca2+, the denaturing temperature of the Alc. tepidum LH1-RC complex dropped accompanied by a blue-shift of the LH1 Qy absorption band. The effect of Ca2+ was also observed in the resonance Raman shift of the C3-acetyl νC═O band of bacteriochlorophyll-a, indicating changes in the hydrogen-bonding interactions between the pigment and LH1 polypeptides. Thermodynamic parameters for the Ca2+-binding to the Alc. tepidum LH1-RC complex indicated that this reaction is predominantly driven by the largely favorable electrostatic interactions that counteract the unfavorable negative entropy change. Our data support a hypothesis that Alc. tepidum may be a transitional organism between mesophilic and thermophilic purple bacteria and that Ca2+ is one of the major keys to the thermostability of LH1-RC complexes in purple bacteria.

Incorporation of spheroidene and spheroidenone into light-harvesting complexes from purple sulfur bacteria

Spheroidene and spheroidenone from the non-sulfur bacterium Rhodobacter (Rba.) sphaeroides were incorporated into diphenylamine (DPA) LH1-RC and LH2 complexes from sulfur bacteria Allochromatium (Alc.) minutissimum and Ectothiorhodospira (Ect.) haloalkaliphila in which carotenoid (Car) biosynthesis was inhibited by ~95%. A series of biochemical characteristics of the modified LH2 complexes was studied (electrophoretic mobility, absorption and CD spectra, Car composition, Car-to-BChl energy transfer and thermal stability). It was found that the electrophoretic mobility of the complexes with incorporated Cars did not change compared to that of the control and DPA-complexes, indicating the absence of any significant change in the structure of LH complexes upon DPA-treatment and subsequent incorporation of Cars. The analysis of fluorescence excitation spectra of the spheroidene-incorporated LH2 complex (LH2:sph) and the spheroidenone-incorporated LH2 complex (LH2:sph-ne) showed that spheroidene and spheroidenone exhibited relatively low efficiencies of energy transfer to BChl, when incorporated into the LH2 DPA-complexes from Alc. minutissimum and Ect. haloalkaliphila, although, they showed high efficiencies, being in their natural state in the LH2 complexes from Rba. sphaeroides. A significant increase in thermostability observed for the LH2:sph and LH2:sph-ne complexes with respect to the LH2 DPA-complexes indicated that the two incorporated Cars stabilized the structure of the LH2 complexes.

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.

Structural Basis for the Unusual Qy Red-Shift and Enhanced Thermostability of the LH1 Complex from Thermochromatium tepidum.

While the majority of the core light-harvesting complexes (LH1) in purple photosynthetic bacteria exhibit a Qy absorption band in the range of 870-890 nm, LH1 from the thermophilic bacterium Thermochromatium tepidum displays the Qy 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 Qy 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) Nature 508, 228], we have determined the crystal structures of the Sr- and Ba-substituted LH1-RC complexes with the LH1 Qy 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.

Carotenoid-to-Bacteriochlorophyll Energy Transfer in the LH1-RC Core Complex of a Bacteriochlorophyll b Containing Purple Photosynthetic Bacterium Blastochloris viridis.

Carotenoid-to-bacteriochlorophyll energy transfer has been widely investigated in bacteriochlorophyll (BChl) a-containing light harvesting complexes. Blastochloris viridis utilizes BChl b, whose absorption spectrum is more red-shifted than that of BChl a. This has implications on the efficiency and pathways of carotenoid-to-BChl energy transfer in this organism. The carotenoids that comprise the light-harvesting reaction center core complex (LH1-RC) of B. viridis are 1,2-dihydroneurosporene and 1,2-dihydrolycopene, which are derivatives of carotenoids found in the light harvesting complexes of several BChl a-containing purple photosynthetic bacteria. Steady-state and ultrafast time-resolved optical spectroscopic measurements were performed on the LH1-RC complex of B. viridis at room and cryogenic temperatures. The overall efficiency of carotenoid-to-bacteriochlorophyll energy transfer obtained from steady-state absorption and fluorescence measurements were determined to be ∼27% and ∼36% for 1,2-dihydroneurosporene and 1,2-dihydrolycopene, respectively. These results were combined with global fitting and target analyses of the transient absorption data to elucidate the energetic pathways by which the carotenoids decay and transfer excitation energy to BChl b. 1,2-Dihydrolycopene transfers energy to BChl b via the S2 → Qx channel with kET2 = (500 fs)(-1) while 1,2-dihydroneurosporene transfers energy via S1→ Qy (kET1 = (84 ps)(-1)) and S2 → Qx (kET2 = (2.2 ps)(-1)) channels.

Temperature dependent LH1 → RC energy transfer in purple bacteria Tch. tepidum with shiftable LH1-Qy band: A natural system to investigate thermally activated energy transfer in photosynthesis

The native LH1-RC complex of the purple bacterium Thermochromatium (Tch.) tepidum has an ultra-red LH1-Qy absorption at 915nm, which can shift to 893 and 882nm by means of chemical modifications. These unique complexes are a good natural system to investigate the thermally activated energy transfer process, with the donor energies different while the other factors (such as the acceptor energy, special pair at 890nm, and the distance/relative orientation between the donor and acceptor) remain the same. The native B915-RC, B893-RC and B882-RC complexes, as well as the LH1-RC complex of Rhodobacter (Rba.) sphaeroides were studied by temperature-dependent time-resolved absorption spectroscopy. The energy transfer time constants, kET−1, are 65, 45, 46 and 45ps at room temperature while 225, 58, 85, 33ps at 77K for the B915-RC, B893-RC, B882-RC and Rba. sphaeroides LH1-RC, respectively. The dependences of kET on temperature have different trends. The reorganization energies are determined to be 70, 290, 200 and 45cm−1, respectively, by fitting kET vs temperature using Marcus equation. The activation energies are 200, 60, 115 and 20cm−1, respectively. The influences of the structure (the arrangement of the 32 BChl a molecules) on kET are discussed based on these results, to reveal how the B915-RC complex accomplishes its energy transfer function with a large uphill energy of 290cm−1.

The origin of the unusual Qy red shift in LH1–RC complexes from purple bacteria Thermochromatium tepidum as revealed by Stark absorption spectroscopy

Native LH1–RC of photosynthetic purple bacteria Thermochromatium (Tch.) tepidum, B915, has an ultra-red BChl a Qy absorption. Two blue-shifted complexes obtained by chemical modification, B893 and B882, have increasing full widths at half maximum (FWHM) and decreasing transition dipole oscillator strength. 77K Stark absorption spectroscopy studies were employed for the three complexes, trying to understand the origin of the 915nm absorption. We found that Tr(∆α) and |∆μ| of both Qy and carotenoid (Car) bands are larger than for other purple bacterial LH complexes reported previously. Moreover, the red shifts of the Qy bands are associated with (1) increasing Tr(∆α) and |∆μ| of the Qy band, (2) the red shift of the Car Stark signal and (3) the increasing |∆μ| of the Car band. Based on the results and the crystal structure, a combined effect of exciton-charge transfer (CT) states mixing, and inhomogeneous narrowing of the BChl a site energy is proposed to be the origin of the 915nm absorption. CT-exciton state mixing has long been found to be the origin of strong Stark signal in LH1 and special pair, and the more extent of the mixing in Tch. tepidum LH1 is mainly the consequence of the shorter BChl–BChl distances. The less flexible protein structure results in a smaller site energy disorder (inhomogeneous narrowing), which was demonstrated to be able to influence |∆μ| and absorption.

Effects of the protonophore carbonyl-cyanide m-chlorophenylhydrazone on intracytoplasmic membrane assembly in Rhodobacter sphaeroides

The effect of carbonyl-cyanide m-chlorophenyl-hydrazone (CCCP) on intracytoplasmic membrane (ICM) assembly was examined in the purple bacterium Rhodobacter sphaeroides. CCCP blocks generation of the electrochemical proton gradient required for integral membrane protein insertion. ICM formation was induced for 8h, followed by a 4-h exposure to CCCP. Measurements of fluorescence induction/relaxation kinetics showed that CCCP caused a diminished quantum yield, a cessation in expansion of the functional absorption cross-section and a 4- to 10-fold slowing in the electron transfer turnover rate. ICM vesicles (chromatophores) and an upper-pigmented band (UPB) containing ICM growth initiation sites, were isolated and subjected to clear-native electrophoresis. Proteomic analysis of the chromatophore gel bands indicated that CCCP produced a 2.7-fold reduction in spectral counts in the preferentially assembled light-harvesting 2 (LH2) antenna, while the RC-LH1 complex, F1FO-ATPase and pyridine nucleotide transhydrogenase decreased by 1.7–1.9-fold. For 35 soluble enzymes, the ratio of 0.99 for treated/control proteins demonstrated that protein synthesis was unaffected by CCCP, suggesting that the membrane complex decline arose from the turnover of unassembled apoproteins. In the UPB fraction, an ~2-fold accumulation was observed for the preprotein translocase SecY, the SecA translocation ATPase, SecD and SecF insertion components, and chaperonins DnaJ and DnaK, consistent with the possibility that these factors, which act early in the assembly process, have accumulated in association with nascent polypeptides as stabilized assembly intermediates.

The open, the closed, and the empty: time-resolved fluorescence spectroscopy and computational analysis of RC-LH1 complexes from Rhodopseudomonas palustris.

We studied the time-resolved fluorescence of isolated RC-LH1 complexes from Rhodopseudomonas palustris as a function of the photon fluence and the repetition rate of the excitation laser. Both parameters were varied systematically over 3 orders of magnitude. On the basis of a microstate description we developed a quantitative model for RC-LH1 and obtained very good agreement between experiments and elaborate simulations based on a global master equation approach. The model allows us to predict the relative population of RC-LH1 complexes with the special pair in the neutral state or in the oxidized state P(+) and those complexes that lack a reaction center.

光合成反応中心と集光アンテナタンパク質との複合体（LH1-RC）の結晶構造

For efficient use of sunlight, the light-harvesting core antenna (LH1) and the reaction center (RC) form a supramolecular complex (LH1-RC) in photosynthetic bacteria. We determined the crystal structure of a LH1-RC complex from a purple bacterium, Thermochromatium tepidum. The structure reveals that the RC is completely surrounded by 16 αβ-heterodimers. Calcium ions are bound at the periplasmic side of LH1. 32 bacteriochlorophyll and 16 spirilloxanthin molecules form an elliptical assembly in LH1. The pigment geometry involved in the absorption characteristics and excitation energy transfer is determined. In addition, ubiquinone channels and lipid molecules are observed in the complex.

Distribution of colored carotenoids between light-harvesting complexes in the process of recovering carotenoid biosynthesis in Ectothiorhodospira haloalkaliphila cells

The processes of recovering colored-carotenoid (Car) biosynthesis in Car-less cells of the purple sulfur bacterium Ectothiorhodospira haloalkaliphila grown with diphenylamine (DPA-cells) have been studied. It has been found that (1) the rate of recovering colored-Car biosynthesis in the lag-phase is far ahead of the growth rate of the cells themselves; (2) several Cars (ζ-carotene, neurosporene etc.) act as intermediates in Car biosynthesis; (3) because filling the “empty” Car pockets in the LH1-RC complexes is faster than in LH2, available spirilloxanthin is preferentially incorporated into the nascent LH1-RC core particles; (4) as a consequence of the resulting lack of spirilloxanthin availability, the biosynthetic intermediates (anhydrorhodovibrin, rhodopin and lycopene) fill the empty nascent LH2 Car pockets. In the present report, we further discuss the process of colored Car incorporation into LH complexes during the recovery of Car biosynthesis in the DPA-cells of Ect. haloalkaliphila.