The oxidation-reduction potentials of electron carriers in chloroplast photosystem I fragments

Oxidation-reduction titrations of several electron carriers found in chloroplast Photosystem I fragments have been performed. The midpoint potential of P700 in these fragments and in chloroplasts has been found to be +520 mV by optical absorbance methods or electron paramagnetic resonance spectroscopy. The copper-containing protein plastocyanin is present in Photosystem I fragments and has a midpoint potential of +320 mV, significantly less positive than the midpoint potential of cytochrome f in the same fragments, which was measured to be +375 mV. Photo-system I fragments contain two b cytochromes, a low-potential form of cytochrome b₅₅₉ (E_m = +110 mV) and cytochrome b₅₆₃ (E_m = ?100 mV).     

Effect of hydrogen peroxide on the fluorescence and the G550 absorption change of spinach chloroplast fragments

Effects of H₂O₂ on the transient phase of fluorescence and thelight-induced absorption change of C550 in the presence of ferricyanidewere studied in spinach chloroplast fragments at room temperature.In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU),the parameter of the variable fluorescence, work integral, wasincreased by the addition of H₂O₂ and the rate of its recoveryin the dark was decreased. The steady-state fluorescence yieldwas decreased by H₂O₂. Essentially the same results were obtainedin the absence of ferricyanide. In the presence of DCMU, H₂O₂ decreased the steady-state absorptionchange of C550 and inhibited its reoxidation in the dark. Thesame effects were observed when H₂O₂ was added to chloroplastfragments in the presence of DCMU and carbonyl cyanide m-chlorophenylhydrazone.From these data we concluded that the fluorescence quencherQ and C550 are not identical. ¹Present address: Department of Biology, Kyushu Dental College,Kitakyushu 803, Japan. (Received June 20, 1974; )     

Studies on the protolytic reactions coupled with water cleavage in photosystem II membrane fragments from spinach

The protolytic reactions of PSII membrane fragments were analyzed by measurements of absorption changes of the water soluble indicator dye bromocresol purple induced by a train of 10 s flashes in dark-adapted samples. It was found that: a) in the first flash a rapid H⁺-release takes place followed by a slower H⁺-uptake. The deprotonation is insensitive to DCMU but is completely eliminated by linolenic acid treatment of the samples; b) the extent of the H⁺-uptake in the first flash depends on the redox potential of the suspension. In this time domain no H⁺-uptake is observed in the subsequent flashes; c) the extent of the H⁺-release as a function of the flash number in the sequence exhibits a characteristic oscillation pattern. Multiphasic release kinetics are observed. The oscillation pattern can be satisfactorily described by a 1, 0, 1, 2 stoichiometry for the redox transitions S_i S_i+1 (i=0, 1, 2, 3) in the water oxidizing enzyme system Y. The H⁺-uptake after the first flash is assumed to be a consequence of the very fast reduction of oxidized Q₄₀₀(Fe³⁺) formed due to dark incubation with K₃[Fe(CN)₆]. The possible participation of component Z in the deprotonation reactions at the PSII donor side is discussed.Abbreviations A protonizable group at the PSII acceptor side - BCP Bromocresol Purple - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - FWHM Full Width at Half Maximum - Q_A, Q_B primary and secondary plastoquinone at PSII acceptor side - Q₄₀₀ redox group at PSII-acceptor side (high spin Fe²⁺) - P680 Photoactive chlorophyll of PSII reaction center - S_i redox states of the catalytic site of water oxidation - Z redox component connecting the catalytic site of water oxidation with the reaction center     

Electron transport in photosystem I in spinach chloroplasts.

We have studied the recovery of the photochemical activity of Photosystem I after the charge separation induced by a flash under conditions where the secondary donors are in the reduced form. The rate-limiting steps are on the donor side. The first step is completed within 400 mus. The second step is much slower (half time approximately equal to 1 ms) and corresponds to the transfer of electrons from plastoquinone. Under our conditions, only one intermediate is involved in electron transfer between the centers and the plastoquinone pool. Electron exchange between the Sytem I centers has been demonstrated.     

Oxidation-reduction reactions between electron transfer components and hydrogen peroxide in photosystem II of chloroplasts

The light-induced oxygen evolution, photoreduction of 2,6-dichlorophenolindophenol (DPIP) and carotenoid photobleaching induced by carbonylcyanide m-chlorophenylhydrazone (CCCP) were investigated withspinach chloroplast fragments in the presence of H₂O₂. Oxygenevolution in the presence of H₂O₂ was not inhibited by CCCPand was only partially inhibited by 5 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea(DCMU) which completely inhibited the Hill reaction with DPIP.The degree of inhibition by DCMU was decreased by a simultaneousaddition of CCCP. Carotenoid photobleaching in the presenceof CCCP was stimulated by H₂O₂. The CCCP-induced carotenoidphotobleaching was completely inhibited by DCMU. However, itwas only partially inhibited by DCMU in the presence of H₂O₂.These data indicate that H₂O₂ donates electrons at a site betweenthe CCCP-sensitive site and the reaction center of photosystemII and is reduced at a site between the DCMU-blocked site andthe reaction center of photosystem II. ¹Present address: Department of Biology, Kyushu Dental College,Kitakyushu 803, Japan. (Received June 20, 1974; )     

Photosystem II reactions in oxidant-treated chloroplast fragments.

Treatment of Photosystem II fragments with the oxidant K₂IrCl₆ destroys approximately 50% of the bulk chlorophyll and results in fragments that are twofold enriched in P680 (the Photosystem II reaction-center chlorophyll) and cytochrome b₅₅₉. The fragments retain a fully competent reaction center, as evidenced by P680 photooxidation and subsequent reduction in a back reaction with the primary electron acceptor ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 5}\text{ms}$ at 25 dgK). The K₂IrCl₆-treated fragments contain no photoactive or chemically detectable C-550 and do not exhibit any variable fluorescence. These results imply that the Photosystem II primary electron acceptor is unaffected by oxidant treatment. It therefore may be concluded that neither C-550 nor the fluorescence quencher, Q, functions as the primary electron acceptor of Photosystem II.     

Light-dependent reduction of hydrogen peroxide by intact spinach chloroplasts

Intact spinach chloroplasts, washed four times in buffered sorbitol to decrease catalase contamination, supported O₂ evolution in the dark at very low rates (less than 2 μmol/mg Chl per h) in the presence of low concentrations of H₂O₂ (0.25 mM); H₂O₂ was not significantly metabolished under these conditions. In the light, washed chloroplasts supported H₂O₂-dependent O₂ evolution at rates of 28–46 μmol/mg Chl per h in the presence of 0.1–0.25 mM H₂O₂; the concentration of H₂O₂ supporting 0.5V_max was estimated to be 25 μM. O₂ evolution in the light was associated with H₂O₂ consumption and ceased after the production of 0.45 mol per mol H₂O₂ consumed. Both O₂ evolution and H₂O₂ consumption were abolished by 5 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Washed intact chloroplasts contained endogenous pools of GSH and ascorbate estimated at 10 and 33 mM, respectively. H₂O₂-dependent O₂ evolution in the light was associated with a decrease in these levels which increased as O₂ evolution gradually ceased. The results are consistent with the hypothesis that H₂O serves as eventual electron donor for the reduction of H₂O₂ in illuminated chloroplasts and that GSH/GSSG and ascorbate/dehydroascorbate serve as intermediate electron carriers. Preincubation of chloroplasts in the dark with 0.1 mM H₂O₂ abolished O₂ evolution in the light.     

Reactions of photosystems I and II in spinach chloroplast fragments treated with ethylene glycol

Photochemical reactions of chloroplast fragments isolated fromspinach leaves were measured in the presence of ethylene glycolor were measured after washing with an ethylene glycol-containingmedium. 2,6-Dichlorophenolindophenol (DPIP) photoreduction,oxygen evolution and oxygen uptake (a photosystem I reaction)were investigated in ethylene glycol-treated chloroplast fragments.By washing with ethylene glycol, oxygen evolution was stronglyinhibited, but oxygen uptake was not much affected by ethyleneglycol washing. Chloroplast fragments in 50% ethylene glycolmaintained a high rate of DPIP photoreduction (85% of the controlactivity in an ethylene glycol-less medium). In 67% ethyleneglycol, DPIP photoreduction mediated by photosystem II was eliminatedand only a small rapid reduction mediated by photosystem I wasobserved. Chloroplast fragments inhibited by ethylene glycolphotoreduced DPIP in the presence of p-aminophenol added asan artificial electron donor to photosystem II. The restoredactivity of DPIP photoreduction was inhibited by 3-(3',4'- dichlorophenyl)-1,1-dimethylurea. (Received September 8, 1970; )     

Photosynthetic electron flow to oxygen and diffusion of hydrogen peroxide through the chloroplast envelope via aquaporins

Light-induced generation of superoxide radicals and hydrogen peroxide in isolated thylakoids has been studied with a lipophilic spin probe, cyclic hydroxylamine 1-hydroxy-4-isobutyramido-2,2,6,6-tetramethylpiperidinium (TMT-H) to detect superoxide radicals, and the spin trap α-(4-pyridyl-1-oxide)-N-tert-butylnitron (4-POBN) to detect hydrogen peroxide-derived hydroxyl radicals. Accumulation of the radical products of the above reactions has been followed using electron paramagnetic resonance. It is found that the increased production of superoxide radicals and hydrogen peroxide in higher light is due to the enhanced production of these species within the thylakoid membrane, rather than outside the membrane. Fluorescent probe Amplex red, which forms fluorescent product, resorufin, in the reaction with hydrogen peroxide, has been used to detect hydrogen peroxide outside isolated chloroplasts using confocal microscopy. Resorufin fluorescence outside the chloroplasts is found to be suppressed by 60% in the presence of the inhibitor of aquaporins, acetazolamide (AZA), indicating that hydrogen peroxide can diffuse through the chloroplast envelope aquaporins. It is demonstrated that AZA also inhibits carbonic anhydrase activity of the isolated envelope. We put forward a hypothesis that carbonic anhydrase presumably can be attached to the envelope aquaporins. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Light-induced absorbance changes at 475-515 nm, and electron flow close to photosystem II in Tris-washed chloroplast fragments

Rapid, reversible light-induced absorbance changes at 475 and515 nm in chloroplast fragments were diminished by washing thefragments with Tris. These diminished absorbance changes wererestoredby the donation of electrons to photosystem II by hydrogen peroxide (Received November 17, 1970; )     

X-ray diffraction studies on photosystem I fragments from a blue-green alga, Anabaena variabilis, and spinach

Photosystem I fragments were prepared from thylakoid membranes of a blue-green alga (Anabaena variabilis) and spinach by treatment with a detergent, Triton X-100. Equatorial X-ray diffraction patterns were recorded on films for oriented specimens of thylakoid membranes and photosystem I fragments. The thylakoid membranes and photosystem I fragments gave essentially the same equatorial diffraction patterns in both Anabaena variabilis and spinach, indicating that the major X-ray scatterers in these thylakoid membranes are the molecular assembly of photosystem I. The equatorial X-ray diffraction from the photosystem I fragments of Anabaena variabilis and spinach extends to the reciprocal space of 1/7 A-1. The diffraction pattern exhibits six to nine distinct maxima though they are diffuse, indicating that the arrangement of the constituent molecules in photosystem I has a definite geometrical regularity. The radial autocorrelation functions indicate that the maximal sizes of photosystem I in these thylakoid membranes are about 100 A, and the geometrical regularity does not correspond to a crystalline order. The X-ray diffraction patterns from photosystem I fragments from Anabaena variabilis and spinach are quite similar to each other, suggesting the possibility that the molecular structures of photosystem I in Anabaena variabilis and spinach have a fundamental similarity. These diffraction patterns, however, are different from that of the chromatophore obtained from a photosynthetic bacterium, Rhodospirillum rubrum.     

Electrochemically produced hydrogen peroxide affects Joliot-type oxygen-evolution measurements of photosystem II

The main technique employed to characterize the efficiency of water-splitting in photosynthetic preparations in terms of miss and double hit parameters and for the determination of S_i (i = 2,3,0) state lifetimes is the measurement of flash-induced oxygen oscillation pattern on bare platinum (Joliot-type) electrodes. We demonstrate here that this technique is not innocent. Polarization of the electrode against an Ag/AgCl electrode leads to a time-dependent formation of hydrogen peroxide by two-electron reduction of dissolved oxygen continuously supplied by the flow buffer. While the miss and double hit parameters are almost unaffected by H₂O₂, a time dependent reduction of S₁ to S_− 1 occurs over a time period of 20 min. The S₁ reduction can be largely prevented by adding catalase or by removing O₂ from the flow buffer with N₂. Importantly, we demonstrate that even at the shortest possible polarization times (40 s in our set up) the S₂ and S₀ decays are significantly accelerated by the side reaction with H₂O₂. The removal of hydrogen peroxide leads to unperturbed S₂ state data that reveal three instead of the traditionally reported two phases of decay. In addition, even under the best conditions (catalase + N₂; 40 s polarization) about 4% of S_− 1 state is observed in well dark-adapted samples, likely indicating limitations of the equal fit approach. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

Heterogeneity in chloroplast photosystem II

Photosystem-two (PSII) in the chloroplasts of higher plants and green algae is not homogeneous. A review of PSII heterogeneity is presented and a model is proposed which is consistent with much of the data presented in the literature. It is proposed that the non-quinone electron acceptor of PSII is preferentially associated with the sub-population of PSII known as PSIIß.Abbreviations and symbols ATP Adenosine triphosphate - Chl Chlorophyll - C₅₅₀ Absorbance bandshift at 550 nm; proportional to [Q_A ^-] - D, D Components involved ir electron donation to P680 - pH Transthylakoid proton gradient - Transthylakoid electrical gradient - DCMU 3-(3,4-Dichlorophenyl)-1,1-dimethylurea referred to as diuron - E _h Oxidation-reduction potential - E _m Cxidation-reduction midpoint potential - EPR Electron paramagnetic resonance - Fm Fluorescence yield when all traps are closed - Fo Fluorescence yield when all traps are open - Fv Variable fluorescence equal to Fm-Fo - Fi Initial fluorescence yield, (usually equivalent to Fo in dark-adapted thylakoids) - Hepes 2-hydroxyethylpiperazine-N-2-ethane sulphonic acid - LHCP Light-harvesting chlorophyll a/b binding protein - PQ Plastoquinone - PSII Photosystem II - P680 Reaction centre chlorophyll of PSII - P₅₁₈ Absorbance bandshift at 518 nm, reflects asymmetric charge distribution across the thylakoid membrane - Q_A, _QH , Q₁ Primary stable plastoquinone electron acceptor of PSII; a quencher of fluorescence - Q _B , B, R Plastoquinone associated with the Q _B -protein, the two-electron gate - Q _D , Q₂, X _a Non-quinone electron acceptor of PSII - X₃₂₀ Absorbance bandshift at 320 nm; semiquinone anion indicator     

Temperature-dependent vibrational and conformational dynamics of photosystem II membrane fragments from spinach investigated by elastic and inelastic neutron scattering

Vibrational and conformational protein dynamics of photosystem II (PS II) membrane fragments from spinach were investigated by elastic and inelastic incoherent neutron scattering (EINS and IINS). As to the EINS experiments, the average atomic mean square displacement values of PS II membrane fragments hydrated at a relative humidity of 57% exhibit a dynamical transition at ~230K. In contrast, the dynamical transition was absent at a relative humidity of 44%. These findings are in agreement with previous studies which reported a "freezing" of protein mobility due to dehydration (Pieper et al. (2008) Eur. Biophys. J. 37: 657-663) and its correlation with an inhibition of electron transfer from Q(A)(-) to Q(B) (Kaminskaya et al. (2003) Biochemistry 42, 8119-8132). IINS spectra of a sample hydrated at a relative humidity of 57% show a distinct Boson peak at ~7.5meV at 20K, which shifts towards lower energy values upon temperature increase to 250K. This unexpected effect is interpreted in terms of a "softening" of the protein matrix along with the onset of conformational protein dynamics as revealed by the EINS experiments. Information on the density of vibrational states of pigment-protein complexes is important for a realistic calculation of excitation energy transfer kinetics and spectral lineshapes and is often routinely obtained by optical line-narrowing spectroscopy at liquid helium temperature. The data presented here demonstrate that IINS is a valuable experimental tool in determining the density of vibrational states not only at cryogenic, but also at nearly physiological temperatures up to 250K. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.