Selective assembly of photosynthetic antenna proteins into a domain-structured lipid bilayer for the construction of artificial photosynthetic antenna systems: structural analysis of the assembly using surface plasmon resonance and atomic force microscopy

Two types of photosynthetic membrane proteins, the peripheral antenna complex (LH2) and the core antenna/reaction center complex (LH1-RC), play an essential role in the primary process of purple bacterial photosynthesis, that is, capturing light energy, transferring it to the RC where it is used in subsequent charge separation. Establishment of experimental platforms is required to understand the function of the supramolecular assembly of LH2 and LH1-RC molecules into arrays. In this study, we assembled LH2 and LH1-RC arrays into domain-structured planar lipid bilayers placed on a coverglass using stepwise combinations of vesicle-to-planar membrane formation and vesicle fusion methods. First, it was shown that assembly of LH2 and LH1-RC in planar lipid bilayers, through vesicle-to-planar membrane formation, could be confirmed by absorption spectroscopy and high resolution atomic force microscopy (AFM). Second, formation of a planar membrane incorporating LH2 molecules made by the vesicle fusion method was corroborated by AFM together with quantitative analysis by surface plasmon resonance (SPR). By combining planar membrane formation and vesicle fusion, in a stepwise manner, LH2 and LH1-RC were successfully organized in the domain-structured planar lipid membrane. This methodology for construction of LH2/LH1-RC assemblies will be a useful experimental platform with which to investigate energy transfer from LH2 to LH1-RC where the relative arrangement of these two complexes can be controlled.     

Excited state dynamics in photosynthetic reaction center and light harvesting complex 1

Key to efficient harvesting of sunlight in photosynthesis is the first energy conversion process in which electronic excitation establishes a trans-membrane charge gradient. This conversion is accomplished by the photosynthetic reaction center (RC) that is, in case of the purple photosynthetic bacterium Rhodobacter sphaeroides studied here, surrounded by light harvesting complex 1 (LH1). The RC employs six pigment molecules to initiate the conversion: four bacteriochlorophylls and two bacteriopheophytins. The excited states of these pigments interact very strongly and are simultaneously influenced by the surrounding thermal protein environment. Likewise, LH1 employs 32 bacteriochlorophylls influenced in their excited state dynamics by strong interaction between the pigments and by interaction with the protein environment. Modeling the excited state dynamics in the RC as well as in LH1 requires theoretical methods, which account for both pigment-pigment interaction and pigment-environment interaction. In the present study we describe the excitation dynamics within a RC and excitation transfer between light harvesting complex 1 (LH1) and RC, employing the hierarchical equation of motion method. For this purpose a set of model parameters that reproduce RC as well as LH1 spectra and observed oscillatory excitation dynamics in the RC is suggested. We find that the environment has a significant effect on LH1-RC excitation transfer and that excitation transfers incoherently between LH1 and RC.     

Long-lived photoinduced charge separation in carbazole–pyrene–viologen system incorporated in Langmuir–Blodgett films of substituted diazacrown ethers

Diaza-18-crown-6 ethers appending two pyrenyl (Py) or two carbazolyl (Cz) groups were synthesized. These macrocyclic compounds form 1:1 host–guest complexes with methyl viologen chloride (MV²⁺), and these complexes were assembled into monolayers by Langmuir–Blodgett technique. The generated assembly involves the general structure of donor–sensitizer–acceptor (Cz–Py–MV²⁺) in space, although any of the photo- and redox-active components are not covalently bonded. Photoirradiation of the pyrenyl group resulted in the charge-separated pair Cz√⁺–Py–MV√⁺ which survived up to hours in a well anaerobic atmosphere. An electrode was fabricated by transferring the L–B film on an ITO glass. The photoinduced voltage of this electrode was measured with a saturated calomel reference electrode in hydroquinone (H₂Q) solution to be ca. 168 mV when the light intensity was 218 mW/cm². This electrode was also used as the light electrode to construct a photogalvanic cell with a platinum electrode as the dark electrode. Irradiation of the light electrode with visible light results in anodic photocurrent, and there is no net chemical change associated with the function of the cell which converts light to electricity.     

Structure–function relationships of the supramolecular assembly of the bacterial photosynthetic antenna complexes in lipid membranes

Appropriate experimental platforms are required to clarify the structure–function relationships of membrane protein assemblies. In photosynthetic bacteria, light-harvesting complex 2 and light-harvesting/reaction center core complex play key roles in capturing and transferring light energy and facilitating subsequent charge separation. These photosynthetic apparatuses form a supramolecular assembly in the photosynthetic membrane. However, the mechanism through which this assembly influences the efficiency of energy conversion remains to be clarified. We review our recent studies that were conducted to evaluate the structure–function relationship of the supramolecular assembly of photosynthetic antenna complexes in various lipid bilayer systems, as well as the construction of novel systems of planar lipid membranes for use as experimental platforms.     

LIGHT-INDUCED ELECTRON TRANSPORT ACROSS SEMICONDUCTOR ELECTRODE/REACTION-CENTER FILM/ELECTROLYTE INTERFACES

Abstract— Reaction center (RC) complexes isolated from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26 were dried as a film onto platinum and semiconductor (SnO₂) electrodes. The light-induced primary charge separation which occurs across the biological complex couples electrically with the SnO₂ but not with the metal electrode on the time scale of observation. As the working electrode in a two-electrode photoelectrochemical cell, RC-coated SnO₂ generated photovoltages as high as 80 mV and photocurrents as high as 0.5µA·cm² when exposed to light of λ >600nm. The number of quinone molecules per RC strongly influences the photovoltage and photocurrent observed. Photo-effects generated by RC electrodes persist after several days of storage; however, the kinetics and polarity of the effects are subject to change. The potential use of RC electrodes lies more as a new probe of photosynthetic electron transport rather than as a solar energy conversion device because modification to the RCs and their environment affect the electrical properties of the cell. An energy-level model is proposed to explain how the photoelectrochemical cell functions.     

Molecular assembly of Zn porphyrin complexes onto a gold electrode using synthetic light-harvesting model polypeptides

Molecular assembly of Zn porphyrin pigments, Zn substituted bacteriochlorophyll a ([Zn]-BChl a) and Zn mesoporphyrin monomer (ZnMPMME) on a gold electrode using synthetic 1α-helix hydrophobic polypeptides which have similar amino acid sequences to the hydrophobic core in the native photosynthetic light-harvesting (LH) 1-β polypeptide from Rhodobacter sphaeroides, has been achieved: this process is dependent on the structures of pigments and polypeptides. Interestingly, an enhanced photoelectric current was observed when ZnMPMME with the LH1 model polypeptide in an α-helical configuration was assembled onto the electrode.     

Structural, spectral and thermal properties of a polymeric nickel(II) complex containing two-dimensional network

The structure of the complex [Ni(hmt)(NCS)₂(H₂O)₂]_n, assembled by hexamethylenetetramine (hmt) and octahedral Ni(II), is reported. Crystal data: Fw 351.07, a=9.885(10) Å, b=12.06(1) Å, c=12.505(8) Å, β=114.41(4)°, V=1357(1) ų, Z=4, space group=C2/c, T=173 K, λ(Mo−K)=0.71070 Å, ρ_calc=1.718 gcm⁻¹, μ=17.44 cm⁻¹, R=0.099, Rw=0.145. The tetrahedral assembling template effect of the hmt molecule is completed by two coordination bonds and two hydrogen interactions. The UV–vis absorption spectrum of this complex [Ni(hmt)(NCS)₂(H₂O)₂]_n with a two-dimensional network is determined in the range of 5000–35000 cm⁻¹ at room temperature. The observed spectrum is discussed and explained perfectly by the scaling radial theory proposed by us. The two-dimensional structure has no apparent effects on the d–d transitions of the central Ni(II) ion. The IR spectrum and the GT curve of the complex were also measured and clearly reflect its structural properties.     

Photoelectric properties of Cz-Py-MV2+ monolayer films

Diaza-18-crown-6-ethers appending two pyrenyl (Py-C) or two carbazolyl (Cz-C) groups form 1 : 1 host–guest complexes with methyl viologen chloride (MV²⁺). These complexes were assembled into monolayers by Langmuir-Blodgett (L-B) technique. The generated assembly involves the general structure of donor-sensitizer-acceptor (Cz-Py-MV²⁺) in space, although any of the photo- and redox-active components are not covalently bonded. This assembly was transferred on an indiumtin oxide (ITO) glass to fabricate an electrode. The photoinduced voltage of this electrode was measured with a saturated calomel reference electrode in hydroquinone (H₂Q) solution to be ca. 168 mV with the light intensity of ca. 218 mW/cm². This electrode was used as the light electrode to construct a photogalvanic cell with a platinum electrode as the dark electrode. Irradiation of the light electrode resulted in anodic photocurrent. The effects of light intensity, bias voltage, concentration of H₂Q and oxygen on the photocurrent were investigated.     

Partition of carotenoid-to-bacteriochlorophyll singlet-energy transfer through two channels in the LH2 complex from Rhodobacter sphaeroides G1C

The excited-state dynamics of both carotenoid (Car) and bacteriochlorophyll (BChl) in the LH2 complex from Rhodobacter sphaeroides G1C were simultaneously probed by subpicosecond time-resolved absorption spectroscopy in the visible and near-infrared regions. By the use of a four excited-state model, where the 1B_u⁺ and 1B_u⁻ states were treated inclusively as the ‘1B_u' state, the time constant and the efficiency partition of Car-to-BChl singlet-energy transfer were determined to be 67–114 fs and 60–74% for the ‘1B_u' channel, and 1.39–1.42 ps and 24–38% for the 2A_g⁻ channel, when a time constant of 170–190 fs was assumed for the 1B_u-to-2A_g⁻ internal conversion.     

Conductive wiring of immobilized photosynthetic reaction center to electrode by cytochrome C

The photosynthetic reaction center (RC) found in photosynthetic bacteria is one of the most advanced photoelectronic devices developed by nature. However, after immobilization on the electrode surface, the efficiency of electron transfer (ET) between the RC and the electrode is relatively low. This inefficiency has limited the possibility of using the RC for technological applications. Here we show that photoinduced electron transfer between the immobilized RC and a gold electrode can be increased by several tens-fold by incorporation of cytochrome c into the RC-self-assembled monolayer (SAM)-electrode complex. The effect does not depend on the initial redox state of the cytochrome and seems to be the result of the formation of a complex between the RC and the cytochrome c serving as an ET wire. This observation opens the possibility for electrochemical analysis of the special pair in the RC protein that is deeply buried inside the protein globe and is barely electrically addressable from the electrode surface.     

Topological characterization and immobilization of a chromatophore membrane from Rhodopseudomonas viridis for application as a photoelectrical device

We characterized topological orientation of bacterial photosynthetic membranes (chromatophores) from Rhodopseudomonas viridis using antibodies against H- and C-subunit of photosynthetic reaction center protein (RC). About 40% of the membranes were unsealed vesicles in the inside-out orientation exposing the cytoplasmic side whereas 20% of those were unsealed vesicles in the right side-out orientation expo-sing the periplasmic side. The residual 20% was in the form of a flat sheet structure. We immobilized the chromatophore membranes onto an indium–tin-oxide (ITO) electrode by the electro-deposition method. The electro-deposited film (ED film) showed photocurrent and photovoltage between different electrodes such as ITO and gold (Au). Addition of buffer salts such as sodium phosphate enhanced very much the photoelectrical response in the ED film.     

Evidence for two light-induced metastable states in Cl3[Ru(NH3)5NO]H2O

Differential Scanning Calorimetry measurements on irradiated Cl₃[Ru(NH₃)₅NO]H₂O reveal the existence of two light-induced long-lived metastable states SI, SII. Irradiation with light in the spectral range 400–500 nm leads to the excitation of SI. For the first time we report experimental evidence for the state SII in this compound, which can be excited by transferring SI into SII with irradiation of light in the spectral range 1000–1200 nm. The excitation and transfer of the metastable states is described and the exponential decays are evaluated according to Arrhenius' law yielding activation energies of E_A(SI)=0.73(3) eV, E_A(SII)=0.66(3) eV and frequency factors of Z(SI)=1 × 10¹² s⁻¹, Z(SII) = 5 × 10¹² s⁻¹.     

Fabrication and Photoelectrochemical Properties of [Ru(bpy)2dppz]2+ on ITO Electrode Associated with the Oxidation of Guanine

<正>Electrochemical assembly of[Ru(bpy)_2dppz]~(2+){bpy=2,2'-bipyridine,dppz=dipyrido[3,2-a:2',3'-c]phenazine} on an ITO electrode in the presence of guanine and photoelectrochemical properties of the assembled layer were investigated.It has been found that[Ru(bpy)_2dppz]~(3+/2+) can be assembled onto the ITO electrode by the method of repetitive voltammetric sweeping,and the assembly is enhanced by guanine.The peak currents of prewaves increase linearly up to a guanine concentration of 0.25 mmol/L.More importantly,upon illumination with 470 nm light source and at an applied potential of 0.2 V,cathodic current for the fabricated layer on the ITO electrode indicate a linear enhancement with the rise of guanine concentration.Meanwhile,[Ru(bpy)_2dppz]~(2+) can be served as an excellent mediator to prompt the oxidation of guanine,and the mediated peak current increases linearly with added guanine concentration from 0.01 to 0.25 mmol/L.In addition,the assembly mechanism of[Ru(bpy)_2dppz]~(2+) on the ITO electrode associated with the oxidation of guanine and the assistance of light irradiation were discussed.     

Studies of interaction between colchicine and bovine serum albumin by fluorescence quenching method

We investigated the interaction between colchicine and bovine serum albumin (BSA) by fluorescence and UV–Vis absorption spectroscopy. In the mechanism discussion, it was proved that the fluorescence quenching of BSA by colchicine is a result of the formation of colchicine–BSA complex; van der Waals interactions and hydrogen bonds play a major role in stabilizing the complex. The modified Stern–Volmer quenching constant K_a and corresponding thermodynamic parameters ΔH, ΔG, ΔS at different temperatures were calculated. The distance r between donor (BSA) and acceptor (colchicine) was obtained according to fluorescence resonance energy transfer (FRET).     

Quantal state-to-state H-atom exchange cross sections of low energy 1H (v= 0) + Br reactions

The dynamics of the IH + Br heavy-light + heavy system is treated using the Born-Oppenheimer-type separation between the light and the heavy nuclear motions. The hydrogenic wavefunctions, that describe the H-nucleus motion for clamped I and Br nuclei, provide the potential energy curves and couplings that govern the I-Br relative motion. The Hatom exchange cross sections in the reactive collision: IH(v= 0,j) + Br → I + HBr(v′,j′) are found to strongly depend upon j and to be large only for v′ = 1 with j′ = 16–20 and for v′ = 2 with j′ = 8–11. The present results are compared to our previous estimates, to results of classical trajectory calculations and to available experimental data.     

Langmuir monolayer properties of the fluorinated-hydrogenated hybrid amphiphiles with dipalmitoylphosphatidylcholine (DPPC)

Surface pressure–area (π–A), surface potential–area (ΔV–A), and dipole moment–area (μ–A) isotherms were obtained for the Langmuir monolayer of two fluorinated-hydrogenated hybrid amphiphiles (sodium phenyl 1-[(4-perfluorohexyl)-phenyl]-1-hexylphosphate (F6PH5PPhNa) and (sodium phenyl 1-[(4-perfluorooctyl)-phenyl]-1-hexylphosphate (F8PH5PPhNa)), DPPC and their two-component systems at the air/water interface. Monolayers spread on 0.02 M Tris buffer solution (pH 7.4) with 0.13 M NaCl at 298.2 K were investigated by the Wilhelmy method, ionizing electrode method and fluorescence microscopy. Moreover, the miscibility of two components was examined by plotting the variation of the molecular area and the surface potential as a function of the molar fraction for the fluorinated-hydrogenated hybrid amphiphiles on the basis of the additivity rule. The miscibility of the monlayers was also examined by construction of two-dimensional phase diagrams. Furthermore, assuming the regular surface mixture, the Joos equation for analysis of the collapse pressure of two-component monolayers allowed calculation of the interaction parameter (ξ) and the interaction energy (−Δ) between the fluorinated-hydrogenated hybrid amphiphiles and DPPC. The observations by a fluorescence microscopy also supported our interpretation as for the miscibility in the monolayer state. Comparing the monolayer behavior between the two binary systems, no remarkable difference was found among various aspects. Among the two combinations, the mole fraction dependence in monlayer properties was commonly classified into two ranges: 0 ≤ X ≤ 0.3 and 0.3 < X ≤ 1. Dependence of the chain length of fluorinated part was reflected for the molecular packing and surface potential.     

Amperometric detection of cytochrome c by capillary electrophoresis at a sol–gel carbon composite electrode

Capillary electrophoresis (CE) was employed for the determination of cytochrome c using a wall-jet amperometric detector consisting a copper(I) oxide-modified sol–gel carbon composite electrode (CCE), which exhibits a sensitive electrocatalytic response for the oxidation of cytochrome c. The optimum conditions of separation and detection are 0.08 M NaOH for the separation solution, 12 kV for separation voltage and +0.60 V versus saturated calomel electrode (SCE) for the detection potential. Calibration was linear over the concentration range 1–600 μM with the limit of detection of 3.4 μM, based on a signal-to-noise ratio (S/N) of 3.     

Binding of brucine to human serum albumin

The feature of brucine binding to human serum albumin (HSA) was investigated via fluorescence and UV/vis absorption spectroscopy. The results revealed that brucine caused the fluorescence quenching of HSA by the formation of brucine–HSA complex. The hydrophobic interaction plays a major role in stabilizing the complex; the binding site number n and apparent binding constant K_A, corresponding thermodynamic parameters the free energy change (ΔG), enthalpy change (ΔH) and entropy change (ΔS) at different temperatures were calculated. The distance r between donor (HSA) and acceptor (brucine) was obtained according to fluorescence resonance energy transfer. The effect of brucine on the conformation of HSA was analyzed using synchronous fluorescence spectroscopy and UV/vis absorption spectroscopy.     

Liquid-state membrane electrode sensitive to bismuth(III)

A liquid ion-exchange electrode containing a tetrachloraethane solution of the complex of bismuth(III) with 5-mercapto-3-(naphthyl-1)-1,3,4-thiadiazol-2-thione is described. The electrode is sensitive to Bi³⁺. The slope of the calibration graph (electrode potential vs. concentration) is 18.7 mV/pBi in the pBi range 6.5–9.5 in ammonium acetate buffer (pH = 4.0). Bivalent cations and Al(III), Fe(III) and Th(IV) do not interfere (K_{Bi³⁺+,M²⁺}<10⁻⁵). The dissociation constant of bismuth acetate has been determined with the aid of the electrode.     

The analysis of serum effects on structure, size and toxicity of DDAB-DOPE and DC-Chol-DOPE lipoplexes contributes to explain their different transfection efficiency

The effect of serum on structural properties of dimethyl-dioctadecyl-ammonium bromide (DDAB)–1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) liposomes and DDAB–DOPE/DNA lipoplexes has been investigated by energy dispersive X-ray diffraction (EDXD) technique, at different cationic lipid/DNA weight ratios (ρ). The role of serum on the size of lipoplexes has also been studied by dynamic light scattering. Lipoplex transfection efficiency (TE) as a function of ρ, and lipoplex toxicity to C6 rat glioma cells have been evaluated in Dulbecco's Modified Eagle Medium (DMEM) with and without serum. A multi-parametric analysis concerning the role of size, structure and cytotoxicity on transfection efficiency contributes to explain the experimental observation that 3β-[N-(N′,N′-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol)–DOPE/DNA transfect C6 cells better than DDAB–DOPE/DNA lipoplexes.