CONFIGURATIONS OF NEUROSPORENE ISOMERS ISOLATED FROM THE REACTION CENTER AND THE LIGHT-HARVESTING COMPLEX OF Rhodobacter spheroides G1C. A RESONANCE RAMAN, ELECTRONIC ABSORPTION, AND 1H-NMR STUDY

Abstract— HPLC analysis of neurosporene extracted from the chromatophore membranes of Rhodobacter spheroides G1C showed two isomeric components 1 and 2. Extract from the light-harvesting complex (LH) gave only component 2, while extract from the reaction center (RC) mainly gave component 1. Both components were isolated, and their configurations were determined by means of (1) resonance Raman, (2) electronic absorption, and (3) ¹H-HMR spectroscopy. The configuration of component 2 originating from LH was determined to be all -trans and that of component 1 from RC was determined to be 15- cis . Thus, our previous configurational prediction of this particular carotenoid bound to RC, by means of resonance Raman spectroscopy, to be 15-cis [Y. Koyama, T. Takii, K. Saiki and K. Tsukida, Photobiochem. Photobiophys. 5 (1983)139–150] is confirmed.     

Molecular assembly of artificial photosynthetic antenna core complex on an amino-terminated ITO electrode

Bacterial photosynthetic membrane proteins, light-harvesting antenna complex (LH1), reaction center (RC), and their combined ‘core’ complex (LH1–RC) are functional elements in the primary photosynthetic events, i.e., capturing and transferring light energy and subsequent charge separation. These photosynthetic units (PSUs) isolated from Rhodospirillum rubrum (Rs. rubrum) were assembled onto an ITO electrode modified with 3-aminopropyltriethoxysilane (APS–ITO). The near IR absorption spectra of PSUs on the assembled electrodes were identical to those of solutions, indicating that the LH1 and LH1–RC core complexes were native on the electrode. Photocurrent response of PSUs on the electrode was examined upon illumination of the LH1 complex at 880 nm. The LH1–RC and a mixed assembly of LH1 and RC exhibited photocurrent response, but not LH1 only, consistent with the function of these PSUs, capturing light energy and transferring electron. This result provides useful methodology for building an artificial fabrication of PSUs on the electrode.     

ADENYLATE KINASE BOUND TO THE CHROMATOPHORE MEMBRANES OF Rhodobactor spheroides G1C

Transformation of adenylates (AMP, ADP and ATP) by washed chromatophore membranes of Rhodobactor spheroides G1C in the dark and in the light indicated the functions of ATPase (ADP + Pi in equilibrium ATP) and of an adenylate kinase (2ADP in equilibrium AMP + ATP). The activity of adenylate kinase of the chromatophores was not inhibited by AP5A, and persisted even after sonication in the presence of EDTA or CaCl2; the results suggested the presence of an adenylate kinase bound to the chromatophore membrane. In search of the enzyme, the supernatant after sonication of the chromatophores in the presence of EDTA was subjected to a molecular sieve and then to ion-exchange HPLC; a fraction with high specific adenylate kinase activity, containing a very sharp peak at 55 kDa, was isolated. Preliminary characterization indicated that it is different from the well-documented water-soluble 33 kDa adenylate kinase.     

Time-dependent changes in the carotenoid composition and preferential binding of spirilloxanthin to the reaction center and anhydrorhodovibrin to the LH1 antenna complex in Rhodobium marinum

Carotenoids were isolated from the cells of Rhodobium marinum, and their structures were determined by mass spectrometry and 1H nuclear magnetic resonance spectroscopy; the carotenoids include lycopene, rhodopin, anhydrorhodovibrin, rhodovibrin and spirilloxanthin. Time-dependent changes in the carotenoid composition in the reaction center (RC) and the light-harvesting complex 1 (LH1) were traced by high-performance liquid chromatography analysis of the extracts. The carotenoid composition changed according to the spirilloxanthin biosynthetic pathway. However, spirilloxanthin having the longest conjugated chain was always preferentially bound to the RC, and anhydrorhodovibrin and other precursors to the LH1.     

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.     

Effect of denaturants on the stability of light-harvesting complex

 The effect of denaturants such as urea and normal alcohols on the formation of light-harvesting (LH) polypeptides/bacteriochlorophyll a (BChla) complex (LH1 complex) in n-octyl-β-D-glucopyranoside (OG) micelle was examined to provide an insight into stability of the complex. The stabilities of the LH1 complex in OG micelle and of the complex in the chromatophore of photosynthetic bacteria were compared by addition of denaturants. The extent of stability of these complexes was monitored by the change in absorbance of Qy band of BChla in these complexes, resulting generally in the blue-shifting of the Qy band from near 870 nm to about 777 nm upon addition of these denaturants. Urea and guanidium hydrochloride (Gnd) showed a relatively weak denaturing effect. Normal alcohols showed stronger denaturing effect, depending on the hydrophobicity of the alcohols. These results imply that the stability of LH1 complex in OG micelle can be largely attributed to the hydrophobic interactions in the complex as well as that of the complex in the chromatophore of photosynthetic bacteria. Received: 23 May 1997 Accepted: 13 September 1997     

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.     

VARIATION IN ELECTRON PARAMAGNETIC RESONANCE SIGNALS OF PHOTOSYNTHETIC SYSTEMS WTTH THE REDOX LEVEL OF THEIR ENVIRONMENT

Abstract— The redox dependence of the light-induced electron paramagnetic resonance signal at g=2 in R. rubrum, R. spheroides and Chromatium chromatophore particles and quantasonie particles from spinach chloroplasts has been determined qualitatively over the range —0.3 to +0.6 V and quantatively over the range +0.3 to ±0.6 V. A light-induced EPR signal has been titrated and demonstrated to have a midpoint potential of +0.44 v at pH 7 and 20°C. Concentration, ionic strength and pH dependence for this transition in R. rubrum chromatophores is reported. In addition to the dark signal which replaces the light signal, in chromatophore material another dark signal, occurring in the seine location as the light signal, has been demonstrated to occur at high potential.
Selective chemical oxidation with K₂lrCl₆ of chromatophore particles from the three bacteria resulted in the removal of some 95 per cent of the absorbance in the near infrared and left the photoactive pigments.
Two light-induced EPR signals were found in quantasome particles by their dependence upon the redox level. Of particular interest is a signal observed at quite high potential (e.g. + 0.60 V). It was demonstrated that oxygen evolution by these quantasonie particles in the presence of K³Fe(cN)₀ occurred at the same rate at +0.55 V as at +0.40 V.     

THE EFFECTS OF PHOTOACTIVATED PROTOPORPHYRIN ON RETICULOCYTE MEMBRANES, INTRACELLULAR ACTIVITIES and HEMOGLOBIN PRECIPITATION

Abstract— Photoactivated protoporphyrin effects were studied on reticulocyte membranes and distinct intracellular activities. Membrane bound (Na ₊ -K ₊)-ATPase activity and incorporation of ₅₅Fe into heme were almost 80% inhibited at a low concentration of protoporphyrin (3 fiM). On the other hand, a much higher protoporphyrin concentration (15 nM) was needed to cause 80% inhibition of protein synthesis. By 15 JXM protoporphyrin and treatment with light, an initial leak of hemoglobin out of the cells was observed. Electron microscopic examination showed that the lytic effects seem to be a result of membrane damage which appeared as holes in the membrane. Heinz-body-like particles of condensed hemoglobin were observed in the protoporphyrin-treated cells. The condensed hemoglobin spheres were shown to be bound to disrupted membranes prepared from photoactivated protoporphyrin-treated reticulocytes     

THE QUANTUM EFFICIENCY FOR THE INTERPHOTOCONVERSION OF THE BLUE and PINK FORMS OF PURPLE MEMBRANE

When the cations bound to purple membrane are removed it turns blue, and when this blue membrane is irradiated its color changes to pink. Irradiation of pink membrane leads to the reformation of blue membrane. We have determined that the quantum efficiency for the formation of pink membrane from deionized blue membrane is 1.6 ± 0.6 ± 10 ⁴ at 0^oC, pH 5.0. We also found that the quantum efficiency for the back photoconversion, i.e. the formation of blue membrane from pink membrane, is 8.8 ± 1.6 ± 10^-3 at 0^oC, 55 times greater than that of the forward photoconversion reaction. The extinction coefficients of the pink membrane and blue membrane were determined to be 44 500 ± 670 cm^-1 M^-1 at 491 nm and 54 760 ± 830 cm^-1 M ^-1 at 603 nm, respectively, assuming light-adapted purple membrane is 63 000 cm^-1 M ^-1 at 568 nm. The quantum efficiency for forming pink membrane from blue membrane is much lower than that for forming the photointermediate of the blue membrane's photocycle. Their relationship is similar to that of light-adaptation and photocycle of the dark-adapted purple membrane.     

THE EFFECTS OF LIQUID HOLDING ON NEAR-UV(300–400 nm) IRRADIATED ESCHERICHIA COLI STRAINS DIFFERING IN NEAR AND FAR-UV RADIATION SENSITIVITY

Abstract— Stationary phase cells from four Escherichia coli strains differing in near- (nur vs. nur ₊) and far-UV (recAl vs. recA₊) radiation sensitivity were subjected to near-UV radiation (NUV) in 0.85% saline. Although the NUV-irradiated cultures yielded increased colony numbers following 24 h of liquid holding (LH), a fluctuation test for each experiment showed that the observed increases were not due to recovery but were in fact due to cell multiplication. The decline in viability observed after NUV with liquid holding using the fluctuation test was equivalent in strains RT2, 3 and 4 while the decline observed with RT1 was less marked. The discrepancy between LH involving cell densities of 10₈-10₉ and 1–4 cells/m/ can be resolved by assuming that with dense cell suspensions, NUV-induced membrane damage leads to leakage or lysis, supplying sufficient nutrients to allow growth of undamaged, surviving cells.     

RESONANCE RAMAN EVIDENCE FOR 15-cis TO MA,-trans PHOTOISOMERIZATION OF SPIRILLOXANTHIN BOUND TO A REDUCED FORM OF THE REACTION CENTER OF Rhodospirillum rubrum SI

The reaction center (RC) of Rhodospirillum rubrum SI, which was prepared by ultrafiltration, showed one peak in molecular-sieve HPLC, but it showed two peaks in diethylaminoethyl (DEAE) ion-exchange HPLC; they were named as RC-α and RC-β in the order of elution, Nonequilibrated isoelectric electrophoresis, together with DEAE ion-exchange HPLC, showed that RC-β is electronically more negative than RC-α. Oxidation of RC-β by addition of ferricyanide caused its transformation into RC-α, while reduction of RC-α by adding ascorbate and subsequent illumination caused its transformation into RC-β. Resonance Raman spectroscopy of the RC at liquid nitrogen temperature detected the all-trans and the 15-cis isomers in a ratio of 1:1, but HPLC analyses of the carotenoid extracted from the RC before and after the Raman measurements detected the pair of isomers in a ratio of 1:6. Thus, the 15-cis to all-trans isomerization takes place during irradiation at liquid nitrogen temperature, while the reverse isomerization takes place in the dark. The isolated RC-α and RC-β exhibited the bleaching of the 868 nm band, and contained the H, M and L subunits and 1.2-1.4 molecules of ubiquinone-10 per RC. Each RC slowly equilibrated in the dark toward a mixture of RC-α and RC-β. Generation of the all-trans isomer in the light was found not in RC-α but in RC-β.     

ULTRASTRUCTURAL STUDIES ON THE MECHANISM OF THE PHOTODYNAMIC THERAPY OF TUMORS

Abstract Balb/c mice bearing a transplanted MS-2 fibrosarcoma were injected with 2.5 mg kg ¹ of either tetra(4-sulfonatophenyl/porphine (TPPS) in phosphate-buffered saline or 0.5 mg kg⁻¹ of Zn²⁺-phthalocyanine (Zn-Pc) incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine. Chromatographic studies showed that TPPS is mainly transported in the serum by globulins and albumin, while Zn-Pc is specifically bound by lipoproteins. Exposure of the injected mice to red light (300 J cm⁻²) caused extensive tumor necrosis. The ultrastructural analysis of tumor specimens taken from mice at 15 h after PDT showed that TPPS photoinduces a preferential necrosis of the neoplastic cells, while Zn-Pc causes severe photodamage to both the vascular system and the neoplastic cells. The different modes of tumor photosensitization by TPPS and Zn-Pc are discussed on the basis of the transport mechanism of the two dyes.     

Synthesis and structural characterization of lanthanide(III) nitrate complexes of a tetraiminodiphenol macrocycle in the solid state and in solution

The lanthanide(III) complexes [Ln(LH2)(NO3)3] 1-11(La-Er), 15(Y) and [Ln(LH2)(NO3)2(H2O)](NO3) 12-14 (Tm-Lu) of the tetraiminodiphenolate macrocycle L2- have been prepared by the transmetallation reaction between [Pb(LH2)(NO3)2] and Ln(NO3)3.nH2O. In these compounds, the uncoordinated imino nitrogens are protonated and are hydrogen bonded to the phenolate oxygens. The X-ray crystal structures of the La (1), Ho (10) and Lu (14) compounds have been determined. Compounds 1 and 10, in which all the three nitrates are bound in bidentate fashion, are isostructural with distorted bicapped square antiprism geometry for the metal centre. In [Lu(LH2)(NO3)2(H2O)](NO3) 14, of the two metal bound nitrates one is bidentate and the other is unidentate, while the metal centre obtains a distorted square antiprism coordination environment. Proton NMR spectra of the paramagnetic lanthanide complexes have been studied in detail. Contributions of contact and pseudo-contact shifts to the lanthanide induced isotropic shifts (LIS) of the macrocycle protons have been separated and good agreement has been obtained between the calculated LIS values and the experimentally observed values. Analysis of the NMR data has led us to conclude that all the complexes in dimethyl sulfoxide solution attain similar configurations. The absorption and emission spectral characteristic of several compounds have been investigated. The complexes of samarium (5) and europium (6) on photoexcitation at 400 nm exhibit well-resolved luminescence spectra at 77 K both in the solid state and a methanol-ethanol (1 : 4) glassy matrix. For the terbium (8) and dysprosium (9) complexes, however, the observed luminescence peaks are less resolved and weak in intensity.     

SOLVENT EFFECT ON SPHEROIDENE IN NONPOLAR AND POLAR SOLUTIONS AND THE ENVIRONMENT OF SPHEROIDENE IN THE LIGHT-HARVESTING COMPLEXES OF Rhodobacter sphaeroides 2.4.1 AS REVEALED BY THE ENERGY OF THE 1Ag−→1Bu+ ABSORPTION AND THE FREQUENCIES OF THE VIBRONICALLY COUPLED C=C STRETCHING RAMAN LINES IN THE 1Ag− AND 1Bu− STATES

Abstract –The ¹A_g^?→¹B_u⁺ electronic absorption band and the vibronically coupled, C=C stretching Raman lines in the ¹A_g^? and 2¹A_g^? states were recorded for spheroidene free in nonpolar and polar solvents as well as for spheroidene bound to the LH1 and LH2 complexes of Rhodobacter sphaeroides 2.4.1. The ¹B_u⁺ energy exhibited a linear dependence on R(n) = (n² - 1)/(n²+ 2) in both nonpolar and polar solvents; the line for polar solvents had a gentler slope and crossed the line for nonpolar solvents at R(n) = 0.3. The above characteristic of polar solvents was ascribed to the electric field generated by fluctuation of the solvent permanent dipoles; it stabilizes the ¹B_u⁺ energy and reduces the polarizability of the solvent. The vibronically coupled, C=C stretching frequencies in the ¹A_g^? and 2¹A_g^? states [ν(A_g) and [ν(2A_g)] also showed similar dependence on R(n), which is explained in terms of vibronic coupling among the ¹A_g^?, 2¹A_g^? and 3¹A_g^? states. The environment of spheroidene in the LH2 and LH1 complexes was assessed on the basis of the ¹B_u⁺ energy and the ν(A_g) and [ν(2A_g) frequencies: Spheroidene in the LH2 complex is located in an environment with high polarizability, while spheroidene in the LH1 complex is located in an environment with lower polarizability.     

THE EFFECT OF TREATMENT OF CHLOROPLAST MEMBRANES WITH GUANIDINE HC1 AND AQUEOUS ACETONE ON THE FLUORESCENCE OF BOUND ANS AND CHLOROPHYLL-a†

Abstract The fluorescence intensity of the extrinsic chromophore 1-anilino-naphthalene-8-sulfonate (ANS) bound to pea chloroplast fragments shows a sigmoidal rise as the pH of the suspending medium is decreased by the addition of HC1. The abrupt increase occurs at pH – 4.5. A 70% decrease in the maximal fluorescence intensity (pH range 3.5-4.5) of bound ANS was observed when soluble chloroplast proteins were removed by washing with water. Extraction of chloroplast membranes with 6 M guanidine-HC1 abolishes the acid–induced enhancement of ANS fluorescence. However, the subsequent removal of lipids (by 80% acetone extraction) from the guanidine-HC1-extracted naked membranes restores the acid-induced fluorescence increase. These results suggest that ANS binds mainly to the surface of the chloroplast membrane and the fluorescence changes of ANS by acidification mainly reflect the changes in the associated proteins. The lack of enhancement of the fluorescence of ANS by acidification of the guanidine-HCl treated membranes and the recovery of the acid-induced fluorescence rise after extraction of the lipids from the guanidine-HCl treated membranes suggest that the boundary lipids somehow prevent the entry of the ANS molecules into the hydrophobic interior of the naked membrane. The lipid-depleted, guanidine-HCl extracted naked membrane fragments do not show any shift in the position of the peak of emission of ANS (λ= 470 nm) upon acidification as the lipid-depleted preparations without guanidine-HCl treatment do (shift from 460 to 470 nm). Divalent cations (Mn²⁺, Ca²⁺, Mg²⁺) also increased ANS fluorescence intensities when added to both types of lipid-depleted chloroplast preparations. A comparative analysis of ANS fluorescence bound to the lipid-depleted and guanidine-HCl treated chloroplast fragments with that of just lipid-depleted fragments shows that the acidification of the latter brings about a greater change in the value of the relative binding sites (n) and the dissociation constant k_d of ANS than the protonation of the former. The role of chloroplast protein and lipid components in the structural changes of the thylakoid membrane imposed by external perturbations is discussed.     

THE USE OF NEUTRAL RED TO MONITOR THE SURFACE POTENTIAL OF THE PURPLE MEMBRANE

Abstract— The binding of neutral red to purple membrane has been studied. The intrinsic p K _a and the apparent p K _a, of bound neutral red were determined by titration and by measuring the binding ratio of neutral red to purple membrane as a function of pH. The surface potential of purple membrane was inferred from the difference between these two p K _as. The H⁺/M412 ratio at different ionic strengths was also measured and compared with the surface potential. The results show that the H⁺/M412 decreased as the surface potential increased due to decreased salt concentrations. However, this correlation holds only for KCl concentrations higher than 30 m M . At lower salt concentrations, the change in surface potential is always less than the variation in the H⁺/M412 ratio.     

R、S双[N-(5-氯邻羟苯亚甲基)-α-苯乙胺]合钯(Ⅱ)的晶体结构及分子绝对构型

旋光性有机化合物的过渡金属配合物在生命现象中占有重要的地他.标题配合物是为此目的而研究的系列配合物之一.按照文献方法合成得到有旋光性质的 Schiff 碱N-(5-氯-邻羟基苯亚甲基)-α-苯乙胺,再与氯化钯反应得到粗产品,用乙醇-氯仿混合溶剂重结晶分别得到桔红色的 R、S 型标题产物.     

ELECTRONIC CONFIGURATIONS OF TRANSITION METAL SANDWICH COMPLEXES

In this paper the electronic configurations of n+1-decker transition metal sandwich com-plexes have been generally discussed by means of the structural rule of transition metalheterocarborans and the numbers of their valence bonding orbitals have been obtained as fol-lows: VBO = 6n + 3-6n + 5,which are not dependent on the properties of transition metal atoms and coordination rings.Then the results of EHMO quantum chemistry calculations of the model skeletons and cor-responding analyses on electronic configurations of actual molecules for double-, triple- andtetra-decker sandwich complexes have been discussed to verify the above formula.