Radical pair interactions in spinach chloroplasts

Time-resolved EPR studies were done on broken spinach chloroplasts under reducing conditions at low temperature (10 K). A dramatic dependence of signal dynamics and lineshape in the g 2.00 region on the reduction state of Photosystem I is demonstrated. Computer simulations of the spin-polarized lineshapes obtained in this work suggest that the primary electron acceptor in Photosystem I, a species known as A₁, could be a chlorophyll a dimer.     

Oxygen activation by isolated chloroplasts from Euglena gracilis. Ferredoxin-dependent function of a fluorescent compound and photosynthetic electron transport close to photosystem.

Oxygen reduction by isolated chloroplast lamellae from spinach, yielding the superoxide free radical in the light, is stimulated by a fluorescent factor (“compound No. 4”, isolated from Euglena gracilis strain Z) in a ferredoxin-dependent reaction. This reaction is not observed with Euglena chloroplasts, although there is a stimulation by compound No. 4 of ferredoxin-dependent oxygen reduction at the expense of NADPH + H⁺ as electron donor in the dark. Evidence is provided that in Euglena chloroplasts in the absence of NADP as electron acceptor a cyclic electron transport is predominating, including photosystem I, ferredoxin, NADP-ferredoxin reductase, and cytochrome₅₅₂. Isolated spinach chloroplast lamellae show a similar “cyclic” electron transport after treatment with digitonin, depending on the addition of the above cofactors. This result might indicate that Euglena chloroplast lamellae show this cyclic electron transport only as an artifact due to the isolation procedure. The results furthermore indicate that the pteridine-like, fluorescent compound No. 4 is not active as the primary electron acceptor of photosystem I; it may however be involved in oxygen activation by Euglena gracilis chloroplasts.     

Flash photolysis electron spin resonance studies of the electron acceptor species at low temperatures in Photosystem I of spinach subchloroplast particles

The light-induced electron spin resonance signals of Photosystem I spinach subchloroplast particles have been studied at approximately 6 °K. Using the technique of flash photolysis-electron spin resonance with actinic illumination at 647 nm, a kinetic analysis of the previously observed bound ferredoxin ESR signals was carried out. Signal I (P700⁺) exhibits a partial light-reversible behavior at 6 °K so it was expected that if the bound ferredoxin is the primary acceptor of Photosystem I, it should also exhibit a partial reversible behavior. However, none of the bound ferredoxin ESR signals showed any such light reversible behavior. A search to wider fields revealed two components which did exhibit the expected kinetic behavior. These components are very broad (about 80 G) and are centered at g = 1.75 and g = 2.07. These two components exhibit the expected characteristics of the primary electron acceptor. A model is presented to account for the reversible and irreversible photochemical changes in Photosystem I. The possible identity of the primary acceptor responsible for these two new components, is discussed in terms of the available information. The primary acceptor may be an iron-sulfur protein, but not of the type characteristic of the bound or water-soluble ferredoxins found so far in chloroplasts.     

On a water soluble factor neutralizing antibodies against the primary acceptor in photosynthetic electron transport of chloroplasts

Summary Rabbits immunized against the thylakoid system of chloroplasts form two different antibodies against the reducing site of photosystem I. One inhibits photosynthetic NADP⁺ and ferredoxin dependent cytochrome c reduction, but not photosynthetic anthraquinone reduction by isolated spinach chloroplasts. The other inhibits all three reductions. Both do not inhibit a Hill reaction with ferricyanide. The presumed antigen moiety against these antibodies was isolated as a water soluble factor released from lyophilized chloroplasts after treatment with diethylether. The heat stable, nondialysable factor has absorption peaks at 262 and 315 m. It is autoxidizable and has the property of a cytochrome c reducing substance. The factor neutralizes the antibody inhibition of photosynthetic anthraquinone and ferredoxin reduction. It is proposed that the soluble factor is the prosthetic group of the primary acceptor of photosystem I.Abbreviations CRS cytochrome c reducing substance - FRS ferredoxin reducing substance - DAD diaminodurene - TMPD N-tetramethyl-p-phenylene-diamine. The work reported on here was supported by Verband der chemischen Industrie — Fonds der chemischen Industrie     

Chlorophyll composition of Photosystems IIα, IIβ and I in tobacco chloroplasts

The antenna composition of the Photosystems II_α, II_β and I was studied in tobacco chloroplasts. Absorbance spectra, recorded at 4 K, were analyzed for the wild type and the mutants Su/su and Su/su var. Aurea, containing higher concentrations of the photosystems. With chloroplasts of Su/su we measured the action spectra of the three photosystems from 625 to 690 nm. Above 675 nm absorption by Photosystem I dominated. This sytem had a maximum at 678 nm and a shoulder at 660 nm. Of the long-wavelength chlorophyll a forms, absorbing at 690, 697 and 705 nm at 4 K, which are generally assigned to Photosystem I, the 697 nm form occurred in an amount of four molecules per reaction center of Photosystem I in each type of chloroplast. The Photosystem II_α spectrum was characterized by maxima at 650 and 672 nm, showing clearly the participation of the chlorophyll a and b containing light-harvesting complex. In the mutants the light-harvesting complex has a chlorophyll a to chlorophyll b ratio of more than 1; the amount of the 672 nm chlorophyll a was normal, whereas the amount of chlorophyll b was markedly decreased in the mutants relative to the wild type. The Photosystem II_β spectrum mainly consisted of a band at 683 nm.     

Stabilization by glutaraldehyde of high-rate electron transport in isolated chloroplasts

Treatment of isolated chloroplasts with glutaraldehyde affects their ability to photoreduce artificial electron acceptors. The remaining rate of O₂ evolution approaches zero with methyl viologen, is low with ferricyanide, but nearly normal with lipophilic Photosystem II acceptors, like oxidized p-phenylenediamine and oxidized diaminodurene. Since Photosystem I donor reactions are also affected, a specific site of inhibition of electron transport to Photosystem I is indicated. At the same time, glutaraldehyde prolongs the longevity of the chloroplasts stored in dark. In control samples the half-life of Photosystem II activity varied between 5 days at 4 °C and 1 day at 25 °C. Glutaraldehyde treatment increased these half times approx. 3-fold. The glutaraldehyde doses required to induce inhibition and stabilization were very similar.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment

A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O₂ evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.     

The role of cytochrome b6 and cytochrome f in cyclic electron flow in a blue-green alga.

Cytochrome b₆ can be both photooxidized and photoreduced by Photosystem I in a cell-free preparation from the blue-green alga Nostoc muscorum. The cytochrome appears to have an oxidation-reduction potential near 0.0 V. The reduction of cytochrome b₆ when ferredoxin is added during Photosystem I illumination suggests that the cytochrome may function as a component of a ferredoxin-catalyzed cyclic electron transport pathway. In the presence of ferredoxin, the addition of ADP in the light results in oxidation of cytochrome b₆ and reduction of cytochrome f, suggesting the existence of a coupling site between the two cytochromes. An acceleration of the rate of the dark reduction of photooxidized cytochrome b₆ also observed on addition of ADP raises the possibility of a second coupling site on the reducing side of cytochrome b₆.     

Energy transfer and the distribution of excitation energy in the photosynthetic apparatus of spinach chloroplasts

Equations are derived from our model of the photochemical apparatus of photosynthesis to show that the yield of energy transfer from Photosystem II to Photosystem I, ?_T(II→Iz), can be obtained from measurements on an individual sample of chloroplasts frozen to ?196 °C by comparing the sum of two specifically defined fluorescence excitation spectra with the absorption spectrum of the sample. Then, given that value of ?_T(II→I), the fraction of the quanta absorbed by the photochemical apparatus which is distributed initially to Photosystem I, α, can be determined as a function of the wavelength of excitation from the same fluorescence excitation spectra. The results obtained in this study of individual samples of chloroplasts frozen to ?196 °C in the absence of divalent cations, namely, that ?_T(II→I) varies from a minimum value of 0.10 when the Photosystem II reaction centers are all open to a maximum value of 0.25 when the centers are all closed and that α has a value of about 0.30 which is almost independent of wavelength for wavelengths shorter than 675 nm (α increases rapidly toward unity at wavelengths longer than 675 nm), agrees quite well with results obtained previously from comparative measurements of chloroplasts frozen to ?196 °C in the presence and absence of divalent cations.     

Photoreduction of ferredoxin-NADP in the presence and absence of ferredoxin-reducing substance (FRS)

Preparations of ferredoxin-reducing substance (FRS) were obtained from spinach chloroplasts within the elution volume range and with the spectral characteristics described by Yocum and San Pietro (8). However, no support was found for the view that FRS is the primary electron acceptor of Photosystem I. The FRS-depleted chloroplast fragments retained their Photosystem I activity, which was not enhanced by the addition of FRS. No evidence was found for a prior photoreduction of FRS by chloroplasts followed by a dark reduction of ferredoxin and NADP by reduced FRS. The FRS-depleted chloroplast fragments were found to retain and to photoreduce bound ferredoxin upon illumination by Photosystem I light at 25°K. These results suggest that the role of a primary electron acceptor of Photosystem I ascribed to FRS may belong to bound ferredoxin.     

p-nitroacetophenoxime N-methylcarbamate,a new electron acceptor in isolated thylakoids

p-Nitroacetophenoxime N-methylcarbamate (MCPNA) is a rather potent inhibitor of the electron transfer in spinach class A chloroplasts. In isolated thylakoids, MCPNA is an electron acceptor at the level of photosystem I (PS I). It inhibits O₂ evolution in the presence of NADP and ferredoxin but not the reduction of ferricyanide. MCPNA is active as an acceptor between 3 μM and 100 μM. At concentrations higher than 300 μM, inhibition of photosystem II (PS II) occurs. MCPNA has no uncoupling effect on photophosphorylation. Reduction of MCPNA by thylakoids in the presence of light is in accordance with the E′_o of this compound (??0.57 V) and is followed by an electron transfer to O₂. This reaction probably explains the inhibitory effect of MCPNA on class A chloroplasts.     

A demonstration of energy transfer from photosystem II to photosystem I in chloroplasts

Photosystem I activity of Tris-washed chloroplasts was measured at room temperature as the rate of photoreduction of NADP and as the rate of oxygen uptake mediated by methyl viologen in both cases using dichlorophenolindophenol plus ascorbate as the source of electrons for Photosystem I. With both assay systems the rate of electron transport by Photosystem I was stimulated approx. 20 % by the addition of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea which caused the Photosystem II reaction centers to close. Photosystem I activity of chloroplasts was measured at low temperature as the rate of photooxidation of P-700. Chloroplasts suspended in the presence of hydroxylamine and 3-(3,4-dichlorophenyl)-1, 1-dimethylurea were frozen to ?196 °C after adaptation to darkness or after a preillumination at room temperature. The Photosystem II reaction centers of the frozen dark-adapted sample were all open; those of the preilluminated sample were all closed. The rate of photooxidation of P-700 at ?196 °C with the preilluminated sample was approx. 25 % faster than with the dark-adapted sample. We conclude from both the room temperature and the low temperature experiments that there is greater energy transfer from Photosystem II to Photosystem I when the Photosystem II reaction centers are closed and that these results are a direct demonstration of spillover.     

Pyridine nucleotides and H2 as electron donors to the respiratory and photosynthetic electron-transfer chains and to nitrogenase in Anabaena heterocysts

The pathways through which NADPH, NADH and H₂ provide electrons to nitrogenase were examined in anaerobically isolated heterocysts. Electron donation in freeze-thawed heterocysts and in heterocyst fractions was studied by measuring O₂ uptake, acetylene reduction and reduction of horse heart cytochrome c. In freeze-thawed heterocysts and membrane fractions, NADH and H₂ supported cyanide-sensitive, respiratory O₂ uptake and light-enhanced, cyanide-insensitive uptake of O₂ resulting from electron donation to O₂ at the reducing side of Photosystem I. Membrane fractions also catalyzed NADH-dependent reduction of cytochrome c. In freeze-thawed heterocysts and soluble fractions from heterocysts, NADPH donated electrons in dark reactions to O₂ or cytochrome c through a pathway involving ferredoxin:NADP reductase; these reactions were only slightly influenced by cyanide or illumination. In freeze-thawed heterocysts provided with an ATP-generating system, NADH or H₂ supported slow acetylene reduction in the dark through uncoupler-sensitive reverse electron flow. Upon illumination, enhanced rates of acetylene reduction requiring the participation of Photosystem I were observed with NADH and H₂ as electron donors. Rapid NADPH-dependent acetylene reduction occurred in the dark and this activity was not influenced by illumination or uncoupler. A scheme summarizing electron-transfer pathways between soluble and membrane components is presented.     

Des activites photosynthetiques sans les complexes pigmentaires CP1 et CP2

Photosynthetic activity in the absence of the CP1 and CP2 pigmentary complexesVarious photochemical activities were tested on chloroplasts of Zea mays that received 4 s of light every 4 h during the culture period. Photosystem I and Photosystem II were functioning, as well as the photosynthetic electron transport. These chloroplasts exhibited upon sodium dodecyl sulphate gel electrophoresis neither Complex 1 (M_r 70 000) generally associated with Photosystem I nor Complex 2 M_r 25 000) generally associated with Photosystem II. Chlorophyll is indeed attached to polypeptides of molecular weight 21 000 and 29 000.These results lead us to question the functional role of chloroplast protein-pigment complexes observed by sodium dodecyl sulphate gel electrophoresis.     

Chemical modification studies of chloroplast membranes. Effects of diazonium coupling on electron transfer in photosystem I

p-Diazonium benzene sulfonate reacts with at least two chloroplast membrane components on the reducing side of Photosystem I leading to inhibition of electron flow from the Photosystem I primary acceptor (X) to ferredoxin, and inhibiting the function of bound ferredoxin-NADP⁺ reductase. While some inhibition of these two components attends p-diazonium benzene sulfonate treatment in the dark, a much more severe inhibition results when p-diazonium benzene sulfonate is given to light-energized membranes.Of particular interest is that electron flux through Photosystem II (3-(3,4-dichlorophenyl)-1, 1-dimethylurea sensitive) is required for potentiating the light-dependent p-diazonium benzene sulfonate inhibition, cyclic electron flow around Photosystem I not being an effective potentiator. We interpret these data as due to Photosystem II-driven conformational changes unmasking additional diazoreactive sites in the bound membrane components.     

Factors influencing photosynthetic enhancement in isolated chloroplasts

Photosynthetic enhancement of oxygen evolution (linked to CO₂ assimilation) in isolated chloroplasts was found to be governed by the supply of ATP. The addition of ATP (but not AMP) abolished enhancement that consistently occurred without added ATP. Enhancement in the H₂O → NADP reaction by chloroplasts was investigated in the light of one recent report that the phenomenon occurs when pure ferredoxin is replaced by a crude preparation (PPNR) and another report that the phenomenon is governed by Mg⁺⁺ concentration. Fractionation of PPNR led to the isolation of a protein factor which when added to pure ferredoxin induced enhancement. However, the rate of NADP reduction with pure ferredoxin and without enhancement was greater than the maximum rate of NADP reduction with enhancement induced by either the protein factor of PPNR. The report that Mg⁺⁺ concentration governs enhancement was not confirmed.     

Rates of vectorial proton transport supported by cyclic electron flow during oxygen reduction by illuminated intact chloroplasts

The light-dependent quenching of 9-aminoacridine fluorescence was used to monitor the state of the transthylakoid proton gradient in illuminated intact chloroplasts in the presence or absence of external electron acceptors. The absence of appreciable light-dependent fluorescence quenching under anaerobic conditions indicated inhibition of coupled electron transport in the absence of external electron acceptors. Oxygen relieved this inhibition. However, when DCMU inhibited excessive reduction of the plastoquinone pool in the absence of oxygen, coupled cyclic electron transport supported the formation of a transthylakoid proton gradient even under anaerobiosis. This proton gradient collapsed in the presence of oxygen. Under aerobic conditions, and when KCN inhibited ribulose bisphosphate carboxylase and ascorbate peroxidase, fluorescence quenching indicated the formation of a transthylakoid proton gradient which was larger with oxygen in the Mehler reaction as electron acceptor than with methylviologen at similar rates of linear electron transport. Apparently, cyclic electron transport occured simultaneously with linear electron transport, when oxygen was available as electron acceptor, but not when methylviologen accepted electrons from Photosystem I. The ratio of cyclic to linear electron transport could be increased by low concentrations of DCMU. This shows that even under aerobic conditions cyclic electron transport is limited in isolated intact chloroplasts by excessive reduction of electron carriers. In fact, P₇₀₀ in the reaction center of Photosystem I remained reduced in illuminated isolated chloroplasts under conditions which resulted in extensive oxidation of P₇₀₀ in leaves. This shows that regulation of Photosystem II activity is less effective in isolated chloroplasts than in leaves. Assuming that a Q-cycle supports a H⁺/e ratio of 3 during slow linear electron transport, vectorial proton transport coupled to Photosystem I-dependent cyclic electron flow could be calculated. The highest calculated rate of Photosystem I-dependent proton transport, which was not yet light-saturated, was 330 mol protons (mg chlorophyll h)^–1 in intact chloroplasts. If H⁺/e is not three but two proton transfer is not 330 but 220 mol (mg Chl H)^–1. Differences in the regulation of cyclic electron transport in isolated chloroplasts and in leaves are discussed.     

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 recorded 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 (τ_I) and Photosystem II fluoresces with a lifetime of 300 ps (τ_II). Fluorescence with a lifetime of 4500 ps (τ_III) may be interpreted as originating from chlorophyll 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 corresponds to the time of energy migration to them from carotenoids.