Anne Joliot

Cyclic electron transfer in plant leaf

The turnover of linear and cyclic electron flows has been determined in fragments of dark-adapted spinach leaf by measuring the kinetics of fluorescence yield and of the transmembrane electrical potential changes under saturating illumination. When Photosystem (PS) II is inhibited, a cyclic electron flow around PSI operates transiently at a rate close to the maximum turnover of photosynthesis. When PSII is active, the cyclic flow operates with a similar rate during the first seconds of illumination. The high efficiency of the cyclic pathway implies that the cyclic and the linear transfer chains are structurally isolated one from the other. We propose that the cyclic pathway operates within a supercomplex including one PSI, one cytochrome bf complex, one plastocyanin, and one ferredoxin. The cyclic process induces the synthesis of ATP needed for the activation of the Benson–Calvin cycle. A fraction of PSI (∼50%), not included in the supercomplexes, participates in the linear pathway. The illumination would induce a dissociation of the supercomplexes that progressively increases the fraction of PSI involved in the linear pathway.

Evidence for two active branches for electron transfer in photosystem I.

All photosynthetic reaction centers share a common structural theme. Two related, integral membrane polypeptides sequester electron transfer cofactors into two quasi-symmetrical branches, each of which incorporates a quinone. In type II reaction centers [photosystem (PS) II and proteobacterial reaction centers], electron transfer proceeds down only one of the branches, and the mobile quinone on the other branch is used as a terminal acceptor. PS I uses iron-sulfur clusters as terminal acceptors, and the quinone serves only as an intermediary in electron transfer. Much effort has been devoted to understanding the unidirectionality of electron transport in type II reaction centers, and it was widely thought that PS I would share this feature. We have tested this idea by examining in vivo kinetics of electron transfer from the quinone in mutant PS I reaction centers. This transfer is associated with two kinetic components, and we show that mutation of a residue near the quinone in one branch specifically affects the faster component, while the corresponding mutation in the other branch specifically affects the slower component. We conclude that both electron transfer branches in PS I are active.

Electron transfer between the two photosystems. I. Flash excitation under oxidizing conditions

The redox changes of cytochrome b-563 (cytochrome b), cytochrome f, plastocyanin and P-700 were measured on dark-adapted chloroplasts after illumination by a series of flashes in oxidizing conditions (0.1 mM ferricyanide). In these conditions, the plastoquinone pool is fully oxidized and the only available plastoquinol are those formed by Photosystem (PS) II reaction. According to the two-electron gate mechanism proposed by Bouges-Bocquet (Bouges-Bocquet, B. (1973) Biochim. Biophys. Acta 314, 250–256), plastoquinol is mainly formed after the second and the fourth flashes. After the second flash, the reoxidation of plastoquinol occurs by a concerted reaction which reduces most of the cytochrome b present and a fraction of PS I donors. Most of these electrons are stored on P-700, which implies a large equilibrium constant between the secondary PS I donors and P-700. One electron is stored on cytochrome b during a time (t12 ≈ 1 s) much longer than the dark interval between flashes. After the fourth flash, a new plastoquinol molecule is formed, which induces the reduction of PS I donors with no corresponding further reduction of cytochrome b. The number of electrons transferred after the fourth flash is larger than that transferred after the second flash although the rate of transfer is lower. To interpret these data, we assume that the plastoquinol formed after the fourth flash is reoxidized by a second concerted reaction: one electron is directly transferred to PS I donors while the other cooperates with the electron stored on cytochrome b to reduce a plastoquinone molecule localized on a site close to the outer face of the membrane. This newly formed plastoquinol crosses the membrane and transfers a second electron to PS I donors. This interpretation resembles a model proposed by Velthuys (Velthuys, B.R. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2765−2769) and which belongs to the modified Q-cycle class of models.

New evidence supporting energy transfer between photosynthetic units.

Abstract The non-exponential character of the fluorescence induction observed in presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea or at low temperature has been previously interpreted in terms of energy transfer between photosynthetic units. Alternative hypotheses have been recently proposed and this problem is discussed on the basis of new experimental results. Several independent methods are used to decrease the concentration of photoactive centers. If the inactive centres are blocked in a non-quenching form, one observes that the number of photons collected per active center increases up to a factor of 3 as the concentration of active centres decreases. However, if the inactive centres are blocked in a quenching form, the number of photons collected per active center remains independent of the concentration of the active centers. From the experiments described in this paper, one can conclude: 1. 1. Each center includes only one photoactive chlorophyll. 2. 2. Energy transfer occurs between three and probably more connected photosynthetic units. 3. 3. One must assume that the photosynthetic units are not identical. This heterogeneity may be due to their size or some structural features.

Etude de la photooxydation de l'hydroxylamine par les chloroplastes d'epinards

Abstract Studies on hydroxylamine photooxidation by spinach chloroplasts By the use of a modulated electrode, an amperometric signal different from the oxygen one has been detected in the presence of hydroxylamine. 1. 1. This signal has been ascribed to an oxidized form of hydroxylamine produced in the light. 2. 2. This photooxidation is sensitized by pigments of Photosystem II. 3. 3. The same photochemical centers are involved in this photooxidation and photolysis of water. 4. 4. Both reactions are associated to the same photosynthetic electron transfer reactions. 5. 5. Both reactions are 3-(3,4-dichlorophenyl)-1,1-dimethylurea sensitive. 6. 6. Contrary to oxygen evolving reaction, the formation of photooxidized hydroxylamine implies no activation phase and occurs in a one-quantum process.

Proton pumping and electron transfer in the cytochrome bf complex of algae

Abstract The electron-transfer reactions and the slow transmembrane electrogenic phase (phase b) have been studied in Photosystem-II-depleted mutant strains of Chlorella sorokiniana under anaerobic conditions. The dark reduction of cytochrome b previously oxidized by a strong illumination is a biphasic process. The first phase, completed in about 20 s, is associated with the reduction of the high-potential form of cytochrome b ; a slower phase, completed in about 10 min, is associated with the reduction of the low-potential form of cytochrome b . The α-bands of these two forms differ slightly. Under repetitive weak flash illumination, i.e., when a fraction of cytochrome b is oxidized prior to each flash, phase b is associated with minor changes in cytochrome b redox changes. In the presence of 2- n -nonyl-4-hydroxyquinoline N -oxide (NQNO), a large reduction of cytochrome b ( t 1 2 ≈ 2 ms ) is followed by a slow reoxidation. Under these conditions, phase b becomes biphasic, a first phase, insensitive to the inhibitor, is associated with cytochrome b reduction while the second phase, dependent upon the inhibitor concentration, is related to cytochrome b oxidation. These results are interpreted in terms of a modified Q-cycle model. A single weak flash given to dark-adapted algae, i.e., when the two b cytochromes are in their reduced state, induces a large cytochrome b oxidation with which is associated a phase b of longer duration but larger amplitude than the one observed under repetitive-flash illumination. Addition of 2- n -nonyl-4-hydroxyquinoline N -oxide inhibits neither the oxidation of cytochrome b nor phase b. These results suggest that a proton pump is coupled to the redox changes of plastoquinone occurring at a site, Z, close to the inner face of the membrane. This model implies that site Z is connected to the outer face of the thylakoid by a proton channel. Thus, depending upon the experimental conditions, two different mechanisms might be involved in the process of proton-pumping by the cytochrome b f complex.

The low-potential electron-transfer chain in the cytochrome bf complex

Abstract The oxidized-minus-reduced spectra of the low- and high-potential cytochromes b6 were measured from 380 to 590 nm under anaerobic conditions in a mutant of Chlorella sorokiniana lacking of Photosystem II (PS II). The two spectra are similar in the Soret band; in the α-band, cytochrome b1 and cytochrome bh peak at 563.4 and 564 nm, respectively. The spectrum of a third component, G, located on the outer face of the membrane and able to exchange electrons with cytochrome bh (Lavergne, J. (1983) Biochim. Biophys. Acta 725, 25–33) was characterized. The oxidized-minus-reduced spectrum of G displays a negative band peaking around 424 nm (probably a double peak) and a broad and small negative band in the green region. This spectrum resembles that of a soluble high-spin cytochrome c′, as characterized in several classes of photosynthetic bacteria. As other cytochromes c′, G binds CO with a low affinity, which inhibits electron exchange with cytochrome bh. The kinetics of reduction of cytochrome bh and cytochrome b1 were measured in algae under anaerobic conditions, following illumination by weak continuous light or saturating flashes. From the kinetic behaviour, the difference between the midpoint potentials of cytochrome bh and cytochrome b1 was estimated at approx. 140 mV. Two populations can be distinguished among cytochrome b f complexes. In a first fraction, the complexes are associated with the carrier G, which very likely takes part in an electron-transfer chain mediating cytochrome b reduction by a stromal reductant. This chain could be involved in cyclic electron flow around Photosystem I. In the second fraction, where G is not attached to the complex, the reduction of the b-cytochromes is a much slower process. Cytochrome bh and G are in equilibrium, the midpoint potential of G being approx. 20 mV higher than that of cytochrome bh. From the study of the PS II-driven photoreduction of cytochrome bh and G (under aerobic conditions) in a mutant which lacks of PS I, we conclude that the affinity of plastosemiquinone for site C is much higher than that of plastoquinol, which makes the properties of this site similar to those of PS II secondary acceptor QB. These results are discussed in terms of a model in which the semiquinone can rapidly move from site Z to site C.

Comparative study of the fluorescence yield and of the C550 absorption change at room temperature.

The C550 absorption change and the fluorescence yield were studied at room temperature in chloroplasts in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and under conditions in which contributions of P-700 and of the electrochromic effect were neglible. 1. The C550 difference spectrum is a typical band shift with an isobestic point close to 550 nm. 2. The maximum amplitude of C550 absorption change is reached upon the first flash of a series of saturating flashes, unlike the maximum fluorescence yield which is attained after several flashes. 3. The comparison of the induction curves of the C550 change and the fluorescence yield in weak light shows that the fluorescence yield is controlled by two quenchers: one of them (Q1), the redox state of which C550 is a probe, is responsible for the major part of the quenching; the other one (Q2), which is less concentrated and less efficient becomes predominant at the end of the fluorescence induction. 4. Quencher Q2 back-reacts faster than quencher Q1. 5. Two alternative models are discussed in which Q1 and Q2 belong either to the same Photosystem II center or to two different photocenters.

Knock-Out of the Genes Coding for the Rieske Protein and the ATP-Synthase δ-Subunit of Arabidopsis. Effects on Photosynthesis, Thylakoid Protein Composition, and Nuclear Chloroplast Gene Expression

In Arabidopsis, the nuclear genes PetC and AtpD code for the Rieske protein of the cytochrome b6/f (cyt b6/f) complex and the δ-subunit of the chloroplast ATP synthase (cpATPase), respectively. Knock-out alleles for each of these loci have been identified. Greenhouse-grown petc-2 and atpd-1 mutants are seedling lethal, whereas heterotrophically propagated plants display a high-chlorophyll (Chl)-fluorescence phenotype, indicating that the products of PetC and AtpD are essential for photosynthesis. Additional effects of the mutations in axenic culture include altered leaf coloration and increased photosensitivity. Lack of the Rieske protein affects the stability of cyt b6/f and influences the level of other thylakoid proteins, particularly those of photosystem II. In petc-2, linear electron flow is blocked, leading to an altered redox state of both the primary quinone acceptor QA in photosystem II and the reaction center Chl P700 in photosystem I. Absence of cpATPase-δ destabilizes the entire cpATPase complex, whereas residual accumulation of cyt b6/f and of the photosystems still allows linear electron flow. In atpd-1, the increase in non-photochemical quenching of Chl fluorescence and a higher de-epoxidation state of xanthophyll cycle pigments under low light is compatible with a slower dissipation of the transthylakoid proton gradient. Further and clear differences between the two mutations are evident when mRNA expression profiles of nucleus-encoded chloroplast proteins are considered, suggesting that the physiological states conditioned by the two mutations trigger different modes of plastid signaling and nuclear response.

Effect of low temperature (−30 to −60°C) on the reoxidation of the Photosystem II primary electron acceptor in the presence and absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea

Abstract The fluorescence yield has been measured on spinach chloroplasts at low temperature (−30 to −60°C) for various dark times following a short saturating flash. A decrease in the fluorescence yield linked to the reoxidation of the Photosystem II electron acceptor Q is still observed at −60°C. Two reactions participate in this reoxidation: a back reaction or charge recombination and the transfer of an electron from Q − to Pool A. The relative competition between these two reactions at low temperature depends upon the oxidation state of the donor side of the Photosystem II center: 1. (1) In dark-adapted chloroplasts (i.e. in States S 0 +S 1 according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C. 2. (2) When chloroplasts are placed in the S 2 +S 3 states by a two-flash preillumination at room temperature, the reoxidation of Q − after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics. 3. (3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O 2 molecule. 4. (4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.