STRUCTURAL RELATIONSHIPS OF THE PHOTOSYSTEM I AND PHOTOSYSTEM II CHLOROPHYLL a/b AND a/c LIGHT-HARVESTING APOPROTEINS OF PLANTS AND ALGAE

Polyclonal antibodies against four different apoproteins of either the chlorophyll (Chl) a/b light-harvesting antenna of photosystem I or II, or a chlorophyll-protein complex homologous to CP26 from Chlamydomonas reinhardtii, crossreact with11–13 thylakoid proteins of Chlamydomonas, Euglena gracilis and higher plants. The number of antigenically-related proteins correlates with the quantity of light-harvesting chlorophyll-protein complex (LHC) gene types that have been sequenced in higher plants. The antibodies also react specifically with Chi a/c-binding proteins of three diatoms and Coccolithophora sp. as determined by immunoblot and Ouchterlony assays. Four to six crossreacting proteins are observed in each chromophyte species and a functional role for some can be deduced by antibody reactivity. It appears that despite major differences in the structures of their pigment ligands, at least some domains of Chl-binding LHC apoproteins have been conserved during their evolution, possibly functioning in protein: protein, as opposed to pigment: protein, interactions in photosynthetic membranes.     

NONLINEAR LASERSPECTROSCOPIC INVESTIGATIONS OF THE PIGMENT-PIGMENT INTERACTION WITHIN THE LIGHT-HARVESTING COMPLEX OF PHOTOSYSTEM II

Using a pump and test beam technique in the frequency domain with pump pulses in the nanosecond time range, the nonlinear transmission properties were investigated at room temperature in photosystem (PS) II membrane fragments and isolated light-harvesting chlorophyll a/b-protein preparations (LHC II preparations). In LHC II preparations and PS II membrane fragments, respectively, pump pulses of 620 nm and 647 nm cause a transmission decrease limited to a wavelength region in the nearest vicinity of the pump pulse wavelength (full width at half maximum ' 0.24 nm). In contrast, at 670 nm neither a transmission decrease nor a narrow band feature were observed. The data obtained for PS II membrane fragments and LHC II preparations at shorter wavelengths (620 nm, 647 nm) were interpreted in terms of excited state absorption of whole pigment-protein clusters within the light-harvesting antenna of photosystem II. The interpretation of the small transmission changes as homogeneously broadened lines led to a transversal relaxation time for chlorophyll in the clusters of about 4 ps.     

INCREASED ENERGY TRANSFER FROM PHYCOBILISOMES TO PHOTOSYSTEM II IN HIGH LIGHT ADAPTED Anabaena cylindrica

Excitation energy transfer from phycobilisomes to photosystem II in high-light adapted cells of Anabaena cylindrica was studied by fluorescence spectroscopy and compared to that of low-light adapted cells. Measurements were made on membrane fragments containing phycobilisomes, photosystem I and II, isolated in 0.75 M K-phosphate. Relative efficiency of 430 to 590 nm light in the excitation of F₆₈₀ chlorophyll fluorescence was compared in low and high light adapted cells, respectively. The values indicate that light energy absorbed by phycobilisomes is transferred to photosystem II antenna chlorophylls with higher efficiency in high-light adapted cells than in low-light adapted cells. Partial dissociation and uncoupling of energy transfer caused by low ion concentration were different in the membrane fragments isolated from the two kinds of cells and indicated a higher aggregation state of pigment-protein complexes of phycobilisomes in high-light adapted A. cylindrica cells.     

PHOTOCHEMICAL GENERATION OF REDUCED QUINONE CATALYSTS OF PHOTOSYSTEM I CYCLIC PHOTOPHOSPHORYLATION ACTIVITY

When reaction mixtures containing 9,10-anthraquinone-2-sulfonate and chloroplasts poisoned with 3–(3,4-dichlorophenyl)-l, l′-dimethylurea were illuminated with white light, photosystem I-catalyzed cyclic photophosphorylation was observed. Illumination of identical reaction mixtures with red light produced no ATP synthesis. This phenomenon is due to photoreduction of the anthraquinone which is supported by the electron donor activity of Tricine buffer. The photoreduction reaction was used to generate reduced catalysts (anthraquinone sulfonate, menadione bisulfite) of photosystem I cyclic photophosphorylation activity. The rates of ATP synthesis obtained by this method (250–300 μmol/h-mg chlorophyll) indicate that sulfonated quinones are efficient mediators of cyclic electron transport around photosystem I. Although the activity catalyzed by these compounds is highly sensitive to dibromothymoquinone, very little decrease in activity is observed with antimycin A.     

LIMITING REACTIONS IN HYDROGEN PHOTOPRODUCTION BY CHLOROPLASTS AND HYDROGENASE

Abstract— Hydrogen was photoproduced from water in a system containing isolated chloroplasts, hy-drogenase, a coupling electron carrier (ferredoxin or methyl viologen), and an oxygen scavenger. The rate and extent of hydrogen production anaerobically was much less than the rate of aerobic electron-carrier reduction by chloroplasts and was not limited by hydrogenase. The limiting reaction in the coupled system was the extent of reduction of methyl viologen anaerobically rather than its oxidation by oxygen produced during the course of the reaction. Inhibition of photosystem II by 3-(3,4dichlorophenyl)-1,1-dimethylurea and addition of a photosystem 1 electron donor did not lead to photoproduction of hydrogen or photoreduction of methyl viologen. Extensive photosystem I hydrogen evolution was obtained when thiols were also present. Platinum asbestos or palladium asbestos replaced hydrogenase in a system coupled to chloroplasts.     

INHIBITION OF ELECTRON TRANSPORT IN CHLOROPLASTS BY CHAOTROPIC AGENTS AND THE USE OF MANGANESE AS AN ELECTRON DONOR TO PHOTOSYSTEM II*

Abstract— Incubating spinach chloroplasts with various chaotropic agents results in inhibition of photosynthetic electron transport between water and Photosystem II similar to the inhibition caused by washing chloroplasts with a high concentration of Tris buffer. Partial restoration of NADP photoreduction and fluorescence of variable yield is achieved by adding hydroquinone or Mn²⁺, either of which donates electrons to Photosystem II in the inhibited chloroplasts. The inhibitory treatments cause the release of Mn from its bound state in the chloroplast, thus allowing the measurement of the ESR signal of Mn²⁺. The ESR measurement is used to follow the photooxidation of Mn²⁺ as it donates electrons to photosystem II.     

CHARGE TRANSFER FROM PHOTOSYSTEM I TO THE CYTOCHROME b/f COMPLEX: DIFFUSION AND MEMBRANE LATERAL HETEROGENEITY

Abstract— We discuss here the minimum requirements for diffusion of a charge carrier between appressed and stroma-exposed membrane regions of chloroplasts based on recent models of the thylakoid membrane and flash-induced kinetic data. We have investigated the kinetics of the transfer of a positive charge from photosystem I to the cytochrome b/f complex in spinach chloroplasts by measuring the light-induced oxidation of cytochrome f. The rate and extent of cytochrome f oxidation were measured spectrophotometrically using either long actinic flashes that induced several turnovers of photosystem I or short actinic flashes that induced a single turnover of photosystem I. In the long actinic flashes, in the electron transfer reaction from water to methyl viologen, we observed the rapid oxidation of all of the cytochrome f present in the membrane. The half-time of the oxidation was 1.0 ± 0.1 ms. The total amount of the cytochrome was determined by chemical difference spectra to be one molecule of cytochrome f per 650 – 30 chlorophyll molecules. Using short actinic flashes we studied the photosystem I-driven electron transfer reaction from duroquinol to methyl viologen in the presence of the inhibitor 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. Under these conditions a single turnover flash induced the oxidation of 62 ± 5% of cytochrome f with a half-time of 240 ± 30 μs. An Arrhenius plot of the temperature dependence of the cytochrome f oxidation rate revealed an activation energy between 16 and 21 kJ/mol, a value consistent with a diffusion-controlled reaction. These kinetic data are considered in the context of two models of the thylakoid membrane.     

EFFECTS OF PHOTOSYSTEM II INHIBITORS ON ELECTRON PARAMAGNETIC RESONANCE SIGNAL II OF SPINACH CHLOROPLASTS

Abstract— In isolated spinach chloroplasts the light-induced electron paramagnetic resonance signal (signal II) associated with the oxygen evolving photosystem (photosystem II) decays slowly and incompletely in the dark. Tris-washing, hydroxylamine, or carbonylcyanide m -chlorophenylhydrazone (CCCP) enhance the decay of signal II, which can still be induced by red (645 nm) but not by far-red (735 nm) radiation. Although 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) alone has no effect on signal II, it blocks the induction of signal II in the presence of hydroxylamine or CCCP. These data suggest that signal II is an indicator of an oxidized intermediate on the water-splitting side of photosystem II.     

PHOTOSYNTHETIC ACTIVITY AND CHLOROPHYLL ABSORPTION SPECTRA OF FRACTIONS FROM THE ALGA,DUNALIELLA*

Abstract— Dunaliella chloroplasts were fractionated according to C. Arntzen et al, Biochim. Biophys. Acta 256 , 85–107, 1972. The initial French-press treatment and differential centrifugation produced Fraction 1 (Fr 1) enriched in photosystem I activity and a heavier Fraction 2 (Fr 2). When Fr 2 was treated with digitonin followed by either gradient or differential centrifugation, two more fractions were recovered: Fr 1 g with a photosystem 1 activity similar to that of Fr 1, and Fr 2 g with very low photosystem II activity. Photosystem II activity was considerably lower in these Dunaliella chloroplasts and fractions than in spinach particles measured under the same conditions, but the relative activities between the fractions were similar to those for spinach. Fr 2 always had greater photosystem II activity than Fr 1, but the digitonin fractions were low and similar in photosystem II activity. Photosystem II activity was measured as the reduction of 2, 6–dichlorophenol indophenol (DCIP) with H₂O, diphenylcarbazide (DPC) or Mn²⁺ as electron donor. The results indicated that exogenous manganous ion competed with H₂O as an electron donor to photosystem II in broken chloroplasts initially, but after 10–15 s of illumination, the Mn³⁺ formed began to reoxidize DCIP and a cyclic reaction ensued. DPC and Mn²⁺ appeared to react at different sites. Computer-assisted curve analysis of the absorption spectrum of each fraction revealed four major component curves representing the absorbing forms of chlorophyll a at 663, 670, 679 and 684 nm seen in numerous other in vivo chlorophyll spectra (C. S. French et al., Plant Physiol. 49 , 421–429, 1972). However, Fr 2g had approx. 20 percent more of Ca663 and Ca670 and 10% more absorption by chl b than Fr 1 which correlated with the difference in photosystem II activity. On the long wavelength side, Fr 2 g had no Ca694 and almost no photosystem I activity. The results are not sufficient to answer the question of whether the photosystem I particle obtained from the original homogenate is significantly similar to or different from the corresponding fraction obtained from Fr 2 with digitonin.     

LIGHT-INDUCED QUENCHING OF PHOTOSYSTEM II FLUORESCENCE AT 77 K

Abstract— Light-induced quenching of the low temperature fluorescence emission from photosystem II (PS II) at 695 nm ( F ₆₉₅) has been observed in chloroplasts and whole leaves of spinach. Photosystem I (PS I) fluorescence emission at 735 nm ( F ₇₃₅) is quenched to a lesser degree but this quenching is thought to originate from PS II and is manifest in a reduced amount of excitation energy available for spillover to PS I. Differential quenching of these two fluorescence emissions leads to an increase in the F ₇₃₅/ F ₆₈₅ ratio on exposure to light at 77 K. Rewarming the sample from -196°C discharges the thermoluminescence Z-band and much of the original unquenched fluorescence is recovered. The relationship between the thermoluminescence Z-band and the quenching of the low temperature fluorescence emission ( F ₆₉₅) is discussed with respect to the formation of reduced pheophytin in the PS II reaction center at 77 K.     

AFFINITY FOR OXYGEN IN PHOTOREDUCTION OF MOLECULAR OXYGEN AND SCAVENGING OF HYDROGEN PEROXIDE IN SPINACH CHLOROPLASTS

Abstract— The apparent K _m for O₂ in the photoreduction of molecular oxygen by spinach class II chloroplasts and photosystem I subchloroplast fragments was determined. In both cases, a value of 2 ∼ 3 μ M O₂ was obtained. The reaction rate constant between O₂ and P-430, the primary electron acceptor of PS I, is estimated to be ∼ 1.5 × 10⁷ M ^-1 s^-1 and the factors affecting the production of superoxide by the photoreduction of O₂ in chloroplasts are discussed. Preliminary evidence is presented indicating the occurrence of an azide-insensitive scavenging system for H₂O₂ in chloroplast stroma.     

HOLE-BURNING SPECTROSCOPY OF ACTIVE AND INACTIVATEE) PHOTOSYSTEM II PARTICLES

This paper reports transient and persistent hole-burning of photosynthetically active as well as chemically reduced and heat inactivated photosystem II particles isolated from cyanobacteria. Transient spectra of active and non-active particles are significantly different. For both, the possible origin of the bottle-neck state is discussed. Persistent holes were ascribed to the antenna complex of photosystem II. From their width the energy transfer rate was estimated to be 4.8 ps at 4.2 K.     

LIGHT-ABSORPTION AND ELECTRON-TRANSPORT BALANCE BETWEEN PHOTOSYSTEM II AND PHOTOSYSTEM I IN SPINACH CHLOROPLASTS

Abstract— The distribution of absorbed light and the turnover of electrons by the two photosystems in spinach chloroplasts was investigated. This was implemented upon quantitation of photochemical reaction centers, chlorophyll antenna size and composition of each photosystem (PS), and rate of light absorption in situ. In spinach chloroplasts, the photosystem stoichiometry was PSIIJPSII_α/PSII_β/PSI= 1.3/0.4/1.0. The number (N) of chlorophyll (a+b) molecules associated with each PS was N(PSII_α)/N(PSII_β)/N(PSI)=230/100/200, i.e. about 65% of all Chl is associated with PSII and about 35% with PSI. Light absorption by PSII in vivo is selectively attenuated at the molecular, membrane and leaf levels, (a) The rate of light absorption by PSII was only 0.85 that of PSI because of the lower rate of light absorption by Chl b as compared to Chl a (approximately 80% of all Chl b in the chloroplast is associated with PSII). (b) The exclusive localization of PSII_α in the membrane of the grana partition regions and of PSI in intergrana lamellae resulted in a differential “sieve effect” or “flattening of absorbance” by the photosystems in the two membrane regions. Due to this phenomenon, the rate of light absorption by PSII was lower than that of PSI by 15-20%. (c) Selective filtering of sunlight through the spinach leaf results in a substantial distortion of the effective absorbance spectra and concomitant attenuation of light absorption by the two photosystems. Such attenuation was greater for PSII than for PSI because the latter benefits from light absorption in the 700-730 nm region. It is concluded that, in spite of its stoichiometric excess in spinach chloroplasts, light absorption by PSII is not greater than that by PSI due to the different molecular composition of the two light-harvesting antenna systems, due to the localization of PSII in the grana, and also because of the light transmission properties through the leaf. The elevated PSII/PSI reaction center ratio of 1.7 and the association of 65% of all Chl with PSII help to counter the multilevel attenuation of light absorption by PSII and ensure a balanced PSII/PSI electron turnover ratio of about 1:1.     

Isolation and purification assay of ex vivo photosystem II D1 protein toward integrated biointeraction analysis

Protein extracts of photosystem II were prepared from leaf chloroplasts of different plant species by fast and nondenaturing methods. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analysis of the proteins obtained showed that the extracts were enriched by D1 proteins, which appeared putatively in association with the 33-kDa oxygen-evolving-complex subunits. In further isolation steps D1 proteins were purified using salt-gradient chromatography (fast protein liquid chromatography) and characterized by western blot and mass spectrometry.     

BIMANES—26. AN ELECTRON TRANSFER REACTION BETWEEN PHOTOSYSTEM I1 AND MONOBROMOBIMANE INDUCES STATIC CHLOROPHYLL a QUENCHING IN SPINACH CHLOROPLASTS

Abstract— Monobromobimane in chloroplasts lowers both the quantum yield of system II photochemistry and the yield of chlorophyll a fluorescence. Illumination of the chloroplasts in the presence of monohromobimane is an absolute prerequisite to the manifestation of this phenomenon, which proceeds via the Photosystem II intermediate, the semiquinone radical anion, Q_A-. The latter transfers an electron to monobromobimane to yield an anion radical (mBBr^·), which may either lose bromide ion to yield a reactive radical (mB^·), or acquire a proton and undergo further reduction, eventually forming syn-(methyl, methyl) bimane. In turn, mB reacts with the protein of the light-harvesting complex, to form a product which acts as static excitation energy quencher in the chlorophyll pigment bed of photosystem 11. Polarographic reduction of monobromobimane shows an adsorption wave at O V and two reduction waves. Prolonged reduction in water at -0.5 V yields syn-(methyl, methyl) bimane (which is further reduced at more negative potentials) and bromide ion. Thus, both electrochemical and chloroplast-induced reduction produce syn-(methyl, methyl) bimane. Monobromobimane may then serve as a Photosystem II activated probe in elucidating the conformation of intrinsic thylakoid membrane polypeptides.     

SINGLET OXYGEN IS NOT PRODUCED IN PHOTOSYSTEM I UNDER PHOTOINHIBITORY CONDITIONS

Abstract— Excess illumination of photosynthetic systems brings about the complex functional and structural damage known as photoinhibition. According to the generally accepted and experimentally confirmed model, photoinhibition involves singlet oxygen production and subsequent oxidative damage in the photosystem II reaction center. However, it was recently suggested that singlet oxygen is not necessarily produced in photosystem II itself but rather in the non-heme iron-containing Fe-S centers of photosystem I (Chung, S.K. & J. Jung, Photochem. Photobiol. 61, 383–389, 1995). Contrary to this suggestion, our electron paramagnetic resonance spectroscopy experiments with the singlet oxygen trap 2,2,6,6-tetramethylpiperidine demonstrate that under photoinhibitory conditions, singlet oxygen is present in thylakoids and photosystem II core complex preparations but is not produced in photosystem I particles.     

PHOTOSYSTEM II HETEROGENEITY IN THE MARINE DIATOM Phaeodactylum tricornutum

Abstract— The kinetics of photosystem II photochemistry are analyzed in the marine diatom Phaeodacfylum tricornutum by measurement of fluorescence induction in cell suspensions treated with 3–(3,4-dichlorophenyl)-1,1-dimethylurea. Photosystem II kinetics are found to be biphasic, the sum of two exponential components, suggesting that biphasic energy conversion in photosystem II may be a general consequence of thylakoid membrane appression. The emission wavelength-dependence of fluorescence induction suggests that the two photosystem II components have different variable fluorescence emission spectra. The slower component exhibits characteristic emission of the diatom light-harvesting complexes while emission from the faster component resembles that of the photosystem II reaction center. Variable fluorescence emission (293 K) at wavelengths > 700 nm is assigned to photosystem II. Application of model equations indicates that the two photosystem II unit types differ primarily in antenna size. A new analytical procedure is presented which eliminates ambiguities in the kinetic analysis associated with the incorrect assignment of the maximal fluorescence yield.     

Conjugated Polymer Nanoparticles to Augment Photosynthesis of Chloroplasts

By coating chloroplasts with conjugated polymer nanoparticles (CPNs), a new bio‐optical hybrid photosynthesis system (chloroplast/CPNs) is developed. Since CPNs possess unique light harvesting ability, including the ultraviolet part that chloroplasts absorb less, chloroplast/CPN complexes can capture broader range of light to accelerate the electron transport rates in photosystem II (PS II), the critical protein complex in chloroplasts, and augment photosynthesis beyond natural chloroplasts. The degree of spectral overlay between emission of CPNs and absorption of chloroplasts is critical for the enhanced photosynthesis. This work exhibits good potential to explore new and facile nanoengineering strategy for reforming chloroplast with light‐harvesting nanomaterials to enhance solar energy conversion.     

QUANTITATION OF PHOTOSYSTEM II ACTIVITY IN SPINACH CHLOROPLASTS. EFFECT OF ARTIFICIAL QUINONE ACCEPTORS

Quantitation of photosystem II (PSII) activity in spinach chloroplasts is presented. Rates of PSII electron-transport were estimated from the concentration of PSII reaction-centers (Chl/PSII = 380:1 when measured spectrophotometrically in the ultraviolet [ΔA₃₂₀] and green [ΔA_540–550] regions of the spectrum) and from the rate of light utilization by PSII under limiting excitation conditions. Rates of PSII electron-transport were measured under the same light-limiting conditions using 2,5-dimethylbenzoquinone or 2,5-dichlorobenzoquinone as the PSII artificial electron acceptors. Evaluation is presented on the limitations imposed in the measurement of PSII electron flow to artificial quinones in chloroplasts. Limitations include the static quenching of excitation energy in the pigment bed by added quinones, the fraction of PSII centers (PSII_β) with low affinity to native and added quinones, and the loss of reducing equivalents to molecular oxygen. Such artifacts lowered the yield of steady-state electron transport in isolated chloroplasts and caused underestimation of PSII electron-transport capacity. The limitations described could explain the low PSII concentration estimates in higher plant chloroplasts (Chl/PSII = 600 ± 50) resulting from proton flash yield and/or oxygen flash-yield measurements. It is implied that quantitation of PSII by repetitive flash-yield methods requires assessment of the slow turnover of electrons by PSII_β and, in the presence of added quinones, assessment of the PSII quantum yield.     

OXYGEN MEDIATED PHOTOSYSTEM I ACTIVITY IN THYLAKOID MEMBRANES MONITORED BY A PHOTOELECTROCHEMICAL CELL

Abstract— A photoelectrochemical cell has been used to monitor the effects of three enzymes on the photocurrent produced by isolated spinach thylakoids. The enzymes were glucose oxidase, superoxide dismutase and catalase. It is shown that all three inhibit the photocurrent to varying degrees. The results demonstrate that electron transport to the working electrode is mediated by oxygen. Further, the activity monitored originated from photosystem I with oxygen as the acceptor and photosystem II/plastoquinone as the donor. Thus, the photoelectrochemical cell constitutes a potential new approach for the monitoring of pseudocyclic electron transport.