The structure and function of eukaryotic photosystem I

Eukaryotic photosystem I consists of two functional moieties: the photosystem I core, harboring the components for the light-driven charge separation and the subsequent electron transfer, and the peripheral light-harvesting complex (LHCI). While the photosystem I-core remained highly conserved throughout the evolution, with the exception of the oxidizing side of photosystem I, the LHCI complex shows a high degree of variability in size, subunits composition and bound pigments, which is due to the large variety of different habitats photosynthetic organisms dwell in. Besides summarizing the most current knowledge on the photosystem I-core structure, we will discuss the composition and structure of the LHCI complex from different eukaryotic organisms, both from the red and the green clade. Furthermore, mechanistic insights into electron transfer between the donor and acceptor side of photosystem I and its soluble electron transfer carrier proteins will be given. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.     

Modification of excitation energy distribution to photosystem I by protein phosphorylation and cation depletion during thylakoid biogenesis in wheat

The effects of protein phosphorylation and cation depletion on the electron transport rate and fluorescence emission characteristics of photosystem I at two stages of chloroplast development in light-grown wheat leaves are examined. The light-harvesting chlorophyll a/b protein complex associated with photosystem I (LHC I) was absent from the thylakoids at the early stage of development, but that associated with photosystem II (LHC II) was present. Protein phosphorylation produced an increase in the light-limited rate of photosystem I electron transport at the early stage of development when chlorophyll b was preferentially excited, indicating that LHC I is not required for transfer of excitation energy from phosphorylated LHC II to the core complex of photosystem I. However, no enhancement of photosystem I fluorescence at 77 K was observed at this stage of development, demonstrating that a strict relationship between excitation energy density in photosystem I pigment matrices and the long-wavelength fluorescence emission from photosystem I at 77 K does not exist. Depletion of Mg²⁺ from the thylakoids produced a stimulation of photosystem I electron transport at both stages of development, but a large enhancement of the photosystem I fluorescence emission was observed only in the thylakoids containing LHC I. It is suggested that the enhancement of PS I electron transport by Mg²⁺-depletion and phosphorylation of LHC II is associated with an enhancement of fluorescence at 77 K from LHC I and not from the core complex of PS I.     

Effect of cadmium on photosystem II activity in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: alteration of O–J–I–P fluorescence transients indicating the change of apparent activation energies within photosystem II

In this study, we evaluated how cadmium inhibitory effect on photosystem II and I electron transport may affect light energy conversion into electron transport by photosystem II. To induce cadmium effect on the photosynthetic apparatus, we exposed Chlamydomonas reinhardtii 24 h to 0–4.62 μM Cd²⁺. By evaluating the half time of fluorescence transients O–J–I–P at different temperatures (20–30°C), we were able to determine the photosystem II apparent activation energies for different reduction steps of photosystem II, indicated by the O–J–I–P fluorescence transients. The decrease of the apparent activation energies for PSII electron transport was found to be strongly related to the cadmium-induced inhibition of photosynthetic electron transport. We found a strong correlation between the photosystem II apparent activation energies and photosystem II oxygen evolution rate and photosystem I activity. Different levels of cadmium inhibition at photosystem II water-splitting system and photosystem I activity showed that photosystem II apparent activation energies are strongly dependent to photosystem II donor and acceptor sides. Therefore, the oxido-reduction state of whole photosystem II and I electron transport chain affects the conversion of light energy from antenna complex to photosystem II electron transport.     

Studies on thylakoid phosphorylation and noncyclic electron transport

The effect of thylakoid phosphorylation on noncyclic electron transport in spinach chloroplasts was investigated by measuring both the reduction of nicotinamide adenine dinucleotide phosphate (NADP) and the steady-state redox level of the primary electron acceptor quinone of photosystem II (Q) during electron flow to NADP. These data are compared with the theoretical predictions for an electron transport model which relates both the redox levels of Q and the photosystem II optical cross section to the overall velocity of noncyclic electron flow. It is demonstrated that transfer of 15-20% of the photosystem II antenna to photosystem I may stimulate electron flow to NADP only if Q is less than 60-70% oxidized (this condition exists with our thylakoids, even at extremely low absorption fluxes, when the illumination is not specifically enriched in photosystem I absorbed wavelengths); in phosphorylated thylakoids the steady-state redox level Q is substantially shifted to a more oxidized one (measurements of this parameter using light of different wavelengths quantitatively support the idea that thylakoid phosphorylation leads to increased photosystem I and decreased photosystem II cross sections); thylakoid phosphorylation leads to stimulated noncyclic electron flow to NADP only when the increased photosystem I antenna is able to bring about large increases in the steady-state level of oxidized Q.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP(+), and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700(+).     

Unexpected changes in photosystem I function in a cytochrome c6-deficient mutant of the cyanobacterium Synechocystis PCC 6803

Cytochrome c6, the product of the petJ gene, is a photosynthetic electron carrier in cyanobacteria, which transfers electrons to photosystem I and which is synthesised under conditions of copper deficiency to functionally replace plastocyanin. The photosystem I photochemical activity (energy storage, photoinduced P700 redox changes) was examined in a petJ-null mutant of Synechocystis PCC 6803. Surprisingly, photosystem I activity in the petJ-null mutant grown in the absence of copper was not much affected. However, in a medium with a low inorganic carbon concentration and with NH4+ ion as nitrogen source, the mutant displayed growth inhibition. Analysis showed that, especially in the latter, the isiAB operon, encoding flavodoxin and CP43', an additional chlorophyll a antenna, was strongly expressed in the mutant. These proteins are involved in photosystem I function and organisation and are proposed to assist in prevention of overoxidation of photosystem I at its lumenal side and overreduction at its stromal side.     

The location of plastocyanin in vascular plant photosystem I

We have studied the binding sites of the electron donor and acceptor proteins of vascular plant photosystem I by electron microscopy/crystallography. Previously, we identified the binding site for the electron acceptor (ferredoxin). In this paper we complete these studies with the characterization of the electron donor (plastocyanin) binding site. After cross-linking, plastocyanin is detected using Fourier difference analysis of two dimensionally ordered arrays of photosystem I located at the periphery of chloroplast grana. Plastocyanin binds in a small cavity on the lumenal surface of photosystem I, close to the center and with a slight bias toward the PsaL subunit of the complex. The recent release of the full coordinates for the cyanobacterial photosystem I reaction center has allowed a detailed comparison between the structures of the eukaryotic and prokaryotic systems. This reveals a very close homology, which is particularly striking for the lumenal side of photosystem I.     

Two molecular forms of pea ferredoxin in the electron transport chain of chloroplasts

The effects of two molecular forms of water-soluble ferredoxin (Fd I and Fd II) on the kinetics of electron transport in bean chloroplasts (class B) were studied. The light-induced redox transitions of the photosystem I reaction center P700 were measured by the intensity of the EPR signal I produced by P700+. Both forms of ferredoxin, Fd I and Fd II, when added to the chloroplasts in catalytic amounts, stimulate the light-induced electron transfer from P700 to NADP+. Nevertheless, Fd I is a better mediator of the back reactions from NADPH to P700+. This electron transfer pathway is sensitive to the cyclic electron transport inhibitor, antimycin A, and to DCMU inhibitor of electron transport between photosystem II and plastoquinone. It may be concluded that the two molecular forms of ferredoxin, Fd I and Fd II, differ in their ability to catalyze cyclic electron transport in photosystem I. The role of Fd I and Fd II in regulation of electron transport at the acceptor site of photosystem I is discussed.     

The thermodynamics and kinetics of electron transfer between cytochrome b6f and photosystem I in the chlorophyll d-dominated cyanobacterium, Acaryochloris marina

We have investigated the photosynthetic properties of Acaryochloris marina, a cyanobacterium distinguished by having a high level of chlorophyll d, which has its absorption bands shifted to the red when compared with chlorophyll a. Despite this unusual pigment content, the overall rate and thermodynamics of the photosynthetic electron flow are similar to those of chlorophyll a-containing species. The midpoint potential of both cytochrome f and the primary electron donor of photosystem I (P(740)) were found to be unchanged with respect to those prevailing in organisms having chlorophyll a, being 345 and 425 mV, respectively. Thus, contrary to previous reports (Hu, Q., Miyashita, H., Iwasaki, I. I., Kurano, N., Miyachi, S., Iwaki, M., and Itoh, S. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13319-13323), the midpoint potential of the electron donor P(740) has not been tuned to compensate for the decrease in excitonic energy in A. marina and to maintain the reducing power of photosystem I. We argue that this is a weaker constraint on the engineering of the oxygenic photosynthetic electron transfer chain than preserving the driving force for plastoquinol oxidation by P(740), via the cytochrome b(6)f complex. We further show that there is no restriction in the diffusion of the soluble electron carrier between cytochrome b(6)f and photosystem I in A. marina, at variance with plants. This difference probably reflects the simplified ultrastructure of the thylakoids of this organism, where no segregation into grana and stroma lamellae is observed. Nevertheless, chlorophyll fluorescence measurements suggest that there is energy transfer between adjacent photosystem II complexes but not from photosystem II to photosystem I, indicating spatial separation between the two photosystems.     

EPR study of electron transport in the cyanobacterium Synechocystis sp. PCC 6803: Oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains

In this work, we investigated electron transport processes in the cyanobacterium Synechocystis sp. PCC 6803, with a special emphasis focused on oxygen-dependent interrelations between photosynthetic and respiratory electron transport chains. Redox transients of the photosystem I primary donor P700 and oxygen exchange processes were measured by the EPR method under the same experimental conditions. To discriminate between the factors controlling electron flow through photosynthetic and respiratory electron transport chains, we compared the P700 redox transients and oxygen exchange processes in wild type cells and mutants with impaired photosystem II and terminal oxidases (CtaI, CydAB, CtaDEII). It was shown that the rates of electron flow through both photosynthetic and respiratory electron transport chains strongly depended on the transmembrane proton gradient and oxygen concentration in cell suspension. Electron transport through photosystem I was controlled by two main mechanisms: (i) oxygen-dependent acceleration of electron transfer from photosystem I to NADP⁺, and (ii) slowing down of electron flow between photosystem II and photosystem I governed by the intrathylakoid pH. Inhibitor analysis of P700 redox transients led us to the conclusion that electron fluxes from dehydrogenases and from cyclic electron transport pathway comprise 20-30% of the total electron flux from the intersystem electron transport chain to P700⁺.     

Ferredoxin and flavodoxin reduction by photosystem I.

Ferredoxin and flavodoxin are soluble proteins which are reduced by the terminal electron acceptors of photosystem I. The kinetics of ferredoxin (flavodoxin) photoreduction are discussed in detail, together with the last steps of intramolecular photosystem I electron transfer which precede ferredoxin (flavodoxin) reduction. The present knowledge concerning the photosystem I docking site for ferredoxin and flavodoxin is described in the second part of the review.     

Quantitation of the rapid electron donors to P700, the functional plastoquinone pool, and the ratio of the photosystems in spinach chloroplasts

Recent studies of chloroplast architecture have emphasized the segregation of photosystem I and photosystem II in different regions of the lamellar membrane. The apparent localization of photosystem II reaction centers in regions of membrane appression and of photosystem I reaction centers in regions exposed to the chloroplast stroma has focused attention on the intervening electron carriers, carriers which must be present to catalyze electron transfer between such spatially separated reaction sites. Information regarding the stoichiometries of these intermediate carriers is essential to an understanding of the processes that work together to establish the mechanism and to determine the rate of the overall process. We have reinvestigated the numbers of photosystem I and photosystem II reaction centers, the numbers of intervening cytochrome b6/f complexes, and the numbers of molecules of the relatively mobile electron carriers plastoquinone and plastocyanin that are actively involved in electron transfer. Our investigations were based on a new experimental technique made possible by the use of a modified indophenol dye, methyl purple, the reduction of which provides a particularly sensitive and accurate measure of electron transfer. Using this dye, which accepts electrons exclusively from photosystem I, it was possible to drain electrons from each of the carriers. Thus, by manipulation of the redox condition of the various carriers and through the use of specific inhibitors we could measure the electron storage capacity of each carrier in turn. We conclude that the ratio of photosystem I reaction centers to cytochrome b6/f complexes to photosystem II reaction centers is very nearly 1:1:1. The pool of rapid donors of electrons to P700 includes not only the 2 reducing equivalents stored in the cytochrome b6/f complex but also those stored in slightly more than 2 molecules of plastocyanin per P700. More slowly available are the electrons from about 6 plastoquinol molecules per P700.     

Hydrogen Photoevolution Indicates an Increase in the Antenna Size of Photosystem I in Chlamydobotrys stellata during Transition from Autotrophic to Photoheterotrophic Nutrition

The changes in the light-harvesting antenna size of photosystem I were investigated in the green alga Chlamydobotrys stellata during transition from autotrophic to photoheterotrophic nutrition by measuring the light-saturation behavior of hydrogen evolution following single turnover flashes. It was found that during autotrophic-to-photoheterotrophic transition the antenna size of photosystem I increased from 180 to 250 chlorophyll. The chlorophyll (a + b)/P700 ratio decreased from 800 to 550. The electron transport of photosystem I measured from reduced 2,6-dichloro-phenolindophenol to methylviologen was accelerated 1.4 times. In the 77K fluorescence spectra, the photosystem II fluorescence yield was considerably lowered relative to the photosystem I fluorescence yield. It is suggested that the increased light-harvesting capacity and redistribution of absorbed excitation energy in favor of photosystem I is a response of photoheterotrophic algae to meet the ATP demand for acetate metabolism by efficient photosystem I cyclic electron transport when the noncyclic photophosphorylation is inhibited by CO₂ deficiency.     

A quantitative model of the domain structure of the photosynthetic membrane

A model is presented that gives a quantitative picture of the distribution of the photosynthetic components in the photosynthetic membrane of higher plants. A salient feature of the model is that most of the pigments are located in the grana where photosystem I and II carry out linear electron transport, whereas the stroma lamellae, which harbour <20% of the pigments, carry out photosystem-I-mediated cyclic electron transport. This arrangement derives from the observation that more pigments are associated with photosystem I, which therefore captures more quanta than photosystem II. The excess pigments associated with photosystem I are thought to be located in the stroma lamellae.     

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.     

Chelator-sensitive in chloroplast electron transport

The effect of various chelators (orthophenanthroline, bathophen-anthroline, bathophenanthroline sulfonate and bathocuproine) on electron transport of spinach chloroplasts has been studied by means of various photosystem I and II reactions. It was found that photosystem II has at least 3 chelator-sensitive sites, photosystem I from 3–4. An uncoupler-affected site was found in each photosystem. In addition, photosystem I had a stimulator site and a soak site. The soak site was sensitive to chelators only after a period of incubation with the chelator.     

Green plant photosystem I binds light-harvesting complex I on one side of the complex

We report a structural characterization by electron microscopy of green plant photosystem I solubilized by the mild detergent n-dodecyl-alpha-D-maltoside. It is shown by immunoblotting that the isolated complexes contain all photosystem I core proteins and all peripheral light-harvesting proteins. The electron microscopic analysis is based on a large data set of 14 000 negatively stained single-particle projections and reveals that most of the complexes are oval-shaped monomers. The monomers have a tendency to associate into artificial dimers, trimers, and tetramers in which the monomers are oppositely oriented. Classification of the dimeric complexes suggests that some of the monomers lack a part of the peripheral antenna. On the basis of a comparison with projections from trimeric photosystem I complexes from cyanobacteria, we conclude that light-harvesting complex I only binds to the core complex at the side of the photosystem I F/J subunits and does not cause structural hindrances for the type of trimerization observed in cyanobacterial photosystem I.     

Functional implications on the mechanism of the function of photosystem II including water oxidation based on the structure of photosystem II

The structure of photosystem I at 3.8 A resolution illustrated the main structural elements of the water-oxidizing photosystem II complex, including the constituents of the electron transport chain. The location of the Mn cluster within the complex has been identified for the first time to our knowledge. At this resolution, no individual atoms are visible, however, the electron density of the Mn cluster can be used to discuss both the present models of the Mn cluster as revealed from various spectroscopic methods and the implications for the mechanisms of water oxidation. Twenty-six chlorophylls from the antenna system of photosystem II have been identified. They are arranged in two layers, one close to the stromal side and one close to the lumenal side. Comparing the structure of the antenna system of photosystem II with the chlorophyll arrangement in photosystem I, which was recently determined at 2.5 A resolution shows that photosystem II lacks the central domain of the photosystem I antenna, which is discussed in respect of the repair cycle of photosystem II due to photoinhibition.     

Loss of phylloquinone in Chlamydomonas affects plastoquinone pool size and photosystem II synthesis

Phylloquinone functions as the electron transfer cofactor at the A(1) site of photosystem I. We have isolated and characterized a mutant of Chlamydomonas reinhardtii, menD1, that is deficient in MenD, which encodes 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase, an enzyme that catalyzes the first specific step of the phylloquinone biosynthetic pathway. The mutant is photosynthetically active but light-sensitive. Analysis of total pigments by mass spectrometry reveals that phylloquinone is absent in menD1, but plastoquinone levels are not affected. This is further confirmed by the rescue of menD1 by addition of phylloquinone to the growth medium. Analysis of electron transfer by absorption spectroscopy indicates that plastoquinone replaces phylloquinone in photosystem I and that electron transfer from A(1) to the iron-sulfur centers is slowed down at least 40-fold. Consistent with a replacement of phylloquinone by plastoquinone, the size of the free plastoquinone pool of menD1 is reduced by 20-30%. In contrast to cyanobacterial MenD-deficient mutants, photosystem I accumulates normally in menD1, whereas the level of photosystem II declines. This decrease is because of reduced synthesis of the photosystem II core subunits. The relationship between plastoquinone occupancy of the A(1) site in photosystem I and the reduced accumulation of photosystem II is discussed.     

A laser flash-induced kinetic analysis of in vivo photosystem I reduction by site-directed mutants of plastocyanin and cytochrome c6 in Synechocystis sp. PCC 6803

In cyanobacteria, plastocyanin and cytochrome c6 are two soluble metalloproteins which can alternately serve as electron donors to photosystem I. From site-directed mutagenesis studies in vitro, it is well-established that both hydrophobic and electrostatic forces are involved in the interaction between the donor proteins and photosystem I. Hence, two isofunctional areas, a hydrophobic one in the north and an acidic one in the east, have been described on the surface of both electron donors. In this work, we have tested the relevance of such kinds of interactions in the photosystem I reduction inside the cell. Several plastocyanin and cytochrome c6 site-directed mutant strains affecting both the acidic and hydrophobic regions of the two metalloproteins, which were previously characterized in vitro, have been constructed. The photosystem I reduction kinetics of the different mutants have been analyzed by laser flash absorption spectroscopy. Relevant differences have been found between the in vitro and in vivo results, mainly regarding the role played by the electrostatic interactions. Adding positive electrostatic charges to the acidic patch of plastocyanin and cytochrome c6 promotes an enhanced interaction with photosystem I in vitro but yields the opposite effect in vivo. These discrepancies are discussed in view of the different environmental conditions, in vitro and in vivo, for the reaction mechanism of photosystem I reduction, namely, differential interaction of the electron donors with the thylakoidal membrane and kinetics of donor exchange.