Localization of polylysine inhibition in a photosystem I subchloroplast particle.

Polylysine was found to increase the efficiency of electron donation from plastocyanin to P700⁺ in highly resolved Photosystem I subchloroplast particles. This increase in efficiency is due to a decrease in the Km for plastocyanin in the presence of polylysine and is similar to results obtained with divalent cations. Cytochrome f photooxidation is observed in the presence of plastocyanin and divalent cations but not in the presence of plastocyanin and polylysine. The results indicate that the binding of polylysine to plastocyanin prevents the reduction of plastocyanin by cytochrome f.     

Plastocyanin as the possible site of photosynthetic electron transport inhibition by glutaraldehyde

Treatment of spinach chloroplasts with glutaraldehyde causes an inhibition in the electron transport chain between the two photosystems. Measurements of O₂ flash yields, pH exchange, and fluorescence induction show that the O₂ evolving apparatus, photosystem II and its electron acceptor pool are not affected. The behavior of P700 indicates that its reduction but not its oxidation, is severely inhibited. Cytochrome f is still reducible by photosystem II but also slowly oxidizable by photosystem I. The sensitivity of isolated plastocyanin to glutaraldehyde further supports the conclusion that glutaraldehyde inhibits at the plastocyanin level and thereby induces a break between P700 and cytochrome f.     

Cytochrome f and plastocyanin kinetics in Chlorella pyrenoidosa II. Reduction kinetics and electric field increase in the 10 ms range

On dark-adapted Chlorella, after one flash, plastocyanin (PC) undergoes reduction with a half-time of 7 ms. After 4 or 5 flashes, the reduction of PC⁺ in the 10 ms range is suppressed, and the level of oxidized plastocyanin increases during the next few flashes before reaching a stationary value. Cytochrome f exhibits approximately the same pattern.The reduction of PC⁺ and cytochrome f⁺ in the 10 ms range is correlated with an increase of the electrice field named phase b (Joliot, P. and Delosme, R., Biochim. Biophys. Acta 357 (1974) 267–284). Both need the presence of a compound R′ in the reduced state. A dark electron transfer involving a carrier of electrons across the membrane, a proton carrier, R′ as terminal reducant, PC⁺ and cytochrome f⁺ as terminal oxidants, would account for this field generation.Cooperation between the electron transfer chains is implied at the level of plastocyanin oxidation. An equilibrium constant of about 2 is observed between cytochrome f and plastocyanin before 1 ms and after 500 ms after the photochemical reactions. We observe that cytochrome f and plastocyanin are not connected from 1 to 100 ms after a photochemical reaction. The equilibrium constant between plastocyanin and P-700 remains large [20] under these conditions.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Kinetic analysis of P-700 photoconversion: Effect of secondary electron donation and plastocyanin inhibition

The kinetics of P-700 photoconversion under weak continuous actinic illumination were quantitatively analyzed to provide information on the relative absorption cross-section σ_PSI of the light-harvesting pigments associated with photosystem I and on the number of electrons stored between the two photosystems in dark-adapted chloroplasts. The theory of chemical kinetics for a system of monomolecular consecutive first-order reactions is reviewed briefly to provide support for the experimental approach taken. A complete inhibition of plastocyanin by cyanide eliminated all secondary electron donation to P-700⁺ and allowed the registration of the exponential (monomolecular) P-700 photoconversion at room temperature. The rate constant K_p-700 of the exponential kinetics was independent of the ionic (± Mg²⁺) and osmotic (± sucrose) strength of the chloroplast suspension medium, and of the oxidation-reduction state of photosystem II. The extent of plastocyanin inhibition in partially inhibited samples was greater under low ionic and low osmotic conditions. In dark-adapted chloroplast samples that were not cyanide treated, the number of electrons stored between the two photosystems was 3.9 ± 0.2 and independent of divalent cations. It is concluded that plastocyanin inhibition by cyanide is favored under low ionic and low osmotic conditions. The Mg²⁺ ion and redox state of photosystem II-independent photoconversion of P-700 does not support significant changes in the spillover of excitation from photosystem II to photosystem I in isolated chloroplasts.     

Effect of trypsin treatment of photosystem I particles on the electron donation to p700

Proteolysis of photosystem I particles had no effect on P700 oxidation but did inhibit the rate of P700⁺ reduction. The V_max values were decreased for both dichlorophenol and plastocyanin, but the K_m values were unaffected indicating that trypsin treatment altered electron transfer rather than the binding of the donor to the photosystem I complex. The salt dependence of P700⁺ reduction was unaffected. The effects of P700⁺ reduction were the same for the preparations of different workers (Shiozawa, Alberte, Thornber 1974 Arch Biochem Biophys 165: 388; and Bengis, Nelson 1975 J Biol Chem 250: 2783).

In both cases, the 70-kilodalton, chlorophyll-containing polypeptide was digested confirming its role in transferring electrons from plastocyanin to P700. The fact that the preparation of Shiozawa et al. lacks subunit (III) but still used plastocyanin as the electron donor rules out a role for this subunit as “the plastocyanin binding protein.” Subunit III was also digested in the Bengis and Nelson preparation.

     

Electron transfer between plastocyanin and P700 in highly-purified photosystem I reaction center complex. Effects of pH, cations, and subunit peptide composition

Treatment of isolated spinach thylakoid fragments with Triton X-100 followed by repeated sucrose density gradient centrifugations and Sephacryl S-300 and DEAE-Sephacel chromatographies yielded a highly purified P700-chlorophyll a protein complex complex which consists of five polypeptides. The protein complex is virtually free of chlorophyll b (Ch1 alpha/Ch1 b greater than 10) with approximately 30 chlorophylls per P700, and contains iron-sulfur centers A, B, and X. At pH values higher than 6, divalent cations, but not monovalent or trivalent cations, efficiently accelerated the electron transfer from reduced spinach plastocyanin to the photooxidized P700 in the P700-chlorophyll alpha protein complex. At pH values lower than 6, the reaction rate drastically increased with decreasing pH with a maximum at about pH 4.3 without cations. Divalent salts as well as monovalent or trivalent salts decreased the P700 reduction rate at low pH, indicating the involvement of electrostatic interaction in those pH regions. The rate of electron transfer from plastocyanin to the photooxidized P700 in the reaction center protein, which consists of only the largest peptide subunit and no iron-sulfur centers, was reduced only 50% at pH 7.0 in the presence of MgCl2 as compared to the case of P700-chlorophyll alpha protein complex. Essentially similar effects of pH and metal ions on this electron transfer reaction were observed as in the case of P700-chlorophyll alpha protein complex. These results strongly suggest that plastocyanin donates electrons directly to the largest peptide of P700-chlorophyll alpha protein complex and the observed effects of pH and cations are mainly due to the interaction between the largest peptide of P700-chlorophyll alpha protein complex and plastocyanin. The four small subunits in the protein complex seemed to have only a minor role in the reaction with plastocyanin.     

Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves

The light-dependent control of photosynthetic electron transport from plastoquinol (PQH₂) through the cytochrome b₆f complex (Cyt b₆f) to plastocyanin (PC) and P700 (the donor pigment of Photosystem I, PSI) was investigated in laboratory-grown Helianthus annuus L., Nicotiana tabaccum L., and naturally-grown Solidago virgaurea L., Betula pendula Roth, and Tilia cordata P. Mill. leaves. Steady-state illumination was interrupted (light-dark transient) or a high-intensity 10 ms light pulse was applied to reduce PQ and oxidise PC and P700 (pulse-dark transient) and the following re-reduction of P700⁺ and PC⁺ was recorded as leaf transmission measured differentially at 810-950 nm. The signal was deconvoluted into PC⁺ and P700⁺ components by oxidative (far-red) titration (V. Oja et al., Photosynth. Res. 78 (2003) 1-15) and the PSI density was determined by reductive titration using single-turnover flashes (V. Oja et al., Biochim. Biophys. Acta 1658 (2004) 225-234). These innovations allowed the definition of the full light response curves of electron transport rate through Cyt b₆f to the PSI donors. A significant down-regulation of Cyt b₆f maximum turnover rate was discovered at low light intensities, which relaxed at medium light intensities, and strengthened again at saturating irradiances. We explain the low-light regulation of Cyt b₆f in terms of inactivation of carbon reduction cycle enzymes which increases flux resistance. Cyclic electron transport around PSI was measured as the difference between PSI electron transport (determined from the light-dark transient) and PSII electron transport determined from chlorophyll fluorescence. Cyclic e⁻ transport was not detected at limiting light intensities. At saturating light the cyclic electron transport was present in some, but not all, leaves. We explain variations in the magnitude of cyclic electron flow around PSI as resulting from the variable rate of non-photosynthetic ATP-consuming processes in the chloroplast, not as a principle process that corrects imbalances in ATP/NADPH stoichiometry during photosynthesis.     

Transient kinetics of the electron transfer between P-700, plastocyanin and cytochrome f in chloroplasts suspended in fluid media at sub-zero temperatures

The kinetics of redox changes of P-700, plastocyanin and cytochrome f in chloroplasts suspended in a fluid medium at sub-zero temperatures have been studied following excitation of the chloroplasts with either a single-turnover flash, a series of flashes or continuous light. The results show that: (1) The kinetics of reduction of P-700⁺ and those of oxidation of plastocyanin are consistent with a bimolecular reaction between these two components as previously suggested (Olsen, L.F., Cox, R.P. and Barber, J. (1980) FEBS Lett. 122, 13–16). (2) Cytochrome f shows heterogeneity with respect to its kinetics of oxidation by Photosystem I. (3) In contrast to the situation when plastoquinol is the electron donor, reduction of cytochrome f by electrons derived from diaminodurene occurs with sigmoidal kinetics that shows a good fit to an apparent equilibrium constant of 12 between the cytochrome and P-700. (4) The rate of electron transfer from plastoquinol to Photosystem I depends on the redox state of the plastoquinone pool. (5) In relation to current ideas about the lateral heterogeneity of Photosystem I and Photosystem II in the thylakoid membrane, the results are consistent with the function of plastocyanin as a mobile carrier of electrons in the intrathylakoid space.     

The kinetics of reactions around the cytochrome bf complex studied in an isolated system

The kinetics of oxidation and reduction of P700, plastocyanin, cytochrome f and cytochrome b-563 were studied in a reconstituted system consisting of Photosystem I particles, cytochrome bf complex and plastocyanin, all derived from pea leaf chloroplasts. Decyl plastoquinol was the reductant of the bf complex. Turnovers of the system were initiated by laser flashes. The reaction between oxidised P700 and plastocyanin was non-homogeneous in that a second-order rate coefficient of c. 5×10^–7 M^–1 s^–1 applied to 80% of the P700⁺ and c. 0.7×10⁷ M^–1 s^–1 to the remainder. In the presence of bf complex, but without quinol, the electron transfer between cytochrome f and oxidised plastocyanin could be described by a second-order rate coefficient of c. 4×10⁷ M^–1 s^–1 (forward), and c. 1.6×10⁷ M^–1 s^–1 (reverse). The equilibrium coefficient was thus 2.5. Unexpectedly, there was little reduction of cytochrome f ⁺ or plastocyanin⁺ by electrons from the Rieske centre. With added quinol, reduction of cytochrome b-563 occurred. Concomitantly, electrons appeared in the oxidised species. It was inferred that either the Rieske centre was not involved in the high-potential chain of electron transfer events, or that, only in the presence of quinol, electrons were quickly passed from the Rieske centre to cytochrome f ⁺. Additionally, the presence of quinol altered the equilibrium coefficient for the cyt f/PC interaction from 2.5 to c. 5. The reaction between quinol and the bf complex was describable by a second-order rate coefficient of about 3×10⁶ M^–1 s^–1. The pattern of the redox reactions around the bf complex could be simulated in detail with a Q-cycle model as previously found for chloroplasts.Abbreviations AQS anthraquinone sulphonate - cyt cytochrome - cyt b-563(H) high-potential cyt b-563 - cyt b-563(L) low potential cyt b-563 - FeS(R) the Rieske protein of the cyt bf complex, containing an Fe₂S₂ centre - PC plastocyanin - PS photosystem - P700 reaction centre in PS I     

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

A 300 μs decay component of ESR Signal I (P-700⁺) in chloroplasts is observed following a 10 μs 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 E_m = 375±10 mV (pH = 7.5). These data suggest the assignment of the 300-μs 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)).     

Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP

The effects of dibromothymoquinone (DBMIB) and methylviologen (MV) on the Chl a fluorescence induction transient (OJIP) were studied in vivo. Simultaneously measured 820-nm transmission kinetics were used to monitor electron flow through photosystem I (PSI). DBMIB inhibits the reoxidation of plastoquinol by binding to the cytochrome b₆/f complex. MV accepts electrons from the FeS clusters of PSI and it allows electrons to bypass the block that is transiently imposed by ferredoxin-NADP⁺-reductase (FNR) (inactive in dark-adapted leaves). We show that the IP phase of the OJIP transient disappears in the presence of DBMIB without affecting F_m. MV suppresses the IP phase by lowering the P level compared to untreated leaves. These observations indicate that PSI activity plays an important role in the kinetics of the OJIP transient. Two requirements for the IP phase are electron transfer beyond the cytochrome b₆/f complex (blocked by DBMIB) and a transient block at the acceptor side of PSI (bypassed by MV). It is also observed that in leaves, just like in thylakoid membranes, DBMIB can bypass its own block at the cytochrome b₆/f complex and donate electrons directly to PC⁺ and P700⁺ with a donation time τ of 4.3 s. Further, alternative explanations of the IP phase that have been proposed in the literature are discussed.     

Electron Transport Reactions in Grana Preparations from Spinach Chloroplasts

Fraction 2 (grana-stack) particles prepared with the French press showed absorbance changes, at room temperature and with sodium ascorbate and methyl-viologen, that were produced by the oxidation of cytochrome b-559. This oxidation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and sensitized by system II of photosynthesis. The oxidation is too slow to account for the rates of the Hill reaction that have been observed with nicotinamide-adenine dinucleotide phosphate (NADP⁺). It appears that this cytochrome is not functioning in the main pathway of electron transport. In the presence of 2,3,5,6-tetramethyl-p-phenylene-diamine (DAD) and ascorbate, light-induced oxidation of cytochrome f took place within 3 msec (or faster) in the grana-stack particles. Treatment with the detergent Triton X-100 disrupted this rapid cytochrome f oxidation as well as the oxidation of cytochrome b-559. Subsequent plastocyanin addition did not restore the rapid oxidation of cytochrome f (nor of cytochrome b-559) but only slow changes of cytochrome f. In view of the fact that these particles contain almost no plastocyanin, it is unlikely that plastocyanin functions in electron transport between cytochrome f and P-700 in the particles derived from the grana-stack regions of the chloroplast.     

Reduction of photosystem I reaction center, P-700, by plastocyanin in stroma thylakoids from spinach: Lateral diffusion of plastocyanin

The reduction kinetics of the photooxidized photosystem I reaction center (P-700⁺) by plastocyanin was studied in the stroma thylakoids prepared by the Yeda press treatment. The kinetics of the P-700⁺ reduction after flash excitation were biphasic and separated into two independent first-order reactions, the fast phase with a half-time of about 4 ms and the slow phase with a half-time of about 18 ms. Only the fast phase of the P-700⁺ reduction was sensitive to KCN and glutaraldehyde treatments of the thylakoids which block the plastocyanin site in the photosynthetic electron flow indicating that the fast phase is mediated by plastocyanin. However, the content of plastocyanin in the stroma thylakoids used was greatly decreased by the Yeda press treatment to only half that of P-700⁺ reduced in the fast phase. This indicates that one plastocyanin molecule turns over more than once in the single turnover of P-700⁺ rather than forming a fixed complex with P-700. On the other hand, the slow phase was not affected by KCN or glutaraldehyde treatment and its apparent rate constant linearly depended on the concentration of reduced dichlorophenolindophenol. These results indicate that the slow phase shows direct reduction of P-700⁺ by dichlorophenolindophenol. A second-order rate constant of 3.96 × 10⁵m^?1 s^?1 was obtained for the slow phase at pH 7.6, 25 °C. Analysis of reaction kinetics in the initial portion of the fast phase indicated initial interaction between P-700⁺ and the reduced plastocyanin and gave a half-time of 0.53 ms for the bimolecular reaction. We assumed the lateral diffusion of plastocyanin on the thylakoid membrane and calculated the two-dimensional diffusion coefficient for plastocyanin from the half-time of the initial reduction of P-700⁺ as about 2 × 10^?9 cm² s^?1.     

Dissection of respiratory and cyclic electron transport in Synechocystis sp. PCC 6803

Cyclic electron transport (CET) is an attractive hypothesis for regulating photosynthetic electron transport and producing the additional ATP in oxygenic phototrophs. The concept of CET has been established in the last decades, and it is proposed to function in the progenitor of oxygenic photosynthesis, cyanobacteria. The in vivo activity of CET is frequently evaluated either from the redox state of the reaction center chlorophyll in photosystem (PS) I, P700, in the absence of PSII activity or by comparing PSI and PSII activities through the P700 redox state and chlorophyll fluorescence, respectively. The evaluation of CET activity, however, is complicated especially in cyanobacteria, where CET shares the intersystem chain, including plastoquinone, cytochrome b₆/f complex, plastocyanin, and cytochrome c₆, with photosynthetic linear electron transport (LET) and respiratory electron transport (RET). Here we sought to distinguish the in vivo electron transport rates in RET and CET in the cyanobacterium Synechocystis sp. PCC 6803. The reduction rate of oxidized P700 (P700⁺) decreased to less than 10% when PSII was inhibited, indicating that PSII is the dominant electron source to PSI but P700⁺ is also reduced by electrons derived from other sources. The oxidative pentose phosphate (OPP) pathway functions as the dominant electron source for RET, which was found to be inhibited by glycolaldehyde (GA). In the condition where the OPP pathway and respiratory terminal oxidases were inhibited by GA and KCN, the P700⁺ reduction rate was less than 1% of that without any inhibitors. This study indicate that the electron transport to PSI when PSII is inhibited is dominantly derived from the OPP pathway in Synechocystis sp. PCC 6803.

     

Plastocyanin redox kinetics in spinach chloroplasts: evidence for disequilibrium in the high potential chain

Reduction kinetics of cytochrome f, plastocyanin (PC) and P₇₀₀ (‘high-potential chain’) in thylakoids from spinach were followed after pre-oxidation by a saturating light pulse. We describe a novel approach to follow PC redox kinetics from deconvolution of 810-860 nm absorption changes. The equilibration between the redox-components was analyzed by plotting the redox state of cytochrome f and PC against that of P₇₀₀. In thylakoids with (1) diminished electron transport rate (adjusted with a cytochrome bf inhibitor) or (2) de-stacked grana, cytochrome f and PC relaxed close to their thermodynamic equilibriums with P₇₀₀. In stacked thylakoids with non-inhibited electron transport, the equilibration plots were complex and non-hyperbolic, suggesting that during fast electron flux, the ‘high-potential chain’ does not homogeneously equilibrate throughout the membrane. Apparent equilibrium constants <5 were calculated, which are below the thermodynamic equilibrium known for the ‘high potential chain’. The disequilibrium found in stacked thylakoids with high electron fluxes is explained by restricted long-range PC diffusion. We develop a model assuming that about 30% of Photosystem I mainly located in grana end-membranes and margins rapidly equilibrate with cytochrome f via short-distance transluminal PC diffusion, while long-range lateral PC migration between grana cores and distant stroma lamellae is restricted. Implications for the electron flux control are discussed.     

Effect of surface potential on P-700 reduction in chloroplasts

The effect of salt addition on the rate of reduction of P-700 oxidized by flash illumination was analyzed. In broken chloroplasts, the rate of P-700 reduction was accelerated by salts of mono-, di- and trivalent cations, with the increasing effectiveness in this order, in the presence of various artificial electron donors or acceptors. The rate was not dependent on the concentration and the valence of anions. On the other hand, in Photosystem I-enriched subchloroplast particles, added KCl did not induce the acceleration of direct reduction of P-700 by reduced DCIP.At low KCl concentrations (below 10 mM), the rate of P-700 reduction was also accelerated by added KCl in sonicated chloroplasts to which purified plastocyanin was added. The curves of dependence of the reduction rate on plastocyanin concentration were not of the Michaelis-Menten type, but sigmoidal. The maximal of P-700 reduction was higher at higher salt concentrations and the half-maximal plastocyanin concentration for P-700 reduction became lower with increasing NaCl concentrations.In broken chloroplasts treated with 50 mM glutaraldehyde, the rate of P-700 reduction was not accelerated by added KCl.The Debye-Hückel theory and the Gouy-Chapman theory were applied to our data to analyze the electrostatic interaction between electron tranfer components on thylakoid membranes. It is suggested that the major factor determining the rate of P-700 reduction is the donation of electrons from plastocyanin to P-700. Most of the observed effect is probably due to the increase in the local concentration or accessibility of plastocyanin to the site of P-700 reduction which is expected when the negative surface potential rises when salt is added.     

Functional homogeneity of P-700 in cyclic and non-cyclic electron transport reactions in thylakoids

The photoinduced turnover of P-700 (the reaction center chlorophyll a of photosystem I) in higher plant thylakoids was examined at room temperature by observation of the kinetics and amplitude of the transmission signal at 700 nm. The concentration of P-700 functional in cyclic and non-cyclic electron transfer reactions was compared. For the cyclic reactions mediated by N-methylphenazonium-p-methosulfate, 2,3,5,6-tetramethylphenylenediamine, 2,6-dichlorophenolindophenol and N,N,N′,N′-tetramethylphenylenediamine and non-cyclic reactions utilizing either methylviologen or NADP⁺ as acceptor, the illuminated steady-state concentration of P-700⁺ was shown to be similar. The data support the concept of a homogeneous pool of P-700 that is capable of interaction in both cyclic and non-cyclic electron transfer reactions and are consistent with previous data obtained in vivo.The amplitude and kinetics of the P-700 signal were found to be very dependent upon the composition of the reaction medium and differences were noted for turnover in the cyclic and non-cyclic reactions. Specifically, at white light saturation, the addition of low concentrations of divalent cations, such as Mg²⁺ or Ca²⁺, had no effect on the signal amplitude during the cyclic reactions, but, in confirmation of previous work, caused an attenuation of the signal amplitude during non-cyclic flow. At low light intensities, the divalent cations caused a similar reduction in redox level of P-700 in the steady-state during non-cyclic flow and also reduced the rate of P-700 photooxidation in the cyclic reactions. The concentration of divalent cation that reduced the signal amplitude of P-700⁺ during non-cyclic flow was compared with that required for the stimulation of the variable component of fluorescence, and it was shown to be similar with half maximal effects at 1 mM Mg²⁺. The observations confirm that divalent cations control non-cyclic electron transport by an activation of Photosystem II in addition to regulating the distribution of excitation energy between the two photosystems.     

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 fragments 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 NADP⁺. 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 α peak at 549 nm. 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.     

A comparative laser-flash absorption spectroscopy study of algal plastocyanin and cytochrome c552 photooxidation by photosystem I particles from spinach.

Laser-flash kinetic absorption spectroscopy has been used to compare the rate constants for electron transfer from reduced plastocyanin and cytochrome c552, obtained from the green alga Monoraphidium braunii, to photooxidized P700 (P700+) in photosystem I (PSI) particles from spinach Sigmoidal protein concentration dependence for the observed electron-transfer rate constants are obtained for both proteins. In the absence of added salts, the P700+ reduction rate increases as the pH decreases from approximately 8 to 5.5, then decreases to pH 3.5, this effect being more pronounced with cytochrome c552 than with plastocyanin. At neutral pH, plastocyanin is a more efficient electron donor to P700+ than cytochrome c552, whereas at pH 5.5, which is closer to physiological conditions, the two redox proteins react with approximately equal rate constants. In the presence of increasing concentrations of added salts, the P700+ reduction rate constants for both proteins increase at pH greater than 5.5, but decrease at pH less than 4. At neutral pH, the observed rate constants for both algal proteins have a biphasic dependence on sodium chloride concentration, increasing in a parallel manner with increasing salt concentration, reaching a maximum value at 50 mM NaCl, then decreasing. A similar biphasic dependence is obtained with magnesium chloride, but in this case the maximum value is reached at salt concentrations ten times smaller, suggesting a specific role for the divalent cations in the electron-transfer reaction.