Computer simulation of interaction of photosystem 1 with plastocyanin and ferredoxin

We designed 3D multiparticle computer models to simulate diffusion and interactions of spinach plastocyanin and ferredoxin with plant photosystem 1 in a solution. Using these models we studied kinetic characteristics of plastocyanin-photosystem 1 and ferredoxin-photosystem 1 complex formation at a variety of ionic strength values. The computer multiparticle models demonstrate non-monotonic dependences of complex formation rates on the ionic strength as the result of long-range electrostatic interactions. Our calculations show that the decrease in the association second-order rate constant at low values of the ionic strength is caused by the protein pairs spending more time in “wrong” orientations which do not satisfy the docking conditions and so do not form the final complex capable of the electron transfer.     

Identification of the plastocyanin binding subunit of photosystem I

Plastocyanin is specifically cross-linked by incubation with N-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) to a subunit of photosystem I in stroma lamellae and in isolated photosystem I complex. SDS-PAGE shows the disappearance of a 18.5 kDa subunit and the appearance of a new 31.5 kDa protein which was recognized by anti-plastocyanin antibodies. The isolated subunit was identified by its N-terminal amino acid sequence as the mature peptide coded by the nuclear gene psaF [Steppuhn et al. (1988) FEBS Lett. 237, 218–224]. P700⁺ was reduced by cross-linked plastocyanin with the same halftime of 13 μs as found in the native complex. This is evidence that cross-linking conserved the orientation of the complex and that the 18.5 kDa subunit provides the conformation of photosystem I necessary for the extremely rapid electron transfer from plastocyanin to P700⁺.     

Photosystem I lacking the PSI-G subunit has a higher affinity for plastocyanin and is sensitive to photodamage

PSI-G is an 11 kDa subunit of PSI in photosynthetic eukaryotes. Arabidopsis thaliana plants devoid of PSI-G have a decreased PSI content and an increased activity of NADP⁺ photoreduction in vitro but otherwise no obvious phenotype [P.E. Jensen, L. Rosgaard, J. Knoetzel, H.V. Scheller, Photosystem I activity is increased in the absence of the PSI-G subunit. J. Biol. Chem. 277, (2002) 2798-2803.]. To investigate the biochemical basis for the increased activity, the kinetic parameters of the reaction between PSI and plastocyanin were determined. PSI-G clearly plays a role in the affinity for plastocyanin since the dissociation constant (K_D) is only 12 μM in the absence of PSI-G compared to 32 μM for the wild type. On the physiological level, plants devoid of PSI-G have a more reduced Q_A. This indicates that the decreased PSI content is due to unstable PSI rather than an adaptation to the increased activity. In agreement with this indication of decreased stability, plants devoid of PSI-G were found to be more photoinhibited both at low temperature and after high light treatment. The decreased PSI stability was confirmed in vitro by measuring PSI activity after illumination of a thylakoid suspension which clearly showed a faster decrease in PSI activity in the thylakoids lacking PSI-G. Light response of the P700 redox state in vivo showed that in the absence of PSI-G, P700 is more reduced at low light intensities. We conclude that PSI-G is involved in the binding dynamics of plastocyanin to PSI and that PSI-G is important for the stability of the PSI complex.     

PSI-O, a new 10-kDa subunit of eukaryotic photosystem I

A novel polypeptide with an apparent molecular mass of 9 kDa was detected after sodium dodecyl sulphate–polyacrylamide gel electrophoresis of Arabidopsis photosystem I (PSI) and was N-terminally sequenced. Corresponding cDNA clones encode a precursor protein of 140 amino acid residues which was imported into isolated intact chloroplasts and processed to the mature protein, designated PSI-O. The mature protein has two transmembrane helices and a calculated mass of 10 104 Da. The PSI-O protein was also shown to be present in PSI isolated from barley and spinach, and was essentially absent in chloroplast grana. Expressed sequences encoding similar proteins are available from many species of plants and green algae.     

Electrogenic reduction of the primary electron donor P700 by plastocyanin in photosystem I complexes

An electrometric technique was used to investigate electron transfer between spinach plastocyanin (Pc) and photooxidized primary electron donor P700 in photosystem I (PS I) complexes from the cyanobacterium Synechocystis sp. PCC 6803. In the presence of Pc, the fast unresolvable kinetic phase of membrane potential generation related to electron transfer between P700 and the terminal iron–sulfur acceptor F_B was followed by additional electrogenic phases in the microsecond and millisecond time scales, which contribute approximately 20% to the overall electrogenicity. These phases are attributed to the vectorial electron transfer from Pc to the protein-embedded chlorophyll dimer P700⁺ within the PsaA/PsaB heterodimer. The observed rate constant of the millisecond kinetic phase exhibited a saturation profile at increasing Pc concentration, suggesting the formation of a transient complex between Pc and PS I with the dissociation constant K_d of about 80 μM. A small but detectable fast electrogenic phase was observed at high Pc concentration. The rate constant of this phase was independent of Pc concentration, indicating that it is related to a first-order process.     

Binding of ferredoxin to algal photosystem I involves a single binding site and is composed of two thermodynamically distinct events

Despite the impressive progress made in recent years in understanding the early steps in charge separation within the photosynthetic reaction centers, our knowledge of how ferredoxin (Fd) interacts with the acceptor side of photosystem I (PSI) is not as well developed. Fd accepts electrons after transiently docking to a binding site on the acceptor side of PSI. However, the exact location, as well as the stoichiometry, of this binding have been a matter of debate for more than two decades. Here, using Isothermal Titration Calorimetry (ITC) and purified components from wild type and mutant strains of the green algae Chlamydomonas reinhardtii we show that PSI has a single binding site for Fd, and that the association consists of two distinct binding events, each with a specific association constant.     

Characterization of photosystem I antenna proteins in the prasinophyte Ostreococcus tauri

Prasinophyceae are a broad class of early-branching eukaryotic green algae. These picophytoplankton are found ubiquitously throughout the ocean and contribute considerably to global carbon-fixation. Ostreococcus tauri, as the first sequenced prasinophyte, is a model species for studying the functional evolution of light-harvesting systems in photosynthetic eukaryotes. In this study we isolated and characterized O. tauri pigment-protein complexes. Two photosystem I (PSI) fractions were obtained by sucrose density gradient centrifugation in addition to free light-harvesting complex (LHC) fraction and photosystem II (PSII) core fractions. The smaller PSI fraction contains the PSI core proteins, LHCI, which are conserved in all green plants, Lhcp1, a prasinophyte-specific LHC protein, and the minor, monomeric LHCII proteins CP26 and CP29. The larger PSI fraction contained the same antenna proteins as the smaller, with the addition of Lhca6 and Lhcp2, and a 30% larger absorption cross-section. When O. tauri was grown under high-light conditions, only the smaller PSI fraction was present. The two PSI preparations were also found to be devoid of the far-red chlorophyll fluorescence (715-730 nm), a signature of PSI in oxygenic phototrophs. These unique features of O. tauri PSI may reflect primitive light-harvesting systems in green plants and their adaptation to marine ecosystems. Possible implications for the evolution of the LHC-superfamily in photosynthetic eukaryotes are discussed.     

Structure and function of wild-type and subunit-depleted photosystem I in Synechocystis

The ability of photosynthetic organisms to use the sun's light as a sole source of energy sustains life on our planet. Photosystems I (PSI) and II (PSII) are large, multi-subunit, pigment–protein complexes that enable photosynthesis, but this intriguing process remains to be explained fully. Currently, crystal structures of these complexes are available for thermophilic prokaryotic cyanobacteria. The mega-Dalton trimeric PSI complex from thermophilic cyanobacterium, Thermosynechococcus elongatus, was solved at 2.5?Å resolution with X-ray crystallography. That structure revealed the positions of 12 protein subunits (PsaA-F, PsaI-M, and PsaX) and 127 cofactors.Although mesophilic organisms perform most of the world's photosynthesis, no well-resolved trimeric structure of a mesophilic organism exists. Our research model for a mesophilic cyanobacterium was Synechocystis sp. PCC6803. This study aimed to obtain well-resolved crystal structures of [1] a monomeric PSI with all subunits, [2] a trimeric PSI with a reduced number of subunits, and [3] the full, trimeric wild-type PSI complex. We only partially succeeded with the first two structures, but we successfully produced the trimeric PSI structure at 2.5?Å resolution. This structure was comparable to that of the thermophilic species, but we provided more detail. The PSI trimeric supercomplex consisted of 33 protein subunits, 72 carotenoids, 285 chlorophyll a molecules, 51 lipids, 9 iron-sulfur clusters, 6 plastoquinones, 6 putative calcium ions, and over 870 water molecules.This study showed that the structure of the PSI in Synechocystis sp. PCC6803 differed from previously described PSI structures. These findings have broadened our understanding of PSI structure.     

Evolution of photosystem I - from symmetry through pseudo-symmetry to asymmetry

The evolution of photosystem (PS) I was probably initiated by the formation of a homodimeric reaction center similar to the one currently present in green bacteria. Gene duplication has generated a heterodimeric reaction center that subsequently evolved to the PSI present in cyanobacteria, algae and plant chloroplasts. During the evolution of PSI several attempts to maximize the efficiency of light harvesting took place in the various organisms. In the Chlorobiaceae, chlorosomes and FMO were added to the homodimeric reaction center. In cyanobacteria phycobilisomes and CP43' evolved to cope with the light limitations and stress conditions. The plant PSI utilizes a modular arrangement of membrane light-harvesting proteins (LHCI). We obtained structural information from the two ends of the evolutionary spectrum. Novel features in the structure of Chlorobium tepidum FMO are reported in this communication. Our structure of plant PSI reveals that the addition of subunit G provided the template for LHCI binding, and the addition of subunit H prevented the possibility of trimer formation and provided a binding site for LHCII and the onset of energy spillover from PSII to PSI.     

O2 reduction by photosystem I involves phylloquinone under steady-state illumination

O₂ reduction was investigated in photosystem I (PS I) complexes isolated from cyanobacteria Synechocystis sp. PCC 6803 wild type (WT) and menB mutant strain, which is unable to synthesize phylloquinone and contains plastoquinone at the quinone-binding site A₁. PS I complexes from WT and menB mutant exhibited different dependencies of O₂ reduction on light intensity, namely, the values of O₂ reduction rate in WT did not reach saturation at high intensities, in contrast to the values in menB mutant. The obtained results suggest the immediate phylloquinone involvement in the light-induced O₂ reduction by PS I.     

Quenching in Arabidopsis thaliana mutants lacking monomeric antenna proteins of photosystem II

The minor light-harvesting complexes CP24, CP26, and CP29 have been proposed to play a key role in the zeaxanthin (Zx)-dependent high light-induced regulation (NPQ) of excitation energy in higher plants. To characterize the detailed roles of these minor complexes in NPQ and to determine their specific quenching effects we have studied the ultrafast fluorescence kinetics in knockout (ko) mutants koCP26, koCP29, and the double mutant koCP24/CP26. The data provide detailed insight into the quenching processes and the reorganization of the Photosystem (PS) II supercomplex under quenching conditions. All genotypes showed two NPQ quenching sites. Quenching site Q1 is formed by a light-induced functional detachment of parts of the PSII supercomplex and a pronounced quenching of the detached antenna parts. The antenna remaining bound to the PSII core was also quenched substantially in all genotypes under NPQ conditions (quenching site Q2) as compared with the dark-adapted state. The latter quenching was about equally strong in koCP26 and the koCP24/CP26 mutants as in the WT. Q2 quenching was substantially reduced, however, in koCP29 mutants suggesting a key role for CP29 in the total NPQ. The observed quenching effects in the knockout mutants are complicated by the fact that other minor antenna complexes do compensate in part for the lack of the CP24 and/or CP29 complexes. Their lack also causes some LHCII dissociation already in the dark.     

Charge recombination in photosystem I at low temperatures kinetics of electron tunneling

Absorption changes accompanying light-induced P-700 oxidation and the decay of P-700+ in the dark were measured in the temperature range 294-5 K over a broad time scale (three to four orders of magnitude). Two qualitatively different types of kinetics for the dark decay of P-700⁺ were observed. In the 294-240K region, a usual exponential kinetics is observed with the rate constant κ = 1 · 10¹⁰ · exp(-16 000/RT) s^?1, with R in cal/mol per degree. Below 220 K, a rather unusual logarithmic or near-logarithmic kinetics are observed. These kinetics can be explained quantitatively if one assumes for the various (P-700+ ··· X^-) pairs a broad rectangular or near-rectangular distribution over the values of the rate constant. The following kinetic equation corresponding to this model was obtained: n_t/n_o = [In(κ_max/κ_min)]-1 - [In(1/κ_min)? In t] where no and nt are respectively the initial concentration of P-700⁺ and its concentration at time t, and kmax and kmin the maximum and minimum values of the rate constant, respectively. The decay processes observed can be ascribed to electron tunneling. Distribution over the values of k can be accounted for by different environments or different mutual orientations of P-700⁺ and X^?, or by different distances between them in the various reacting pairs.The corresponding distribution function was reconstructed from the experimentally measured P-700+-decay curves. The rate of tunneling was found to be temperature dependent. In the 160-80-K region, the temperature dependence corresponds to an activation energy of 2.9 kcal/mol. Below 80 K, new modes of P-700+ decay with lower activation energy become operative. The tunneling distance for the majority of the (P-700+ ··· X^?) pairs was estimated from the EPR linewidth of P-700+ to exceed 13.2 A.     

Structure, function and regulation of plant photosystem I

Photosystem I (PSI) is a multisubunit protein complex located in the thylakoid membranes of green plants and algae, where it initiates one of the first steps of solar energy conversion by light-driven electron transport. In this review, we discuss recent progress on several topics related to the functioning of the PSI complex, like the protein composition of the complex in the plant Arabidopsis thaliana, the function of these subunits and the mechanism by which nuclear-encoded subunits can be inserted into or transported through the thylakoid membrane. Furthermore, the structure of the native PSI complex in several oxygenic photosynthetic organisms and the role of the chlorophylls and carotenoids in the antenna complexes in light harvesting and photoprotection are reviewed. The special role of the ‘red’ chlorophylls (chlorophyll molecules that absorb at longer wavelength than the primary electron donor P700) is assessed. The physiology and mechanism of the association of the major light-harvesting complex of photosystem II (LHCII) with PSI during short term adaptation to changes in light quality and quantity is discussed in functional and structural terms. The mechanism of excitation energy transfer between the chlorophylls and the mechanism of primary charge separation is outlined and discussed. Finally, a number of regulatory processes like acclimatory responses and retrograde signalling is reviewed with respect to function of the thylakoid membrane. We finish this review by shortly discussing the perspectives for future research on PSI.     

The Lhca antenna complexes of higher plants photosystem I

The Lhca antenna complexes of photosystem I (PSI) have been characterized by comparison of native and recombinant preparations. Eight Lhca polypeptides have been found to be all organized as dimers in the PSI-LHCI complex. The red emission fluorescence is associated not only with Lhca1-4 heterodimer, but also with dimers containing Lhca2 and/or Lhca3 complexes. Reconstitution of Lhca1 and Lhca4 monomers as well as of the Lhca1-4 dimer in vitro was obtained. The biochemical and spectroscopic features of these three complexes are reported. The monomers Lhca1 and Lhca4 bind 10 Chls each, while the Chl a/b ratio is lower in Lhca4 as compared to Lhca1. Three carotenoid binding sites have been found in Lhca1, while only two are present in Lhca4. Both complexes contain lutein and violaxanthin while β-carotene is selectively bound to the Lhca1-4 dimer in substoichiometric amounts upon dimerization. Spectral analysis revealed the presence of low energy absorption forms in Lhca1 previously thought to be exclusively associated with Lhca4. It is shown that the process of dimerization changes the spectroscopic properties of some chromophores and increases the amplitude of the red absorption tail of the complexes. The origin of these spectroscopic features is discussed.     

Fluorescence decay kinetics of mutants of corn deficient in photosystem I and photosystem II

The fast fluorescence decay kinetics of two photosynthetic mutants of corn (Zea mays) have been compared with those of normal corn. The fluorescence of normal corn can be resolved into three exponential decay components of lifetime 900–1500 ps (slow), 300–500 ps (middle) and 50–120 ps (fast), the yields of which are affected by light intensity and Mg²⁺ levels. The Photosystem II-(PS II)-defective mutant hcf-3 has similar decay lifetimes (approx. 1200, 450 and 100 ps) but is not affected by light intensity, reflecting the absence of PS II charge recombination. However, yields do respond to Mg²⁺ in a fashion typical of normal corn, which may be correlated with the presence of normal levels of light-harvesting chlorophyll a + b complex (LHCP). The PS I mutant hcf-50 also shows three-component decay kinetics. In conjunction with the results on the LHCP-deficient mutant of barley presented in a recent paper (Karukstis, K.K. and Sauer, K. (1984) Biochim. Biophys. Acta 766, 148–155), these data suggest that the slow component of normal chloroplasts is kinetically controlled by the decay processes of the LHCP and that the energy comes from one of two sources: (a) charge recombination in the reaction centre or (b) energy transferred within or between LHCP units only. The fast component appears to originate from both PS I and PS II. The complex response of the middle component to cations and light intensity, and its presence in all of the mutants, suggests that it also may have multiple origins.     

Association of chlorophyll a/c2 complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c₂ proteins were found. The most common complexes have Chl a/c₂ complexes at both sides of the PSI core monomer and have dimensions of about 17 × 24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c₂ light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c₂ proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c₂ proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

Energy equilibration and primary charge separation in chlorophyll d-based photosystem I reaction center isolated from Acaryochloris marina

Primary photochemistry in photosystem I (PS I) reaction center complex from Acaryochloris marina that uses chlorophyll d instead of chlorophyll a has been studied with a femtosecond spectroscopy. Upon excitation at 630 nm, almost full excitation equilibration among antenna chlorophylls and 40% of the excitation quenching by the reaction center are completed with time constants of 0.6(±0.1) and 4.9(±0.6) ps, respectively. The rise and decay of the primary charge-separated state proceed with apparent time constants of 7.2(±0.9) and 50(±10) ps, suggesting the reduction of the primary electron acceptor chlorophyll (A₀) and its reoxidation by phylloquinone (A₁), respectively.     

The photosystem I trimer of cyanobacteria: molecular organization, excitation dynamics and physiological significance

The photosystem I complex organized in cyanobacterial membranes preferentially in trimeric form participates in electron transport and is also involved in dissipation of excess energy thus protecting the complex against photodamage. A small number of longwave chlorophylls in the core antenna of photosystem I are not located in the close vicinity of P700, but at the periphery, and increase the absorption cross-section substantially. The picosecond fluorescence kinetics of trimers resolved the fastest energy transfer components reflecting the equilibration processes in the core antenna at different redox states of P700. Excitation kinetics in the photosystem I bulk antenna is nearly trap-limited, whereas excitation trapping from longwave chlorophyll pools is diffusion-limited and occurs via the bulk antenna. Charge separation in the photosystem I reaction center is the fastest of all known reaction centers.