Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments.

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP+). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, I1/2, is defined as the actinic light intensity that, at a given concentration of electron donro, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, I1/2 is compared with another parameter, equivalent to I1/2, that is obtained independently of the reaciton-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow. The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP+ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.     

Laser-flash-activated electron paramagnetic resonance studies of primary photochemical reactions in chloroplasts.

Electron paramagnetic resonance studies of the primary reactants of Photosystems I and II have been conducted at cryogenic temperatures after laser-flash activation with monochromatic light.P-700 photooxidation occurs irreversibly in chloroplasts and in Photosystem I fragments after activation with a 730 nm laser flash at a temperature of 35 degrees K. Flash activation of chloroplasts or Photosystem II chloroplast fragments with 660 nm light results in the production of a free-radical signal (g = 2.002, linewidth approximately 8 gauss) which decays with a half-time of 5.0 ms at 35 degrees K. The half-time of decay is independent of temperature in the range of 10-77 degrees K. This reversible signal can be eliminated by preillumination of the sample at 35 degrees K with 660 nm light (but not by 730 nm light), by preillumination with 660 nm light at room temperature in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) plus hydroxylamine, or by adjustment of the oxidation-reduction potential of the chloroplasts to - 150 mV prior to freezing. In the presence of ferricyanide (20-50 mM), two free-radical signals are photoinduced during a 660 nm flash at 35 degrees K. One signal decays with a half-time of 5 ms, whereas the second signal is formed irreversibly. These results are discussed in terms of a current model for the Photosystem II primary reaction at low temperature which postulates a back-reaction between P-680+ and the primary electron acceptor.     

The site of KCN inhibition in the photosynthetic electron transport pathway

Treatment of chloroplasts with high concentrations of KCN inhibits reactions which involve Photosystem I (e.g. electron transport from water or diaminodurene to methylviologen), but not those assumed to by-pass Photosystem I (e.g. electron transport from water to quinonediimides). The spectrophotometric experiments described in this paper showed that KCN inhibits the oxidation of cytochrome f by far-red light without blocking its reduction by red light. Both optical and EPR experiments indicated that KCN does not inhibit the photooxidation of P700 but markedly slows down the subsequent dark decay (reduction). Reduction of P700 by Photosystem II is prevented by KCN. It is concluded that KCN blocks electron transfer between cytochrome f and P700, i.e. the reaction step which is believed to be mediated by plastocyanin. In KCN-poisoned chloroplasts the slow dark reduction of P700 following photooxidation is greatly accelerated by reduced 2,6-dichlorophenolindophenol or by reduced N-methylphenazonium methosulfate (PMS), but not by diaminodurene. It appears that the reduced indophenol dye and reduced PMS are capable of donating electrons directly to P700, at least partially by-passing the KCN block.     

Study of the acceptor portion of photosystem I according to the temperature relationships of P700 photoconversions

The functioning of the acceptor part of photosystem I was studied by temperature dependence of time course of light induced absorbtion changes at 700 nm of digitonin chloroplast fragments, enriched by photosystem I. Partial irreversibility of P700 photooxidation at low temperatures and appearance of two components (rapid and slow) in the time course of P700+ dark reduction reflect the contribution of different acceptors in electron transport. Thermoinactivation of fragments incubation at acid pH or treatment by glutaraldehyde cause complete inhibition of irreversible P700 photooxidation and slow dark reduction of P700+ at -170 degrees. The slow component of P700+ reduction and irreversible photooxidation of P700 are ascribed to contribution of secondary ferredoxin acceptors. The accurence of rapid component of P700+ dark reduction in light induced signal of treated fragments indicate that this component is due to recombination of reduced primary acceptor and P700+. Because only one electron transport takes at -170 degrees, the occurence of rapid and slow components in dark decay kinetics of P700+ suggests, that secondary acceptors of some reaction centers are incapable to reduction at -170 degrees. The shape of temperature dependence curve of the slow P700+ reduction component is interpreted as an indication of the tunneling electron transport.     

Energy transfer from photosystem II to photosystem I in Porphyridium cruentum

Rates of photooxidation of P-700 by green (560 nm) or blue (438 nm) light were measured in whole cells of porphyridium cruentum which had been frozen to -196 degrees C under conditions in which the Photosystem II reaction centers were either all open (dark adapted cells) or all closed (preilluminated cells). The rate of photooxidation of P-700 at -196 degrees C by green actinic light was approx. 80% faster in the preilluminated cells than in the dark-adapted cells. With blue actinic light, the rates of P-700 photooxidation in the dark-adapted and preilluminated cells were not significantly different. These results are in excellent agreement with predictions based on our previous estimates of energy distribution in the photosynthetic apparatus of Porphyridium cruentum including the yield of energy transfer from Photosystem II to Photosystem I determined from low temperature fluorescence measurements.     

Light-dependent development of photosynthetic competence in Scenedesmus mutant No. 8.

The heterotrophically grown, P-700-free mutant No. 8 of Scenedesmus obliquus is unable to carry out photosynthesis. Yet, chloroplast particles isolated from the alga reduced ferricyanide. They also reduced methyl viologen in the presence of the artificial donor reduced 2,6-dichlorophenol indophenol with a low yield but an appreciable saturation rate. NADP reduction or P-700 turn-over could not be detected. When grown mixotrophically, the mutant showed increasing P-700 activity with a concomitant increase in the rate of photosynthesis. Both activities were lost again when the algae were returned to darkness. Isolated chloroplast particles showed a good P-700 turn-over and reasonable rates of NADP reduction. The data suggest that the mutation occurred at a site preceding the formation of the pigment. The results on the photochemical activities are discussed in the light of reports concerning the involvement of P-700 in linear electron transport.     

The role of the membrane-bound iron-sulphur centres A and B in the photosystem I reaction centre of spinach chloroplasts.

Photosystem I particles prepared from spinach chloroplast using Triton X-100 were frozen in the dark with the bound iron-sulphur Centre A reduced. Illumination at cryogenic temperatures of such samples demonstrated the photoreduction of the second bound iron-sulphur Centre B. Due to electron spin-electron spin interaction between these two bound iron-sulphur centres, it was not possible to quantify amounts of Centre B relative to the other components of the Photosystem I reaction centre by simulating the line-shape of its EPR spectrum. However, by deleting the free radical signal I from the EPR spectra of reduced Centre A alone or both Centres A plus B reduced, it was possible to double integrate these spectra to demonstrate that Centre B is present in the Photosystem I reaction centre in amounts comparable to those of Centre A and thus also signal I (P-700) and X. Oxidation-reduction potential titrations confirmed that Centre A had Em congruent to -550 mV, Centre B had Em congruent to -585 mV. These results, and those presented for the photoreduction of Centre B, place Centre B before Centre A in the sequence of electron transport in Photosystem I particles at cryogenic temperatures. When both A and B are reduced, P-700 photooxidation is reversible at low temperature and coupled to the reduction of the component X. The change from irreversible to reversible P-700 photooxidation and the photoreduction of X showed the same potential dependence as the reduction of Centre B with Em congruent to -585 mV, substantiating the identification of X as the primary electron acceptor of Photosystem I.     

Correlation of reaction-center chlorophyll (P-700) oxidation and bound iron-sulfur protein photoreduction in chloroplast Photosystem I at low temperatures

The extent of P-700 photooxidation at 18 °K has been followed in three different chloroplast preparations (unfractionated chloroplasts and two preparations enriched in Photosystem I). More than 90% of P-700⁺ formation in all preparations was eliminated by the addition of sodium dithionite at pH 10. Photoreduction of a bound chloroplast iron-sulfur protein was also decreased by at least 90% under similar conditions. Electron paramagnetic resonance spectra of the chloroplast preparations in the presence of dithionite showed chemical reduction of bound iron-sulfur protein under conditions where primary photochemistry is eliminated. These results indicate that P-700 photooxidation is concomitant with photoreduction of a bound iron-sulfur protein and that this iron-sulfur protein functions as the primary electron acceptor of Photosystem I.     

Correlation of redox levels of component electron carriers with total electron flux in an electron-transport system. P-700 and the photoreduction of NADP+ in chloroplast fragments

A mathematical analysis is described which measures the effects of actinic light intensity and concentration of an artificial electron donor on the steady-state light-induced redox level of a reaction-center pigment (e.g. P-700) and on the overall light-induced electron flux (e.g. reduction of NADP⁺). The analysis led to a formulation (somewhat similar to the Michaelis-Menten equation for enzyme kinetics) in which a parameter, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, is defined as the actinic light intensity that, at a given concentration of electron donor, renders the reaction-center pigment half oxidized and half reduced. To determine the role of a presumed reaction-center pigment, $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ is compared with another parameter, equivalent to $\text{I}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, that is obtained independently of the reaction-center pigment by measuring the effect of actinic light intensity and concentration of electron donor on the overall electron flow.The theory was tested and validated in a model system with spinach Photosystem I chloroplast fragments by measurements of photooxidation of P-700 and light-induced reduction of NADP⁺ by reduced 2,6-dichlorophenolindophenol. A possible extension of this mathematical analysis to more general electron-transport systems is discussed.     

Flash photolysis-electron spin resonance studies of photosystem I. A fast reduction of component of P-700+.

A 300 mus decay component of ESR Signal I (P-700+) in chloroplasts is observed following a 10 mus actinic xenon flash. This transient is inhibited by treatments which block electron transfer from Photosystem II to Photosystem I (e.g. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), KCN and HgCl2). The fast transient reduction of P-700+ can be restored in the case of DCMU or DBMIB inhibition by addition of an electron donor couple (2,6-dichlorophenol indophenol (Cl2Ind)/ascorbate) which supplies electrons to cytochrome f. However, this donor couple is inefficient in restoring electron transport in chloroplasts which have been inhibited with the plastocyanin inactivators, KCN and HgCl2. Oxidation-reduction measurements reveal that the fast P-700+ reduction component reflects electron transfer from a component with Em = 375 +/- 10 mV (pH = 7.5). These data suggest the assignment of the 300-mus decay kinetics to electron transfer from cytochrome f (Fe2+) to P-700+, thus confirming the recent observations of Haehnel et al. (Z. Naturforsch. 26b, 1171-1174 (1971)).     

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.     

Concentration-dependent effects of salicylaldoxime on chloroplast reactions

Salicylaldoxime has been found to have a variety of concentration-dependent effects on chloroplast activities. At low concentrations (< 10 mM), salicylaldoxime reversibly inhibits all reactions which involve Photosystem II. Since the DCMU-insensitive silicomolybdate Hill reaction is also inhibited, one site of inhibition is definitely located before the DCMU-sensitive site, possibly before the photoact. The inhibition kinetics and the response of chloroplast fluorescence may indicate another site in the DCMU-sensitive region. At almost exactly the same concentrations (< 10 mM), salicylaldoxime uncouples phosphorylation reversibly, whether it is supported by Photosystem II or by Photosystem I. At higher concentrations (approx. 20 mM) salicylaldoxime inhibits Photosystem II irreversibly, uncouples irreversibly, and begins to cause changes in chloroplast light scattering which could be manifestations of membrane damage. At very high concentrations (approx. 45 mM) salicylaldoxime irreversibly inhibits Photosystem I activity in the region of plastocyanin. This is indicated by the ability of salicylaldoxime to inhibit the photooxidation of cytochrome f but not the photooxidation of P-700.     

Primary photochemistry of Photosystem I in chloroplasts. Dynamics of reversible charge separation in open reaction centers at 25 K

The reduction rate of oxidized reaction center chlorophyll of Photosystem I after laser-flash excitation at 25 K has been determined for D-144 subchloroplast fragments and chloroplasts. A maximum of 40% of Photosystem I reaction centers undergo irreversible charge separation (P-700, Cluster A: P-700⁺, Cluster A^?) at 25 K, a percentage which is independent of laser-flash intensity. The remaining reaction centers in chloroplasts and D-144 fragments undergo reversible charge separation with biphasic recombination. Similar amplitudes and time constants (chloroplasts, 49 μs (61%); D-144 fragments, 90 μs (67%)) were obtained for the fast component, while the slower component differed considerably in time (chloroplasts, 2.9 ms; D-144 fragments, 170 ms). It is known that Fe-S Cluster A is photoreduced in less than 1 ms at 25 K. Data obtained support a model for Photosystem I involving a single intermediate in the decay path between the reduced primary electron acceptor (A^?₁) and P-700⁺ and a second intermediate in the decay path between a reduced secondary electron acceptor and P-700⁺. Dual laser-flash experiments to determine rate constants for these processes are included.     

Photochemical properties of a photosystem I subchloroplast fragment.

Treatment of spinach chloroplast fragments with the detergent lauryl dimethylamine oxide, followed by column chromatography on DEAE-cellulose, leads to the isolation of a Subchloroplast fragment that is enriched in Photosystem I. The spectrum of the lauryl dimethylamine oxide fragments, characterized by maxima at 418, 435, and 671 nm, shows the absence of chlorophyll b. The fragments contain 1 molecule of P700 per 40 chlorophyll molecules but have no cytochromes. The P700 in the fragments is photochemically active at both room temperature and liquid helium temperature. The fragments contain the primary electron acceptor of Photosystem I, as evidenced by the low-temperature photoreduction of a bound iron-sulfur protein. The fragments are able to catalyze noncyclic electron transfer from ascorbate to oxygen but not to the electron acceptor NADP.     

Electron paramagnetic resonance saturation studies of P-700+ reaction center chlorophyll in plant photosynthesis

Electron paramagnetic resonance (EPR) power saturation and saturation recovery methods have been used to determine the spin lattice, T1, and spin-spin, T2, relaxation times of P-700+ reaction-center chlorophyll in Photosystem I of plant chloroplasts for 10 K less than or equal to T less than or equal to 100 K. T1 was 200 mus at 100 K and increased to 900 mus at 10 K. T2 was 40 ns at 40 K and increased to 100 ns at 10 K. T1 for 40 K less than or equal to T less than or equal to 100 K is inversely proportional to temperature, which is evidence of a direct-lattice relaxation process. At T = 20 K, T1 deviates from the 1/T dependence, indicating a cross relaxation process with an unidentified paramagnetic species. The individual effects of ascorbate and ferricyanide on T1 of P-700+ were examined: T1 of P-700+ was not affected by adding 10 mM ascorbate to digitonin-treated chloroplast fragments (D144 fragments). The P-700+ relaxation time in broken chloroplasts treated with 10 mM ferricyanide was 4-times shorter than in the untreated control at 40 K. Ferricyanide appears to be relaxing the P-700+ indirectly to the lattice by a cross-relaxation process. The possibility of dipolar-spin broadening of P-700+ due to either the iron sulfur center A or plastocyanin was examined by determining the spin-packet linewidth for P-700+ when center A and plastocyanin were in either the reduced or oxidized states. Neither reduced center A nor oxidized plastocyanin was capable of broadening the spin-packet linewidth of P-700+ signal. The absence of dipolar broadening indicates that both center A and plastocyanin are located at a distance at least 3.0 nm from the P-700+ reaction center chlorophyll. This evidence supports previous hypotheses that the electron donor and acceptor to P-700 are situated on opposite sides of the chloroplast membrane. It is also shown that the ratio of photo-oxidized P-700 to photoreduced centers A and B at low temperature is 2 : 1 if P-700 is monitored at a nonsaturating microwave power.     

Photochemical properties of a Photosystem II subchloroplast fragment

1. The Photosystem II fraction (D-10) obtained by incubation of spinach chloroplasts with digitonin was further purified by incubation with Triton X-100. The resulting Photosystem II subchloroplast fragment (DT-10) contained 1 mole of cytochrome b-559 per 170 moles of chlorophyll. It lacked cytochrome f and cytochrome b₆ and its content of P700 was low.

2. The DT-10 fragment showed only traces of photochemical activity with water as electron donor, but it was active in a Photosystem II reaction with 2,6-dichlorophenolindophenol as electron acceptor and diphenyl carbazide as donor. Photoreduction of NADP⁺ with diphenyl carbazide as donor was negligible. There was some photoreduction of NADP⁺ with ascorbate plus 2,6 dichlorophenolindophenol as donor but this activity could be accounted for by contamination with Photosystem I. These results are consistent with the Z-scheme of photosynthesis with Photosystems I and II operating in series for the reduction of NADP⁺ from water. DT-10 subchloroplast fragments showed a light-induced rise in fluorescence yield at 20 °C in the presence of diphenyl carbazide. A light-induced fluorescence increase also was observed at 77 °K.

3. During the preparation of the DT-10 fragment, the high potential form of cytochrome b-559 was largely converted to a form of lower potential and C-550 was converted to the reduced state. A photoreduction of C-550 was observed at liquidnitrogen temperature, provided the C-550 was oxidised with ferricyanide prior to cooling. Some photooxidation of cytochrome b-559 was obtained at 77 °K if the preparation was reduced prior to cooling, but the degree of photooxidation was variable with different preparations. C-550 does not appear to be identical with the primary fluorescence quencher, Q.

4. Photosystem I subchloroplast fragments (D-144) released by the action of digitonin were compared with Photosystem I fragments (DT-144) released from D-10 fragments by Triton X-100. There were no significant differences between D-144 and DT-144 fragments either in chlorophyll a/b ratio or in P700 content.     

Light-dependent development of photosynthetic competence in Scenedesmus mutant no. 8

The heterotrophically grown, $\text{P-700-}\text{free}$ mutant No. 8 of Scenedesmus obliquus is unable to carry out photosynthesis. Yet, chloroplast particles isolated from the alga reduced ferricyanide. They also reduced methyl viologen in the presence of the artificial donor reduced 2,6-dichlorophenol indophenol with a low yield but an appreciable saturation rate. NADP reduction or $\text{P-700}$ turn-over could not be detected.When grown mixotrophically, the mutant showed increasing $\text{P-700}$ activity with a concomitant increase in the rate of photosynthesis. Both activities were lost again when the algae were returned to darkness. Isolated chloroplast particles showed a good $\text{P-700}$ turn-over and reasonable rates of NADP reduction.The data suggest that the mutation occurred at a site preceding the formation of the pigment. The results on the photochemical activities are discussed in the light of reports concerning the involvement of $\text{P-700}$ in linear electron transport.     

Quantitative EPR studies of the primary reaction of Photosystem I in chloroplasts

Quantitative electron paramagnetic resonance studies of the primary event associated with Photosystem I in chloroplasts have been carried out at 25 °K. After illumination of either whole chloroplasts or Photosystem I subchloroplast fragments (D-144) with 715-nm actinic light at 25 °K, equal spin concentrations of oxidized P700 and reduced bound iron-sulfur protein (bound ferredoxin) have been measured. Quantitative determination of the concentration of these two carriers by EPR spectroscopy after illumination at low temperature indicates that Photosystem I fragments are enriched in P700 and the bound iron-sulfur protein as compared with unfractionated chloroplasts. These results indicate that P700 and the bound iron-sulfur protein function as the donor-acceptor complex of chloroplast Photosystem I.     

Effects of natural shade on soybean thylakoid membrane composition

The effect of natural shade on chloroplast thylakoid membrane activity and composition was examined for soybean (Glycine Max. cv. Young) grown under field conditions. Plots with high (10 plants m^–1 row) or low (1 plant m^–1 row) plant density were established. Expanding leaves were tagged at 50, 58 and 65 days after planting (DAP). At 92 DAP, tagged leaves were used as reference points to characterize canopy light environments and isolate thylakoid membranes. Light environments ranged from a photosynthetic photon flux density (PPFD) of 87% of full sun to a PPFD of 10% of full sun. The decline in PPFD was accompanied by an increase in the far-red/red (735 nm/645 nm) ratio from 0.9 to approximately six. The major effects of shade on chloroplast thylakoid membranes were a reduction in chloroplast coupling factor and a shift in light-harvesting capacity from Photosystem I to Photosystem II. Photosynthetic electron transport capacity was not affected by differences in PPFD, but was 20 to 30% higher in the 1 plant m^–1 row treatment. The plant density effect on electron transport was associated with differences in plastocyanin concentration, suggesting that plastocyanin is a limiting factor in soybean. Shade did not have a significant effect on the concentration of Photosystem II, Cyt b₆f, or Photosystem I complexes.Abbreviations CF₁ chloroplast coupling factor - DAP days after planting - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCIP 2,6-dichlorophenolindophenol - FR/R far-red/red - PBS 10 mM sodium phosphate (pH 7.0), 150 mM NaCl - PPFD photosynthetic photon flux density - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - TBS 20 mM Tris-HCl (pH 7.5), 500 mM NaCl - TTBS 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 0.05% (w/v) polyoxyethylenesorbitan monolaurate (Tween-20) The US Government right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.The US Government right to retain a non-exclusive, royalty-free licence in and to any copyright is acknowledged.     

Effect of NADP on light-induced cytochrome changes in membrane fragments from a blue-green alga.

The effect of NADP+ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragements prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP+ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH2) to NDAP+. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH2 as the electron donor, NADP+ had no effect on the light-induced redox changes of cytochromes: with or without NADP+, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an alpha peak at 549nm. With 664 nm illumination and water as the electron donor, NADP+ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b559. Cytochrome b559 appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP+) for the electron flow from water.