Difference picosecond spectroscopy of pigment-protein complexes of photosystem I from higher plants

Using a difference picosecond spectrophotometer with a time resolution of 10 ps, we investigated excitation energy transfer and charge separation in pigment-protein complexes of Photosystem I from bean leaves (chlorophyll/P-700 = 60). Under 20 ps excitation at 650 or 667 nm, the difference absorption spectra in the spectral region 600–720 nm were measured. They are associated with transition of antenna chlorophylls into singlet excited states and P-700 photooxidation. It was shown that the excited states in the whole inhomogeneous antenna were generated within 10 ps and deactivated with three-component kinetics, the t_1/e values being 20–45, 100–300 and over 500 ps. Formation of P-700⁺ has a rise time of 15–30 ps. The fast component of the depletion of the antenna excited states is suggested to be due to transfer of excitation energy from antenna pigments to reaction centers and its trapping. The kinetics of the fast component is independent of excitation energy and a redox state of P-700.     

Photoelectric studies of the transmembrane charge transfer reactions in photosystem I pigment-protein complexes

The results of studies of charge transfer in cyanobacterial photosystem I (PS I) using the photoelectric method are reviewed. The electrogenicity in the PS I complex and its interaction with natural donors (plastocyanin, cytochrome c(6)), natural acceptors (ferredoxin, flavodoxin), or artificial acceptors and donors (methyl viologen and other redox dyes) were studied. The operating dielectric constant values in the vicinity of the charge transfer carriers in situ were calculated. The profile of distribution of the dielectric constant along the PS I pigment-protein complex (from plastocyanin or cytochrome c(6) through the chlorophyll dimer P700 to the acceptor complex) was estimated, and possible mechanisms of correlation between the local dielectric constant and electron transfer rate constant were discussed.     

Molecular arrangement of pigment-protein complex of photosystem 1

The circular dichroism (CD) method was applied to study the molecular organization of P700, antenna chlorophyll and protein of photosystem 1 complexes (CP1), isolated from chloroplasts under mild treatment with Triton X-100. Analysis of CD spectra and protein: chlorophyll: P700 ratios for CP1 complexes that were different in their chlorophyll content indicate that CP1 preparations can be considered as a mixture of CP1-RC, containing P700 (10–20%), and CP1-LH without P700 (80–90%). Both types of complexes contain approximately 25 chlorophyll molecules, and the destruction of their spatial organization with detergents represents a cooperative transition. The rate of chlorophyll destruction in CP1-LH is much higher than that in CP1-RC. In both complexes a 65 kDa polypeptide predominates, whose secondary structure (typical for / proteins) is stable to Triton X-100 and does not depends on the chlorophyll content. Chlorophyll seems to be grouped in clusters (5–7 molecules) in the hydrophobic cores of 2–3 parallel / domains of the 65 kDa protein. Only one of the clusters in CP1-RC includes P700; on P700 photooxidation the change of its interaction with the nearest pigment environment results in a complicated shape of the light-induced CD spectra.Abbreviations PS1 photosystem 1 - CP1 pigment-protein complex of PS1 - Chl chlorophyll a - CP1-140 CP1 with ratio Ch1:P700 140 - RC reaction center - LH light-harvesting pigment - CP1-RC CP1, containing P700 - CP1-LH CP1 without P700 (containing LH) - CD circular dichroism - SDS sodium dodecyl sulfate Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

Effect of DNAase on fluorescent properties of pigment-protein complexes, contained in photosystem II

It was found that chlorophyll fluorescence spectra and spectra of fluorescence excitation of pigment-protein complexes of photosystem II are affected by treatment with DNase. Pigment-protein complexes were isolated from pea thylakoid membranes. Spectra were measured at room temperature. It was shown that the treatment with DNase leads to a 30% increase in fluorescence yield at excitation in chlorophyll absorption bands in the fraction containing CP47, CP43, and CP29, and also in the fraction containing reaction center complexes with minor contaminations of light-harvesting complexes. Upon excitation at 260-300 nm and in the region of 500 nm, a diminishing of fluorescence yield takes place. These results suggest that pigments and/or pigment-protein complexes are bound to nucleic acids. This association, by influencing the pigment properties, can participate in the photoregulation of biochemical reactions through changes in the thermal dissipation of excited chlorophyll molecules.     

Analysis of the pigment stoichiometry of pigment-protein complexes from barley (Hordeum vulgare). The xanthophyll cycle intermediates occur mainly in the light-harvesting complexes of photosystem I and photosystem II.

The carotenoid zeaxanthin has been implicated in a nonradiative dissipation of excess excitation energy. To determine its site of action, we have examined the location of zeaxanthin within the thylakoid membrane components. Five pigment-protein complexes were isolated with little loss of pigments: photosystem I (PSI); core complex (CC) I, the core of PSI; CC II, the core of photosystem II (PSII); light-harvesting complex (LHC) IIb, a trimer of the major light-harvesting protein of PSII; and LHC IIa, c, and d, a complex of the monomeric minor light-harvesting proteins of PSII. Zeaxanthin was found predominantly in the LHC complexes. Lesser amounts were present in the CCs possibly because these contained some extraneous LHC polypeptides. The LHC IIb trimer and the monomeric LHC II a, c, and d pigment-proteins from dark-adapted plants each contained, in addition to lutein and neoxanthin, one violaxanthin molecule but little antheraxanthin and no zeaxanthin. Following illumination, each complex had a reduced violaxanthin content, but now more antheraxanthin and zeaxanthin were present. PSI had little or no neoxanthin. The pigment content of LHC I was deduced by subtracting the pigment content of CC I from that of PSI. Our best estimate for the carotenoid content of a LHC IIb trimer from dark-adapted plants is one violaxanthin, two neoxanthins, six luteins, and 0.03 mol of antheraxanthin per mol trimer. The xanthophyll cycle occurs mainly or exclusively within the light-harvesting antennae of both photosystems.     

Interaction of the photosystem 1 reaction center with antenna chlorophyll in a pigment-protein complex isolated from pea chloroplasts

Using a specially developed phosporoscopic attachment to spectropolarimeter, light induced spectra of circular dichroism (CD) in region 600-750 nm were measured for a pigment protein complex of photosystem 1 (PC-1) isolated from pea chloroplast (chlorophyll : P700 = 40). Minor components at 672 and 678 nm are observed in light induced spectra besides the components of dimer splitting of P700 Qy transition at 691 and 698 nm. Haussian deconvolution of light induced CD spectra of P700 and low temperature CD spectrum of PC-1 indicates that minor components are due to forms of antenna chlorophylls Chl672 and Chl678, rotational strength of that is changed by 2-4% as a result of P700 oxidation. Long term incubation of PC-1 with Triton X-100 inhibits P700 and destroys longwave optically active chlorophyll forms. A strong relation between dichroic density of 693 nm band in CD spectrum of PC-1 and the value of light induced absorption change at 698 nm could be used to determine P700 concentration on the basis of CD spectrum of PC-1. Such a relation shows that Chl693 is an important component of photo-system 1 reaction center. It is suggested that P700 is not an isolated dimer but it is included in the local complex from 8-10 chlorophyll molecules (Chl672, Chl678, Chl686, Chl693).     

Probing the kinetics of photosystem I and photosystem II fluorescence in pea chloroplasts on a picosecond pulse fluorometer.

Picosecond fluorescence kinetics of pea chloroplasts have been investigated at room temperature using a pulse fluorometer with a resolution time of 10-11 s. Fluorescence has been excited by both a ruby and neodymium-glass mode-locked laser and has been reocrded within the 650 to 800 nm spectral region. We have found three-component kinetics of fluorescence from pea chloroplasts with lifetimes of 80, 300 and 4500 ps, respectively. The observed time dependency of the fluorescence of different components on the functional state of the photosynthetic mechanism as well as their spectra enabled us to conclude that Photosystem I fluoresces with a lifetime of 80 ps (tauI) and Photosystem II fluoresces with a lifetime of 300 ps (tauII). Fluorescence with a lifetime of 4500 ps (tauIII) may be interpreted as originating from chlorophill monomeric forms which are not involved in photosynthesis. It was determined that the rise time of Photosystem I and Photosystem II fluorescence after 530 nm photoexcitation is 200 ps, which corrsponds to the time of energy migration to them from carotenoids.     

Pigment analysis of chloroplast pigment-protein complexes in wheat

Pigment-protein complexes separated from wheat (Triticum aestivum L. selection ND96-25 by two gel electrophoresis techniques were analyzed by high-performance liquid chromatography for chlorophylls and carotenoids. The two techniques are compared, and pigment analyses are given for the major reaction centers and light-harvesting complexes. Reaction centers contain mostly chlorophyll a, carotene, and lutein, whereas light-harvesting complexes contain chlorophyll a, chlorophyll b, lutein, and neoxanthin. The amounts of violaxanthin are variable.     

Polypeptide composition of the purified photosystem II pigment-protein complex from spinach.

The Photosystem II pigment-protein complex, the chlorophyll alpha-protein comprising the reaction center of Photosystem II, was prepared from EDTA-treated spinach chloroplasts by digitonin extraction, sucrose-gradient centrifugation, DEAE-cellulose column chromatography, and isoelectrofocussing on Ampholine. The dissociated pigment-protein complex exhibits two polypeptide subunits that migrate in SDS-polyacrylamide gel with electrophoretic mobilities corresponding to molecular weights of approximately 43,000 and 27,000. the chlorophyll was always found in the free pigment zone at the completion of the electrophoresis. Heat-treatment of the sample (100 degrees C, 90 s) for electrophoresis caused association of the two polypeptides into large aggregates. It is concluded that these two polypeptides, 43,000 and 27,000, are valid structural or functional components of Photosystem II pigment-protein complex.     

Strong-light photoinhibition treatment accelerates the changes of protein secondary structures in triton-treated photosystem I and photosystem II complexes

Changes in the protein secondary structure and electron transport activity of the Triton X-100-treated photosystem I (PSI) and photosystem II (PSII) complexes after strong illumination treatment were studied using Fourier transform-infrared (FT-IR) spectroscopy and an oxygen electrode. Short periods of photoinhibitory treatment led to obvious decreases in the rates of PSI-mediated electron transport activity and PSII-mediated oxygen evolution in the native or Triton-treated PSI and PSII complexes. In the native PSI and PSII complexes, the protein secondary structures had little changes after the photoinhibitory treatment. However, in both Triton-treated PSI and PSII complexes, short photoinhibition times caused significant loss of -helical content and increase of -sheet structure, similar to the conformational changes in samples of Triton-treated PSI and PSII complexes after long periods of dark incubation. Our results demonstrate that strong-light treatment to the Triton-treated PSI and PSII complexes accelerates destruction of the transmembrane structure of proteins in the two photosynthetic membranes.     

Prompt and delayed fluorescence in pigment-protein complexes of a green photosynthetic bacterium

Excitation spectra were measured at 4 K of bacteriochlorophyll a fluorescence in reaction center containing pigment-protein complexes obtained from the green photosynthetic bacterium Prosthecochloris aestuarii. Excitation spectra for the longest-wave emission (838 nm) showed bands of bacteriochlorophyll a, carotenoid, and of a pigment with absorption bands at 670, 438 and possibly near 420 nm, which is probably identical to an unidentified porphyrin described in the preceding paper (Swarthoff, T., Kramer, H.J.M. and Amesz, J. (1982) Biochim. Biophys. Acta 681, 354–358). At room temperature the longest-wave emission is stimulated by a magnetic field, which indicates that at least part of the emission is delayed fluorescence brought about by a reversal of the primary charge separation. Below about 150 K no stimulation was observed. The excitation spectra for short-wave emission (828 nm) were very similar to the absorption spectrum of the isolated antenna bacteriochlorophyll a-protein complex, and showed bands of bacteriochlorophyll a only. This indicates that two forms of the antenna protein exist that are spectroscopically similar: a soluble form that is released by treatment with guanidine hydrochloride and a bound form that remains attached to the reaction center complex. The bands of the antenna complexes were weak in the excitation spectra of the 838 nm fluorescence, which indicates that the efficiency of energy transfer to the reaction center complex is low.     

Analysis of absorption spectra changes induced by temperature lowering on phycobilisomes,thylakoids and chlorophyll-protein complexes

Using fourth derivative analysis, differences between room and low temperature absorption spectra were studied. The positions of most absorption bands of the water-soluble, accessory pigment complex, the phycobilisome, remained unchanged after cooling. The stability of the wavelength positions of chlorophyll a forms in vivo as a function of temperature (Gulyaev, B.A. and Litvin, F.F. (1967) Biofizika 12, 845–854) was generally confirmed. The wavelength positions of all chlorophyll a forms in the P-700 chlorophyll a protein complex were unchanged when the preparations were cooled to ?196°C. Likewise, with other chlorophyll-containing materials: the light-harvesting chlorophyll $\text{a}\text{b}$ protein complex and the thylakoids of higher plants, algae, and cyanobacteria, the wavelength positions of most chlorophyll a forms were stable upon cooling. An exception was a 680 nm chlorophyll a band which was generally split at low temperature into two bands with the materials investigated. An interpretation of the multiplicity of chlorophyll spectral forms and the spectral changes induced by cooling for these forms is given using exciton theory and the energy-coupling variation of chlorophyll a molecules.     

Shifts of the bacteriochlorophyll absorption band at 880 nm in chromatophores and subchromatophore pigment-protein complexes from Rhodospirillum rubrum]

The redox potential dependency of the light-induced absorption changes of bacteriochlorophyll in the chromatophores and subchromatophore particles from Rhodospirillum rubrum has been studied. The highest values of the absorption changes due to the bleaching of P870 and the blue shift of P800 are observed within the redox potential range of 360--410. At the potential values below 300 mV the 880 nm band of bacteriochlorophyll shifts to shorter wavelengths in the subchromatophore particles and to longer wavelengths in the chromatophores. Redox titration revealed that the red and blue shifts of 880 nm bacteriochlorophyll band are caused by the functioning of a non-identified component (X) which has an oxidation -- reduction midpoint potential close to 340 mV (n = 1) within the pH range of 6,0--7,6. The Em for this component decreases by 60 mV/pH unit within the pH range of 7.6--9,2. The results obtained suggest that the red shift is due to the transmembrane, while the blue shift -- to the local intramembrane electric field. The generation of both the transmembrane and local intramembrane electric fields apparently depends on redox transitions of the component X.     

Variability of light-induced circular dichroism spectra of photosystem I complexes of cyanobacteria

Circular dichroism (CD) spectra of photosystem I (PSI) complexes of the cyanobacteria Thermosynechococcus elongatus, Arthrospira platensis and Synechocystis sp. PCC 6803 were studied. CD spectra of dark-adapted PSI trimers and monomers, measured at 77 K, show common bands at 669–670(+), 673(+), 680(−), 683–685(−), 696–697(−), 702(−) and 711(−) nm. The intensities of these bands are species specific. In addition, bands at 683–685(−) and 673(+) nm differ in intensity for trimeric and monomeric PSI complexes. CD difference spectra (P700⁺–P700) of PSI complexes at 283 K exhibit conservative bands at 701(−) and 691(+) nm due to changes in resonance interaction of chlorophylls in the reaction center upon oxidation of P700. Additional bands are observed at 671(−), 678(+), 685(−), 693(−) nm and in the region 720–725 nm those intensities correlate with intensities of analogous bands of antenna chlorophylls in dark-adapted CD spectra. It is suggested that the variability of CD difference spectra of PSI complexes is determined by changes in resonance interaction of reaction center chlorophylls with closely located antenna chlorophylls.     

The cooperativity phenomena in a pigment-protein complex of light-harvesting antenna revealed by picosecond absorbance difference spectroscopy

A model of the cooperative changes in optical properties of light-harvesting bacteriochlorophyll molecules of complex B890 in response to the absorption of light quanta is proposed. According to the model, each antenna chromophore may persist in either of two optically non-excited states, R and T. The occurrence of at least one excitation per complex causes all optically non-excited chromophores of the complex to be converted from state R to state T. The theory is shown to be in good agreement with experimental ‘light curves’ (ΔAvs intensity of picosecond excitation pulse) for the ‘minor’ and ‘major’ signals of light-harvesting bacteriochlorophylls of complex B890 from Chromatium minutissimum.     

固氮红细菌(Rhodobacter azotoforman)色素蛋白复合体的分离纯化与特性

【目的】为揭示不产氧光合细菌产氢菌株色素蛋白复合体(PPC)色素组成和含量与光合放氢的关系奠定基础。【方法】以PPC特征光谱为检测指标,采用硫酸铵分级分离、DEAE-纤维素层析、吸收光谱和SDS-PAGE等方法进行了固氮红细菌(Rhodobacter azotoformans,R.azotoformans)R7产氢菌株PPC的分离纯化、纯度分析和鉴定;采用表面增强激光解吸电离离子飞行时间质谱、HPLC-MS和荧光光谱法对其中一种PPC进行了组成分析和能量传递活性测定。【结果】从R7菌株获得了3种纯化的PPC,1种为反应中心与中心捕光色素蛋白复合体(RC-LH1),2种为外周捕光色素蛋白复合体(LH2),其中一种LH2的吸收光谱具有异常的423nm强吸收峰,其蛋白的两种亚基的分子量分别为5356.8Da和5697.8Da,类胡萝卜素属球形烯系,分子量为562Da,激发光能够从类胡萝卜素向细菌叶绿素以及细菌叶绿素向细菌叶绿素传递。【结论】固氮红细菌产氢菌株含有2种不同光谱特性的LH2,其中一种具有新光谱特性。     

Analysis of absorption spectra changes induced by temperature lowering on phycobilisomes, thylakoids and chlorophyll-protein complexes.

Using fourth derivative analysis, differences between room and low temperature absorption spectra were studied. The positions of most absorption bands of the water-soluble, accessory pigment complex, the phycobilisome, remained unchanged after cooling. The stability of the wavelength positions of chlorophyll a forms in vivo as a function of temperature (Gulyaev, B.A. and Litvin, F.F. (1967) Biofizika 12, 845--854) was generally confirmed. The wavelength positions of all chlorophyll a forms in the P-700 chlorophyll a protein complex were unchanged when the preparations were cooled to -196 degrees C. Likewise, with other chlorophyll-containing materials: the light-harvesting chlorophyll a/b protein complex and the thylakoids of higher plants, algae, and cyanobacteria, the wavelengths positions of most chlorophyll a forms were stable upon cooling. An exception was a 680 nm chlorophyll a band which was generally split at low temperature into two bands with the materials investigated. An interpretation of the multiplicity of chlorophyll spectral forms and the spectral changes induced by cooling for these forms is given using exciton theory and the energy-coupling variation of chlorophyll a molecules.     

Pigment compositions,spectral properties,and energy transfer efficiencies between the xanthophylls and chlorophylls in the major and minor pigment-protein complexes of photosystem II

Absorption, fluorescence, and fluorescence excitation spectra have been measured from CP26, CP29, and monomeric and trimeric LHCIIb light-harvesting complexes isolated from Photosystem II subchloroplast particles from spinach. The complexes were purified using a combination of isoelectric focusing and sucrose gradient ultracentrifugation. The chlorophyll (Chl) and xanthophyll pigment compositions were measured using high-performance liquid chromatography (HPLC). Using the pigment compositions from the HPLC analysis as a starting point, the absorption spectral profiles of the complexes have been reconstructed from the individual absorption spectra obtained for each of the pigments. Also, the fluorescence excitation spectra of the complexes have been deconvoluted. The data reveal the energy transfer efficiencies between Chl b and Chl a and between specific xanthophylls and Chl a in the complexes. The spectral analyses reveal the underlying features of the highly congested spectral profiles associated with the complexes and are expected to be beneficial to researchers employing spectroscopic methods to investigate the mechanisms of energy transfer between the pigments bound in these complexes.     

Spectroscopic resolution of the picosecond reduction kinetics of the secondary electron acceptor A1 in photosystem I

Forward electron transfer in photosystem I from Synechocystis sp. PCC 6803 has been studied in the picosecond time range with transient absorption spectroscopy in the blue and near-UV spectral regions. From the direct measurement, at 380-390 nm, of the reduction kinetics of the phylloquinone secondary acceptor A1 and from the absence of spectral evolution between 100 ps and 2 ns, we conclude that electron transfer, from the chlorophyll a primary acceptor A0, to A1 occurs directly and completely with a time constant of about 30 ps.