Reduction Of P700 Research Articles

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.

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.

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 HgCl 2, 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 b 6 - 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 b 6 - 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.

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 × 10 5 m −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 cm 2 s −1.

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.

Transient kinetics of the reaction between cytochrome c-552 or plastocyanin and P-700 in subchloroplast particles

We have investigated the kinetics of reduction of P-700 in Photosystem I-enriched subchlroplast particles by cytochrome c-552 from Euglena and by plastocyanin, and the effects of cations on these reactions. In both cases, the results can be explained in terms of a bimolecular reaction between P-700 + and the electron donor. Addition of low concentrations of mono-, di- or polyvalent cations stimulate, whereas addition of high concentrations of cations inhibit these reactions. The effects of cations can be interpreted as effects on the membrane surface potential of the Photosystem I particles, as shown previously (Tamura, N., Yamamoto, Y. and Nishimura, M. (1980) Biochim. Biophys. Acta 592, 536–545). The maximal second-order rate constants obtained for the reduction of P-700 + are 9 · 10 7 M −1 · s −1 if cytochrome c-552 is the electron donor, and 1.5 · 10 8 M −1 · s −1 when the donor is plastocyanin. Using the latter rate constant, we are able to demonstrate that the apparently biphasic kinetics of reduction of P-700 + observed in intact chloroplasts show a good fit to those expected for a bimolecular reaction between P-700 + and plastocyanin.

Studies on P-700 photo-oxidation and reduction in Photosystem I subchloroplast particles

The photochemical oxidation and reduction of P-700 were studied in digitonin- and in sodium dodecyl sulphate (SDS)-Photosystem I (PS I) particles in the presence of ascorbate. In digitonin-PS I particles, reduction of P-700 + occurs by the bound iron-sulphur protein ( P-430) and by ascorbate. The relative contribution of these back reactions depends on the length of the exposure to light and on the temperature and pH of the reaction medium. Experiments performed under anaerobic conditions demonstrate that some endogenous component may serve as the electron acceptor of P-430 −. The rate of the latter reaction is also dependent upon the temperature and pH of the sample. At pH 9 and lower temperatures the rate of this reaction is so much reduced that the reduction of P-700 + by ascorbate, which increases rapidly at high pH, can be observed even during illumination. The effects of secondary electron acceptors and of the presence of SDS on the absorption changes due to P-700 are also reported. Low concentrations of SDS are shown to retard the back reaction of P-700 + with P-430 −. Studies with SDS-PS I particles (CP I) confirm the absence of the iron-sulphur centres in this preparation. Three larger P-700-chlorophyll a-protein complexes prepared by mild electrophoresis in the presence of SDS plus Triton X-100, however, still contain P-430.

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.

Evidence for complexed plastocyanin as the immediate electron donor of P-700

The reduction of P-700 by its electron donors shows two fast phases with half-times of 20 and 200 μs in isolated spinach chloroplasts. We have studied this electron transfer and the oxidation kinetics of cytochrome f. Incubation of chloroplasts with KCN or HgCl 2 decreased the amplitude of the 20 μs phase. This provides evidence for a function of plastocyanin as the immediate electron donor of P-700. At low concentrations of salt and sugar the fast phases of P-700 + reduction were largely inhibited. Increasing concentrations of MgCl 2, KCl and sorbitol (up to 5, 150 and 200 mM, respectively) were found to increase the relative amplitudes of the fast phases to about one-third of the total P-700 signal. Addition of both 3 mM MgCl 2 and 200 mM sorbitol increased the relative amplitude of the 20 μs phase to 70%. The interaction between P-700 and plastocyanin is concluded to be favoured by a low internal volume of the thylakoids and compensation of surface charges of the membrane. The half-time of 20 μs was not changed when the amplitude of this phase was altered either by salt and sorbitol, or by inhibition of plastocyanin. This is evidence for the existence of a complex between plastocyanin and P-700 with a lifetime long compared to the measuring time. The 200 μs phase exhibited changes in its half-time that indicated the participation of a more mobile pool of plastocyanin. Cytochrome f was oxidized with a biphasic time course with half-times of 70–130 μs and 440–860 μs at different salt and sorbitol concentrations. The half-time of the faster phase and a short lag of 30–50 μs in the beginning of the kinetics indicate an oxidation of cytochrome f via the 20 μs electron transfer to P-700. An inhibition of this oxidation by MgCl 2 suggests that the electron transfer from cytochrome f to complexed plastocyanin is not controlled by negative charges in contrast to that from plastocyanin to P-700.

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.

The effect of some inhibitors of photosynthetic electron transport on the kinetics of redox changes of the reaction centre chlorophyll of photosystem I (P700) and of cytochrome f at sub-zero temperatures.

1 The effect of five inhibitory treatments on the extent of photooxidation of the reaction centre chlorophyll of photosystem I (P700) and cytochrome f in saturating continuous light and on the kinetics of their subsequent dark reduction was studied in spinach chloroplasts suspended at–17°C in a fluid medium containing 50 % (v/v) ethanediol: water. Under these conditions the turnover time of the uninhibited electron transport system is of the order of seconds. 2 Treatment with KCN or polylysine inhibited the photooxidation of cytochrome f and the reduction of P700, but not the reduction of the cytochrome or P700 oxidation. These observations are those expected if inhibition is at the level of plastocyanin and this component mediates electron flow between cytochrome f and P700. 3 Inhibition with bathophenanthroline or 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone had no effect on the photooxidation of either cytochrome f or P700. In contrast the rate of dark reduction of both components was reduced in parallel by both inhibitors. This suggests a site of inhibition common to the reduction pathways of both cytochrome f and P700. 4 HgCl2 had the same inhibitory effects as polylysine and KCN but in addition the reduction of cytochrome f was inhibited. It is suggested that this inhibitor may act at more than one site. 5 In most cases cytochrome f reduction showed first-order kinetics and in no case was there any evidence for a lag in the reduction. Inhibitors preventing electron flow from plastocyanin to P700 caused no increase in the rate of cytochrome f reduction. These results do not fit the expected properties of a linear scheme of electron transfer from plastoquinol to P700 via cytochrome f and plastocyanin.

Chloroplast cytochrome b-563, hydrophobic environment and lack of direct reaction with ferredoxin.

1. The reaction of electron donors with cytochrome b-563 in chloroplasts was studied by investigating their effects on the rate of its reduction in the presence of dithionite, which reacts only slowly with the cytochrome. The relative effects of 9,10-anthraquinone and 9,10-anthraquinone 2-sulphonate in the presence of dithionite suggested that the site of attack of redox reagents was protected behind a hydrophobic barrier from the external medium. 2. Ferredoxin had no measurable effect on the rate of reduction of the cytochrome in the presence of dithionite. 3. The reduction of pigment P700 in the dark after illumination in the presence of the inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea was stimulated by a combination of NADPH and ferredoxin, although NADPH alone had little effect. The same combination was unable to reduce cytochrome b-563 at a measurable rate. 4. It is concluded that the cytochrome is unlikely to be part of a linear pathway of electron flow between ferredoxin and pigment P700.

Interaction of plastocyanin and P700 in PSI reaction center particles from C. reinhardi and spinach

Highly resolved Photosystem I (PSI) reaction center particles, solubilized from spinach and C. reinhardi chloroplasts with Triton X-100, catalyze very poorly the reduction of P700 by ascorbate at mildly acidic to alkaline pHs (pH 5–8). (Photooxidation of P700 means conversion of P700 to P700 +; dark reduction of P700 is the reverse reaction.) The interaction between plastocyanin (PC) and P700 is assessed by measuring the stimulation of the rate of P700 reduction upon addition of PC at submicromolar concentrations. At a ratio of 2.6 PC per P700, about 20- to 25-fold stimulation of the rate of P700 reduction was observed in the presence of MgCl 2 or CaCl 2. Efficient interaction between PC and P700 occurred at near neutral pH (optimal pH = 6.6–6.8) and exhibited a preferential requirement for divalent metal cations. Monovalent cations are less effective in promoting transfer of electrons from PC to P700. At alkaline pHs (greater than 8.5) an accelerated rate of the PC-independent reduction of P700 was observed. Further, the PC-independent reaction at pH 9.4 showed a strong requirement for divalent cations. In contrast, monovalent cations are only partially effective in promoting the PC-independent reduction of P700 by ascorbate.

Subunit Structure of Chloroplast Photosystem I Reaction Center

Purified Photosystem I reaction center preparation catalyzed a plastocyanin-dependent cytochrome f photooxidation. Euglena cytochrome 552 replaced plastocyanin in all photochemical reactions catalyzed by the purified reaction center and was also photooxidized. A reaction center preparation lacking Subunit III was obtained by means of Triton X-100 treatment and DEAE-cellulose chromatography or sucrose gradient centrifugation. This preparation was incapable of cytochrome f photooxidation or of NADP photoreduction in the presence of plastocyanin. However, the reaction center lacking Subunit III photoreduced NADP when plastocyanin was replaced by N-methylphenazonium-3-sulfonate, serving as direct electron donor for P700. It was concluded that Subunit III mediates reduction of P700+ by plastocyanin. Mild treatment with sodium dodecyl sulfate yielded reaction center preparations which were partially depleted in Subunits II, III, IV, V, and VI. The NADP photoreduction activity and EPR-detectable bound ferredoxin were correlated to the presence of Subunits IV, V, and VI in the depleted preparations. Subunits IV, V, and VI might mediate photoreduction of soluble ferredoxin by the primary electron acceptor, in the reducing site of Photosystem I. The P700 reaction center (Subunit I) was further purified. Preparations with chlorophyll a to P700 ratios below 40 were obtained with the use of higher concentrations of Triton X-100, which caused antenna chlorophyll a to be partially solubilized. Removal of the sdlubilized chlorophyll by DEAE-cellulose chromatography resulted in preparations which contained 10 to 20 chlorophyll a molecules/P700. It is suggested that a unit of about 20 chlorophyll a molecules is specifically associated with the reaction center protein (Mr = 70,000) in the vicinity of P700. A model has been presented which depicts probable organization of the Photosystem I reaction center in the thylakoid membrane.

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, I 1 2 , 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, I 1 2 is compared with another parameter, equivalent to I 1 2 , 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 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 HgCl 2). 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 (Cl 2Ind)/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 HgCl 2. 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 (Fe 2+) to P-700 +, thus confirming the recent observations of Haehnel et al. (Z. Naturforsch. 26b, 1171–1174 (1971)).

Antagonistic relationships between electron transport and P700 in chloroplasts and intact algae.

The effects of divalent salts and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) upon nicotinamide adenine dinucleotide phosphate (NADP) reduction and P700 in isolated chloroplasts are described and compared with the influence of DCMU on oxygen evolution and P700 in intact cells. Most experiments were carried out with a steady-state relaxation spectrometer. A kinetic mechanism for the estimation of P700 fluxes is proposed and experimentally tested. Good agreement between theory and experiment was found. Concurrent measurements of P700 and NADP reduction revealed two antagonisms: addition of divalent cations caused an increase in the yield of NADP reduction and a decrease in the yield of P700. Conversely, in the presence of Mg2+ low concentrations of DCMU decreased the yield of NADP reduction and increased the yield of P700. Aging of the chloroplasts at 30 degrees C exerted a similar effect. With far-red actinic light, Mg2+ stimulated the yield of NADP reduction without affecting the flux. Also, in the absence of Mg2+, DCMU inhibited both reactions although P700 required a higher herbicide concentration for fractional inhibition than NADP reduction. In the presence of Mg2+, chloroplasts resembled intact algae in which a high rate of oxygen evolution was accompanied by little P700 turn-over. Titration with DCMU decreased the rate of photosynthesis and increased P700 flux. On the whole, the data suggest that P700 relaxing in 20 msec is not directly involved in linear electron transport.

The properties of cytochrome f and P700 in chloroplasts suspended in fluid media at sub-zero temperatures.

1. The properties of P700 and cytochrome f have been studied at sub-zero temperatures in chloroplasts suspended in a medium containing 50% (v/v) ethylene glycol. The dark reduction of these components after a period of illumination provided information about the rate-limiting step of photosynthetic electron transport under these conditions. 2. The oxidation of P700 on illumination in the presence of methyl viologen and its subsequent dark reduction can be observed at -35 degrees C. This cycle of reactions could be repeated many times. The rate of reduction was increased by NH4Cl and reduction was inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea. 3. The oxidation and reduction of cytochrome f could also be observed under similar conditions. The activation energies for the reduction of cytochrome f and P700 are similar (about 75 kJ mol-1) and the reduction of cytochrome f is also inhibited by dichlorophenyldimethylurea and stimulated by NH4Cl. 4. The reduction of both cytochrome f and P700 seemed to follow first-order kinetics, but the t1/2 for the redcution of the cytochrome was at least three times that for the reduction of P700 at the same temperature. It was concluded that the results were only compatible with a model in which the main pathway of electrons from plastoquinone to P700 involved cytochrome f if the equilibrium constant between the cytochrome and P700 was very much less than that expected from their redox potentials.

The P700-chlorophyll a-protein : Isolation and some characteristics of the complex in higher plants

A P700-chlorophyll a-protein complex has been purified from several higher plants by hydroxylapatite chromatography of Triton X-100-dissociated chloroplast membranes. The isolated material exhibits a red wavelength maximum at 677 nm, major spectral forms of chlorophyll a at 662, 669, 677, and 686 nm, a chlorophyll/P700 ratio of 40–45 1 , and contains only chlorophyll a and β-carotene of the photosynthetic pigments present in the chloroplast. The spectral characteristics and composition of the higher plant material are homologous to those of the P700-chlorophyll a-protein previously isolated from blue-green algae; however, unlike the blue-green algal component, cytochromes f and b 6 are associated with the higher plant material. Evidence is presented that a chlorophyll a-protein termed “Complex I” which can be isolated from sodium dodecyl sulfate extracts of chloroplast membranes is a spectrally altered form of the eucaryotic P700-chlorophyll a-protein. The isolation procedure described in this paper is a more rapid technique for obtaining the heart of photosystem I than presently exists; furthermore, the P700 photooxidation and reduction kinetics in the fraction are improved over those in other isolated components showing the same enrichment of P700. It appears very probable that the heart of photosystem I is organized in the same manner in all chlorophyll a-containing organisms.

FLASH PHOTOLYSIS‐ELECTRON SPIN RESONANCE STUDIES OF THE DYNAMICS OF PHOTOSYSTEM I IN GREEN‐PLANT PHOTOSYNTHESIS—II. INTACT AND BROKEN SPINACH CHLOROPLASTS*

Abstract—The transient oxidation and subsequent reduction of P700+ in spinach chloroplasts has been monitored by flash photolysis‐electron spin resonance spectroscopy in the presence of various donors and acceptors. In general, the results obtained correlate well with results on Photosystem I subchloroplast particles, with two major differences. For Type A and B intact chloroplasts in the presence of 3‐(3, 4‐dichlorophenyl)‐1, 1‐dimethylurea (DCMU), the electron acceptor methyl viologen has no effect on the decay kinetics. This phenomenon is interpreted in terms of a functioning cyclic electron flow path around Photosystem I. Also, the photoresponse of Signal I depends on the length of the photolyzing flash. This is interpreted in terms of the existence of a primary electron donor to P700+ with a transfer time of ˜ 10 μs.