Photoreactions of cytochrome b-559 and cyclic electron flow in Photosystem II of intact chloroplasts

The high potential cytochrome b-559 of intact spinach chloroplasts was photooxidized by red light with a high quantum efficiency and by far-red light with a very low quantum efficiency, when electron flow from water to Photosystem II was inhibited by a carbonyl cyanide phenylhydrazone (FCCP or CCCP). Dithiothreitol, which reacts with FCCP or CCCP, reversed the photooxidation of cytochrome b-559 and restored the capability of the chloroplasts to photoreduce CO₂ showing that the FCCP/CCCP effects were reversible. The quantum efficiency of cytochrome b-559 photooxidation by red or far-red light in the presence of FCCP was increased by 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone which blocks oxidation of reduced plastoquinone by Photosystem I. When the inhibition of water oxidation by FCCP or CCCP was decreased by increased light intensities, previously photooxidized cytochrome b-559 was reduced. Red light was much more effective in photoreducing oxidized high potential cytochrome b-559 than far-red light. The red/far-red antagonism in the redox state of cytochrome b-559 is a consequence of the different sensitivity of the cytochrome to red and far-red light and does not indicate that the cytochrome is in the main path of electrons from water to NADP. Rather, cytochrome b-559 acts as a carrier of electrons in a cyclic path around Photosystem II. The redox state of the cytochrome was shifted to the oxidized side when electron transport from water became rate-limiting, while oxidation of water and reduction of plastoquinone resulted in its shifting to the reduced side.     

A highly active oxygen-evolving Photosystem II preparation from synechococcus lividus

A PS II preparation highly active in oxygen generation was prepared from the cyanophyte, Synechococcus lividus. This preparation was enriched in Hill reaction activity, manganese, cytochrome b-559, and possessed only trace amounts of cytochrome b-563. This non-phosphorylating, visually clear preparation appears to be a promising system for the detailed study of Photosystem II.     

Characteristics of the Photosystem II reaction centre. II. Electron donors

The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.     

The role of the high potential form of the cytochrome b559: Study of Thermosynechococcus elongatus mutants

Cytochrome b559 is an essential component of the photosystem II reaction center in photosynthetic oxygen-evolving organisms, but its function still remains unclear. The use of photosystem II preparations from Thermosynechococcus elongatus of high integrity and activity allowed us to measure for the first time the influence of cytochrome b559 mutations on its midpoint redox potential and on the reduction of the cytochrome b559 by the plastoquinone pool (or Q_B). In this work, five mutants having a mutation in the α-subunit (I14A, I14S, R18S, I27A and I27T) and one in the β-subunit (F32Y) of cytochrome b559 have been investigated. All the mutations led to a destabilization of the high potential form of the cytochrome b559. The midpoint redox potential of the high potential form was significantly altered in the αR18S and αI27T mutant strains. The αR18S strain also showed a high sensitivity to photoinhibitory illumination and an altered oxidase activity. This was suggested by measurements of light induced oxidation and dark re-reduction of the cytochrome b559 showing that under conditions of a non-functional water oxidation system, once the cytochrome is oxidized by P680⁺, the yield of its reduction by Q_B or the PQ pool was smaller and the kinetic slower in the αR18S mutant than in the wild-type strain. Thus, the extremely positive redox potential of the high potential form of cytochrome b559 could be necessary to ensure efficient oxidation of the PQ pool and to function as an electron reservoir replacing the water oxidation system when it is not operating.     

Variability in the synthesis of cytochromes f and b-563 during the cell cycle of Euglena gracilis

The study of the successive formation of the photosynthetic apparatus in Euglena gracilis (Brandt, P. and Von Kessel, B. (1983) Plant Physiol. 72, 616–619) was extended to the determination of the stage-specific synthesis of cytochrome $\text{b}\text{f}$ complex during the cell cycle of this alga. Most of the cytochrome f (33 kDa) has properties of an intrinsic membrane protein, but part of it is soluble. Cytochrome b-563 (18 kDa) is only intrinsic. The intensity of binding the intrinsic cytochromes in the thylakoids depends on the developmental stage of the organism. The light-independent synthesis of cytochrome f takes place prior to the assembly of the chlorophyll-protein complex I (CP I). Immediately after this assembly of CP I, cytochrome b-563 is synthesized in the light. Hence, the ratio cytochrome b-563/cytochrome f changes during the cell cycle of E. gracilis. The physiological implication of presumably non-complexed cytochrome f and of complex-bound cytochromes f and b-563 on the stage-specific efficiency of photosynthesis of E. gracilis is discussed.     

On the question of the identity of cytochrome b-560 in thylakoid stromal membranes

Stromal membranes enriched in PS I contain a low potential cytochrome with a reduced -band peak close to 560 nm. The identity of this cytochrome component has been ascribed either to a low potential form of the Photosystem II cytochrome b-559 or to a different cytochrome with a reduced -band of 560 nm. The half-bandwidth of the 560 nm component in stromal membranes is identical to that of purified cytochrome b-559. Western blots show that the stromal membranes contain an amount of PS II cytochrome b-559 -subunit that is more than sufficient to account for the cytochrome b-560 detected spectrophotometrically in these membranes. These immunochemical data and the similarity of (i) the spectral peaks, and (ii) the redox properties of low potential PS II cytochrome b-559 and the b-560 component, suggest that the simplest inference is that the cytochrome b-560 protein in stromal membranes is identical to the PS II cytochrome b-559.Abbreviations: A absorbance - cyt cytochrome - DCBQ 2,5-dichloro-p-benzoquinone - E_mx midpoint potential at pH x - hbw half-bandwidth - LP low potential - MD menadiol - MES 2-(N-morpholino)ethanesulfonic acid - MHQ methylhydroquinone - PS I-PS II photosystems I, II - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis     

Cytochrome function in the cyclic electron transport pathway of chloroplasts

Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the α-bands of cytochrome b₆ and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b₆ and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH₄Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift, with formation half-time of about 2 ms. The inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b₆ and f by factors of ten or more.These observations indicate that cytochrome b₆, plastoquinone and cytochrome f participate in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 μmol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO₂ fixation.     

The fate of cytochrome b559during anaerobic photoinhibition and its recovery processes

The function of the cytochrome b₅₅₉, a Photosystem II (PS II) reaction center ubiquitous component is not yet known. Cytochrome b₅₅₉appears in a high (HP) or low (LP) potential form. The HP form is converted into the LP form during aerobic photoinhibition. It has been proposed before that this conversion, assumed to be reversible, ascribes protection against light stress of PS II by redirecting electron flow within PS II thus avoiding charge recombination of the primary radical pair and related oxidative damage. Here, we have used an experimental system allowing to assay the relation between the cytochrome b₅₅₉redox potential shift, its reversibility and protection against light induced PS II inactivation. Under anaerobic conditions fast reversible photoinactivation of PS II in isolated spinach thylakoids is observed accompanied by monomerisation of PS II. Monomers did not dissociate further into PS II sub-particles and did not migrate out of the grana partitions as observed in aerobic photoinactivation. The anaerobic photoinactivation is accompanied by an increase in the cytochrome b₅₅₉LP/HP ratio. However, despite recovery of PS II activity and partially of its dimeric form in darkness under aerobic conditions, no reversal of the cytochrome b₅₅₉redox potential shift accompanied these processes. Re-exposure of reactivated thylakoids having an increased PS II population in the LP form of the cytochrome b₅₅₉to strong illumination under aerobic conditions, did not result in a measurable protection of PS II as compared to control thylakoids. While it is possible that cytochrome b₅₅₉may play a protective role against light stress in PS II, the results presented here do not indicate that the increase in the ratio LP/HP form is involved in this process.     

Gel electrophoresis of chloroplast membranes of mutants of Chlamydomonas reinhardii which have impaired Photosystem II function and lack photosynthetic cytochromes

Photosystem (PS) II-enriched particles or chloroplast fragments of the wild type and of three nonphotosynthetic mutants of Chlamydomonas reinhardii, which lack chloroplast cytochromes, were analyzed by lithium dodecyl sulfate polyacrylamide gel electrophoresis at 4°C to locate which chlorophyll complexes and which proteins are associated with cytochrome b-559. Two mutants, Fl 39 and Fl 50, have previously been shown to contain, respectively, 3.6- and 2.7-times less hydroquinone-reducible high-potential cytochrome b-559 than the wild type. They have impaired PS II functions. In the presence of ADRY agents: carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p) or 2-(3,4,5-trichloro)-anilino-3,5-dinitrothiophene (ANT 2s), Fl 50 carried out photo-oxidation of cytochrome b-559 with half the amplitude of that of the wild type. No photo-oxidation was observed with Fl 39. We show here that in both these mutants chlorophyll-protein complexes CP III, CP IV and CP V were missing. There were traces of the corresponding apoproteins (45 000, 42 000 and 33 000 daltons, respectively) in Fl 50 but none in Fl 39. In addition, a 19 000 dalton protein was missing in Fl 39 and present in a very small amount in Fl 50. In another mutant, Fl 9, previously characterized as lacking both cytochromes b-563 and c-553 with a normal cytochrome b-559 content, CP III-CP V and the 19 000 dalton protein were detected. CP I (110 000 daltons) and CP II (24 000 daltons) were present in all strains. These observations confirmed the close relationship between deficiencies in cytochrome b-559, lack of CP III and CP IV and anomalies in the photochemistry of PS II. They provided additional evidence that CP V and a 19 000 dalton protein are also involved in this PS II photochemistry. Staining of the gels with 3,3′,5,5′-tetramethylbenzidine and H₂O₂ allowed us to distinguish clearly four heme protein bands having peroxidase activity. Three of these bands (45 000, 42 000 and 19 000 daltons), which were shown in wild-type, Fl 39 and Fl 50 preparations but not in Fl 9, appeared related to cytochromes b-563 and c-553. The fourth heme protein (14 000 daltons) occurred in wild type and Fl 9 but was missing in Fl 39 and Fl 50; it appeared related to cytochrome b-559.     

Light-induced redox changes of cytochrome b-559

Dark incubation of spinach or pea chloroplasts with 10 μm carbonylcyanide m-chlorophenylhydrazone (CCCP) had a negligible effect either on the redox state or the redox potential of the high potential form of cytochrome b-559 (cytochrome b-559_hp). A similar result was obtained with spinach chloroplasts on incubation with 3.3 μm carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), but pea chloroplasts showed a decrease of 10–20% in the amount of reduced cytochrome b-559.Light-induced redox changes of cytochrome b-559 were not observed in untreated spinach chloroplasts. In the presence of CCP or FCCP, cytochrome b-559 was photooxidized both in 655 nm actinic light and in far-red light. Addition of the plastoquinone antagonist, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) to CCCP- or FCCP-treated chloroplasts had only a small effect on the photooxidation of cytochrome b-559 in 655 light, but it completely inhibited the oxidation in far-red light.Electron flow from water to 2,3′,6-trichlorophenolindophenol was partly inhibited by CCCP or FCCP, but the degree of inhibition does not appear to be sufficient to account for the photooxidation of cytochrome b-559.The photooxidation of cytochrome b-559 by 655 nm light at liquid nitrogen temperature was not influenced by prior treatment of the chloroplasts at room temperature with CCCP, DBMIB, or CCCP + DBMIB.The results cannot be explained by the presence of two independent pools of cytochrome b-559 in CCCP-treated chloroplasts, one photooxidized by Photosystem II and the other photooxidized by Photosystem I and photoreduced by Photosystem II.     

Cytochromes and prenylquinones in preparations of cytoplasmic and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans

The cytochrome and prenylquinone compositions were compared for cytoplasmic membranes and thylakoid membranes from the cyanobacterium (blue-green alga) Anacystis nidulans. Reduced-minus-oxidized difference absorption spectra at ?196°C indicated that the thylakoid membranes contained photosynthetic cytochromes such as cytochrome ?, cytochrome b-559 and cytochrome b₆, while cytochromes c-549 and c-552 were detected spectrophotometrically only after their release by sonic oscillation. The cytoplasmic membrane preparation contained one or two low-potential cytochrome(s) with α-band maxima at 553 and 559 nm at ?196°C, which differed from the cytochromes in the thylakoid membranes. A cytochrome specific to the cytoplasmic membranes was also found by heme-staining after lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Both types of membranes contained the three prenylquinones plastoquinone-9, phylloquinone and 5′-monohydroxyphylloquinone, but in different proportions.     

Restoration of the high potential form of cytochrome b-559 through the photoreactivation of Tris-inactivated oxygen-evolving center

Restoration of a high potential (HP) form of cytochrome b-559 (Cyt b-559) from a low potential (LP) form was the primary process in the reconstitution of O₂-evolving center during the photoreactivation of Tris-inactivated chloroplasts. In normal chloroplasts, about 0.5 to 0.7 mol of Cyt b-559 was present in the HP form per 400 chlorophyll molecules. However, the HP form was converted to the LP form when the O₂-evolving center was inactivated by 0.8 M alkaline Tris-washing (pH 9.1). The inactivation was reversible and both the Cyt b-559 HP form and the O₂-evolving activity were restored by incubating the inactivated chloroplasts with weak light, Mn²⁺, Ca²⁺ and an electron donor (photoreactivation). The recovery of the HP form preceded the recovery of O₂-evolving activity. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) did not inhibit the recovery of the HP form. Thus, the recovery of Cyt b-559 HP form was the primary reaction in the photoreactivation, which was stimulated by the light-induced redox reaction of the PS-II core center.Abbreviations ASC ascorbate - BSA bovine serum albumin - Chl chlorophyll - Cyt b-559 HP form high potential form of cytochrome b-559 - Cyt b-559 LP form low potential form of cytochrome b-559 - Cyt b-559 VLP form very low potential form of cytochrome b-559 - Cyt f cytochrome f - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCPIP 2,6-dichlorophenol indophenol - Hepes N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid - HQ hydroquinone - SHN chloroplast-preparation medium containing 0.4 M sucrose, 50 mM Hepes-Na (pH 7.8) and 20 mM NaCl - PS-II Photosystem II     

The relationship between the activity of chloroplast Photosystem II and the midpoint oxidation-reduction potential of cytochrome b-559

The role of cytochrome b-559 in Photosystem II reactions has been investigated using hydroxylamine treatment of chloroplast membranes. Incubation of chloroplasts with hydroxylamine in darkness resulted in inhibition of water oxidation and a decrease in the amplitude of cytochrome b-559 reducible by hydroquinone. The loss of water oxidizing activity perfectly correlated with the decrease in amplitude of cytochrome b-559 reduction. Potentiometric titration of cytochrome b-559 after hydroxylamine treatment revealed a component with E_m7.8 at +240 mV in addition to a lower potential species at +90 mV. This compared to control chloroplasts in which cytochrome b-559 exists in the typical high potential state, E_m7.8 = +383 mV, in addition to some of the low potential (E_m7.8 = +77 mV) form. Photosystem II activity could be further inhibited by incubation with hydroxylamine in the light. In these chloroplasts only low rates of photooxidation of artificial electron donors were observed compared to ‘dark’ chloroplasts. In addition, the hydroxylamine light treatment caused a further change in cytochrome b-559 redox properties; a single component, E_m7.8 = 90 mV is seen in titration curves. The role of cytochrome b-559 in Photosystem II functioning is discussed on the basis of these observations which suggest a dependence of photooxidizing ability of Photosystem II on the redox properties of this cytochrome.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.     

The use of photoactivated hetero-bifunctional reagents to cross-link cytochrome c to proteins that bind it by electrostatic interactions.

Lysine residues of horse heart cytochrome c have been modified with N-5-azido-2-nitrobenzoyloxysuccinimide (ANB-NOS) and ethyl N-5-azido-2-nitrobenzoylaminoacetimidate (ANB-AI), reagents that attach nitroaryl azides onto the surface of proteins by amide and amidine linkages, respectively. When acting as an electron acceptor for yeast cytochrome b₂, modification of cytochrome c with ANB-NOS increases the K_m for the reaction by 2-fold, while modification with ANB-AI has little effect on the K_m. The V_max for the reduction of cytochrome c by cytochrome b₂ is reduced by the attachment of both compounds to cytochrome c. When the modified cytochromes c were illuminated with phosvitin, cytochrome b₅, and cytochrome c peroxidase, cross-linked species were formed which could be resolved by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. In each case the amidine derivatives of cytochrome c modified with ANB-AI showed more cross-linking than the amide derivatives of cytochrome c modified with ANB-NOS. When the modified cytochromes c were present in a 3-fold excess of phosvitin, cross-linked products containing 1, 2, and 3 molecules of cytochrome c covalently attached to phosvitin were observed. Photolysis of the modified cytochromes c in the presence of cytochrome b₅, resulted in the formation of a cross-linked 1:1 complex between the two cytochromes as well as higher order aggregates containing up to 5 molecules of cytochrome c plus cytochrome b₂. When cytochrome c peroxidase was illuminated with the modified cytochromes c, the predominant cross-linked product was a 1:1 complex between the two heme proteins. However, a cross-linked species was detected in small amounts with the apparent composition of 2 molecules of cytochrome c and 1 of the peroxidase. Also, a procedure is described for the synthesis of ANB-AI with ¹⁴C in the imidocarbon which is ultimately derived from ¹⁴CN.     

Purification of cytochrome b-559 from oxygen-evolving Photosystem II preparations of spinach and maize

A rapid and simple procedure is presented for the purification of chloroplast cytochrome b-559. The method is based on the protocol devised by Garewal and Wasserman (Garewal, H.S. and Wasserman, A.R. (1974) Biochemistry 13, 4063–4071), which we have modified to eliminate the requirement for a lengthy electrophoretic step. Novel features of our method include: the use of oxygen-evolving Photosystem II preparations (Kuwabara, T. and Murata, N. (1982) Plant Cell Physiol. 23, 533–539) as the starting material; isocratic elution of cytochrome b-559 from a DEAE-cellulose column (yielding the protein in a pure state); and a simple column procedure for removal of excess Triton X-100. The procedure has been applied to both spinach and maize (Zea mays L.). Purified cytochromes b-559 from these species have similar optical spectra and mobility during gel electrophoresis under native conditions. Lithium dodecyl sulfate polyacrylamide gel electrophoresis of cytochrome b-559 from both spinach and maize reveals a major polypeptide band (apparent molecular mass = 9 kDa), and two minor bands (apparent molecular masses = 10 kDa and 6 kDa).     

Inhibitory effect of α-tocopherol and its derivatives on bovine heart succinate-cytochrome c reductase

α-Tocopherol and its derivatives inhibit succinate-cytochrome c reductase activity at a concentration of 0.5 μmol/mg protein in 50 mM phosphate buffer, pH 7.4, containing 0.4 % sodium cholate when α-tocopherol is predispersed in sodium cholate solution. The inhibitory site is located at the cytochrome b-c₁ region. Succinate-ubiquinone reductase activity of succinate-cytochrome c reductase was not impaired by treatment with α-tocopherol. The α-tocopherol-inhibited succinate-cytochrome c reductase activity can be reversed by the addition of ubiquinone and its analogs. When ubiquinone- and phospholipid-depleted succinate-cytochrome c reductase was treated with α-tocopherol followed by reaction with a fixed amount of 2,3-dimethoxy-6-methyl-5-(10-bromodecyl)-1,4-benzoquinone and phospholipid, the amount of α-tocopherol needed to express the maximal inhibition was only 0.3 μmol/mg protein. When ubiquinone- and phospholipid-depleted enzyme was treated with a given amount of α-tocopherol and followed by titration with 2,3-dimethoxy-6-methyl-5-(10-bromodecyl)-1,4-benzoquinone, restoration of activity was enhanced at low concentrations of ubiquinone analog, indicating that α-tocopherol can serve as an effector for ubiquinone. The maximal binding capacity of α-[¹⁴C]tocopherol, dispersed in 50 mM phosphate buffer containing 0.25% sodium cholate, pH 7.4, to succinate-cytochrome c reductase was shown to be 0.68 μmol/mg protein. A similar binding capacity, based on cytochrome b content, was observed in submitochondrial particles. Binding of α-tocopherol to succinate-cytochrome c reductase not only caused an inhibition of enzymatic activity but also caused a reduction of cytochrome c₁ in the absence of substrate, a phenomenon analogous to the removal of phospholipids from the enzyme preparation. Furthermore, binding of α-tocopherol to succinate-cytochrome c reductase decreased the rate of reduction of cytochrome b by succinate. Since electron transfer from succinate to ubiquinone was not affected by α-tocopherol treatment, the decrease in reduction rate of cytochrome b by succinate must be due to a change in environment around cytochrome b. These results as well as the fact that reactivation of α-tocopherol-inhibited enzyme requires only low concentrations of ubiquinone were used to explain the inhibitory effect as a result of a change in protein conformation and protein-phospholipid interaction rather than the direct displacement of ubiquinone by α-tocopherol. This deduction was further supported by the fact that no ubiquinone was released from succinate-cytochrome c reductase upon treatment with α-tocopherol.     

Electron transfer between the two photosystems. II. Equilibrium constants

(1) The equilibrium constants for the redox reactions occurring between Photosystem (PS) I donors were measured on chloroplasts, dark-adapted in the presence of sodium ascorbate and 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU) and then illuminated by d.c. light. The equilibrium constant for the electron transfer between plastocyanin and P-700 is close to 1 and the overall equilibrium constant between cytochrome f and P-700 is about 2.3. As these equilibrium constants do not depend upon the intensity of the d.c. beam, the low values we measured cannot be due to kinetic limitations. (2) The equilibrium constants were measured also in the absence of DCMU using chloroplasts in oxidizing conditions (ferricyanide or far red illumination) illuminated by a saturating flash. During the course of the reduction of PS I donors by plastoquinol molecules formed by the flash, the equilibrium constants are higher than in the preceding conditions: the value for plastocyanin to P-700 is close to 5, and that for cytochrome f to P-700 is about 25. (3) The variations of these equilibrium constants are tentatively interpreted as being due to mutual electrostatic interactions between cytochrome b and f which are included in the same complex. This model implies that the perturbation of the redox properties of cytochrome f by a positive charge located on cytochrome b is identical to the perturbation of the redox properties of cytochrome b by a positive charge located on cytochrome f.     

Cytochrome distribution across chloroplast thylakoid membranes. Controlled proteolysis of inside-out and right-side-out vesicles

The transverse distribution of chloroplast cytochromes b-559 (high and low potentials), b-563 and f in pea thylakoid membranes was studied by the effects of trypsin and pronase on inside-out and right-side-out thylakoid vesicles. The high potential (HP) form of cytochrome b-559 was degraded to a low potential (LP) form most rapidly in right-side-out vesicles. In either type of vesicle there was no overall loss of the cytochrome from the membrane. This suggests that the haem group is buried in the membrane but that the cytochrome environment is most labile at the outer surface. Cytochrome b-563 was unaffected by trypsin and only slightly degraded by pronase in inverted vesicles. However, pronase caused the loss of an M_r 1000, non-haem fraction from the cytochrome f polypeptide in inside-out vesices only. The total cytochrome f content (measured spectrophotometrically and by staining polyacrylamide gels for haem associated peroxidase activity) decayed only slightly in either type of vesicle. These observations suggest that cytochrome f is, in part, exposed to the intrathylakoid lumen, whilst its haem group is retained in a more hydrophobic region.