The carboxyl-terminal region of the spinach PsaD subunit contains information for its specific assembly into plant thylakoids

The assembly of the multi-subunit membrane-protein Photosystem I (PS I) complex involves incorporation of peripheral proteins into the complex. Here we studied assembly of the PsaD subunit of the cyanobacterial and plant PS I into the thylakoid membranes. We generated partial and chimeric psaD genes from which labeled proteins were synthesized in vitro. Assembly of these proteins into the cyanobacterial or plant thylakoids was assayed. The deletion of leader sequence and N-terminal extension of spinach prePsaD did not inhibit its assembly into spinach or cyanobacterial thylakoids. Addition of these sequences to the cyanobacterial PsaD did not enable it to assemble into plant thylakoids. Moreover, these additions significantly decreased the ability of the chimeric proteins to assemble into cyanobacterial thylakoids. In contrast, when the carboxyl-terminal half of cyanobacterial PsaD was replaced by the corresponding region of the spinach PsaD, the chimeric protein could assemble into both spinach and cyanobacterial thylakoids. Therefore, information in the carboxyl-terminal region of spinach PsaD is crucial for its assembly into plant thylakoids.Abbreviation prePsaD precursor of the PsaD subunit of PS I     

Antenna function of a chlorophyll ab protein complex of Photosystem I

The functional role of a chlorophyll $\text{a}\text{b}$ complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, K_P-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll $\text{a}\text{b}$ complex and the other lacking the complex. K_P-700 for the former preparation is half of that of the preparation which has the chlorophyll $\text{a}\text{b}$ complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll $\text{a}\text{b}$ complex. Experiments involving reconstitution of the chlorophyll $\text{a}\text{b}$ complex with the antenna-depleted PS I preparation indicate a substantial recovery of the K_P-700 rate. These results demonstrate that the chlorophyll $\text{a}\text{b}$ complex functions as a light-harvesting antenna in PS I.     

Evidence that the low-potential (−700 mV) electron acceptor (X) in Photosystem I has two iron-sulphur centres

The Photosystem I reaction centre contains two groups of iron-sulphur centres: Fe-S_A and Fe-S_B with redox potentials between ?510 and ?590 mV, and Fe-S_X with redox potential about ?700 mV. Spin quantitation (Heathcote, P., Williams-Smith, D.L. and Evans, M.C.W. (1978) Biochem. J. 170, 373–378) and Mössbauer spectroscopy (Evans, E.H., Dickson, D.P.E., Johnson, C.E., Rush, J.D. and Evans, M.C.W. (1981) Eur. J. Biochem. 118, 81–84) did not show unequivocally whether Fe-S_X has one or two centres. Experiments are described which support the proposal that Fe-S_X has two centres. Fe-S_X can be photoreduced irreversibly by 210 K illumination of dithionite-reduced samples or reversibly by 7.5 K illumination of these samples. The amplitude of the Fe-S_X signal reversibly induced by illumination at 7.5 K is never more than 50% of the amplitude of the signal when Fe-S_X is prereduced by room temperature illumination or by 210 K illumination. Approx. half of the Fe-S_X is rapidly reduced by 210 K illumination, the remainder more slowly. The extent of reversible Fe-S_X reduction and P-700 photooxidation is little affected by the fast reduction of about half of the Fe-S_X. Subsequent reduction of the remaining Fe-S_X is paralleled by loss of the reversible photoreaction.     

Genetic variation in soybean photosynthetic electron transport capacity is related to plastocyanin concentration in the chloroplast

Fifteen ancestral genotypes of United States soybean cultivars were screened for differences in photosynthetic electron transport capacity using isolated thylakoid membranes. Plants were grown in controlled environment chambers under high or low irradiance conditions. Thylakoid membranes were isolated from mature leaves. Photosynthetic electron transport was assayed as uncoupled Hill activity using 2,6-dichlorophenolindophenol (DCIP). Soybean electron transport activity was dependent on genotype and growth irradiance and ranged from 6 to 91 mmol DCIP reduced [mol chlorophyll]^–1 s^–1. Soybean plastocyanin pool size ranged from 0.1 to 1.3 mol plastocyanin [mol Photosystem I]^–1. In contrast, barley and spinach electron transport activities were 140 and 170 mmol DCIP reduced [mol chlorophyll]^–1 s^–1, respectively, with plastocyanin pool sizes of 3 to 4 mol plastocyanin [mol Photosystem I]^–1. No significant differences in the concentrations of Photosystem II, plastoquinone, cytochrome b₆f complexes, or Photosystem I were observed. Thus, genetic differences in electron transport activity were correlated with plastocyanin pool size. The results suggested that plastocyanin pool size can vary significantly and may limit photosynthetic electron transport capacity in certain species such as soybean. Soybean plastocyanin consisted of two isoforms with apparent molecular masses of 14 and 11 kDa, whereas barley and spinach plastocyanins each consisted of single polypeptides of 8 and 12 kDa, respectively.Abbreviations DAP days after planting - DCIP 2,6-dichlorophenolindophenol - LiDS lithium dodecyl sulfate - PPFD photosynthetic photon flux density (mol photons m^–2 s^–1) - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.     

Effect of trypsin on D1/D2-cytochrom b 559 Photosystem 2 reaction center complex and reaction center from Rhodopseudomonas viridis

Proteolytic enzyme (trypsin) was used to structurally alter the RCs isolated from plant and bacterium as a way of probing the relation between structure (chromophore-apoprotein interactions) and function (photochemical activity). It was found that neither spectral characteristics (absorption spectrum, the 4th derivative of absorption spectrum) nor photochemical activity (pheophytine photoreduction, P680 photooxidation, etc.) were changed dramatically in D₁/D₂/cytochrom b ₅₅₉ PS 2 reaction center complex digested with trypsin. The PS 2 RC treated with trypsin migrates by one green band during electrophoresis with dodecylmaltoside. The peptides with a molecular mass higher than 3–4 kDa were not separated from PS 2 RC. These data indicate that digestion of D1 and D2 proteins does not disturb yet the conformation of peptides or their interactions in so-called core of RC and the native state of pigments. In contrast to that, the RC from Rhodopseudomonas viridis treated with enzyme has changed absorption spectrum and lost photochemical activity. The stability of the bacterial RC increased after exchange of LDAO by dodecylmaltoside.Abbreviations Chl chlorophyll a - Cyt cytochrome - DPC diphenylcarbazide - Dodecylmaltoside dodecyl--D-maltoside - LDAO lauryldimethylamino oxide - Pheo pheophytine - PS 2 Photosystem 2 - RC reaction center - SiMo silicomolybdate - SD sodium dodecyl sulfate     

Kok's oxygen clock: What makes it tick? The structure of P680 and consequences of its oxidizing power

New insights in the structure of P680, the primary electron donor in Photosystem II, are summarized and the implications of its oxidizing power for energy transfer and singlet oxygen production are discussed.Abbreviations BChl bacteriochlorophyll - Chl chlorophyll - LD linear dichroism - Pheo pheophytin - PS II Photosystem II     

Turtle hemoglobin: evidence of a T-state oxyhemoglobin

The 270 MHz ¹H NMR spectra of rabbit skeletal long and short S2 were indistinguishable at 20°C and 30°C and contained only a small proportion of sharp peaks associated with flexible regions. At 60°C both proteins were denatured and had essentially identical spectra. At 40°C and 50°C the long S2 spectrum contained a marginally greater proportion of sharp peaks, representing not more than 25 residues/chain. Our results are consistent with the presence of a small hinge in long S2 but do not support its containing an extensive region which provides contractile force by a helix—coil transition.     

On the origin of photosynthesis as inferred from sequence analysis

Sequence alignments between membrane-spanning segments of pheophytin-quinone-type photosynthetic reaction centers, FeS-type photosynthetic reaction centers, core chlorophyll-proteins of PS II, chlorophyll t a/t b-containing antenna proteins of plants and light-harvesting complexes of purple bacteria led us to postulate a large common ancestral pigment-carrying protein with more than 10 membrane spans. Its original function as a UV-protector of the primordial cell is discussed. It is conceivable that a purely dissipative photochemistry started still in the context of the UV-protection. We suggest that mutations causing the t loss of certain porphyrin-type pigments led to the acquisition of redox cofactors and paved the way for a gradual transition from dissipative to productive photochemistry.     

Structural comparison of cytochrome c2and cytochrome c6

Photosynthesis Research - This review compares the three-dimensional structures of the solublec-type cytochromes that functionally link membrane-bound energy transducingcomplexes in algal,...     

A routine method to determine the chlorophyll a,pheophytin a and β-carotene contents of isolated Photosystem II reaction center complexes

The most simple way in which the stoichiometry of chlorophyll a, pheophytin a and -carotene in isolated Photosystem II reaction center complexes can be determined is by analysis of the spectrum of the extracted pigments in 80% acetone. We present two different calculation methods using the extinction coefficients of the purified pigments in 80% acetone at different wavelengths. One of these methods also accounts for the possible presence of chlorophyll b. The results are compared with results obtained with HPLC pigment analysis, and indicate that these methods are suitable for routine determination of the pigment stoichiometry of isolated Photosystem II reaction center complexes.     

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.     

Acceptor side photoinhibition in PS II: On the possible effects of the functional integrity of the PS II donor side on photoinhibition of stable charge separation

Photoinhibition under aerobic and anaerobic conditions was analyzed in O₂-evolving and in Tris-treated PS II-membrane fragments from spinach by measuring laser-flash-induced absorption changes at 826 nm reflecting the transient P680⁺ formation and the chlorophyll fluorescence lifetime. It was found that anaerobic photoinhibitory treatment leads in both types of samples to the appearence of two long-lived fluorescence components with lifetimes of 7 ns and 16 ns, respectively. The extent of these fluorescence kinetics depends on the state of the reaction center (open/closed) during the fluorescence measurements: it is drastically higher in the closed state. It is concluded that this long-lived fluorescence is mainly emitted from modified reaction centers with singly reduced Q_A(Q_A ^-). This suggests that the observation of long-lived fluorescence components cannot necessarily be taken as an indicator for reaction centers with missing or doubly reduced and protonated Q_A (Q_AH₂). Time-resolved measurements of 826 nm absorption changes show that the rate of photoinhibition of the stable charge separation (P680^*Q_A P680⁺Q_A ^-), is nearly the same in O₂-evolving and in Tris-treated PS II-membrane fragments. This finding is difficult to understand within the framework of the Q_AH₂-mechanism for photoinhibition of stable charge separation because in that case the rate of photoinhibition should strongly depend on the functional integrity of the donor side of PS II. Based on the results of this study it is inferred, that several processes contribute to photoinhibition within the PS II reaction center and that a mechanism which comprises double reduction and protonation of Q_A leading to Q_AH₂ formation is only of marginal – if any – relevance for photoinhibition of PS II under both, aerobic and anaerobic, conditions.     

Phylogenetic Analyses of the Core Antenna Domain: Investigatingthe Origin of Photosystem I

Phototrophy, the conversion of light to biochemical energy, occurs throughout the Bacteria and plants, however, debate continues over how different phototrophic mechanisms and the bacteria that contain them are related. There are two types of phototrophic mechanisms in the Bacteria: reaction center type 1 (RC1) has core and core antenna domains that are parts of a single polypeptide, whereas reaction center type 2 (RC2) is composed of short core proteins without antenna domains. In cyanobacteria, RC2 is associated with separate core antenna proteins that are homologous to the core antenna domains of RC1. We reconstructed evolutionary relationships among phototrophic mechanisms based on a phylogeny of core antenna domains/proteins. Core antenna domains of 46 polypeptides were aligned, including the RC1 core proteins of heliobacteria, green sulfur bacteria, and photosystem I (PSI) of cyanobacteria and plastids, plus core antenna proteins of photosystem II (PSII) from cyanobacteria and plastids. Maximum likelihood, parsimony, and neighbor joining methods all supported a single phylogeny in which PSII core antenna proteins (PsbC, PsbB) arose within the cyanobacteria from duplications of the RC1-associated core antenna domains and accessory antenna proteins (IsiA, PcbA, PcbC) arose from duplications of PsbB. The data indicate an evolutionary history of RC1 in which an initially homodimeric reaction center was vertically transmitted to green sulfur bacteria, heliobacteria, and an ancestor of cyanobacteria. A heterodimeric RC1 (=PSI) then arose within the cyanobacterial lineage. In this scenario, the current diversity of core antenna domains/proteins is explained without a need to invoke horizontal transfer.This article contains online-only supplementary material.Reviewing Editor: Dr. W. Ford Doolittle     

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl₂ selectively destroys Fe–S center B (F_B). The function of electron acceptors in F_B-less PS I particles was studied by following the decay kinetics of P700⁺ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t _1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t _1/20.25 ms). In F_B-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in F_B-less particles, electron transfer can proceed normally at room temperature from F_X to F_A and that the charge recombination between P700⁺ and F_X ^-/A₁ ^- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of F_B. Even in the presence of 60% glycerol, F_B-less particles can transfer electrons to F_A at room temperature as efficiently as untreated particles.Abbreviations DCIP 2, 6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur center A, B and X, respectively - PMS phenazine methosulfate     

Temperature dependence of cytochrome photooxidation and conformational dynamics of Chromatium reaction center complexes

A temperature dependence of multiheme cytochrome c oxidation induced by a laser pulse was studied in photosynthetic reaction center preparations from Chromatium minutissimum. Absorbance changes and kinetic characteristics of the reaction were measured under redox conditions where one or all of the hemes of the cytochrome subunit are chemically reduced (E _h =+300 mV or E _h =–20 to -60 mV respectively). In the first case photooxidation is inhibited at temperatures lower than 190–200 K with the rate constant of the photooxidation reaction being practically independent on temperature over the range of 300 to 190 K (k=2.2×10⁵ s^-1). Under reductive conditions (E _h =–20 to -60 mV) lowering the temperature to 190–200 K causes the reaction to slow from k=8.3×10⁵ s^-1 to 2.1×10⁴ s^-1. Under further cooling down to the liquid nitrogen temperature, the reaction rate changes negligibly. The absorption amplitude decreases by 30–40% on lowering the temperature. A new physical mechanism of the observed critical effects of temperature on the rate and absorption amplitude of the multiheme cytochrome c oxidation reaction is proposed. The mechanism suggests a close interrelation between conformational mobility of the protein and elementary electron tunneling act. The effect of freezing conformational motion is described in terms of a local diffusion along a random rough potential.     

Perfusion chromatography—a new procedure for very rapid isolation of integral photosynthetic membrane proteins

The biochemical isolation of pure and active proteins or chlorophyll protein complexes has been crucial for elucidating the mechanism of photosynthetic energy conversion. Most of the proteins involved in this process are embedded in the photosynthetic membrane. The isolation of such hydrophobic integral membrane proteins is not trivial, and involves the use of detergents often combined with various time-consuming isolation procedures. We have applied the new procedure of perfusion chromatography for the rapid isolation of photosynthetic membrane proteins. Perfusion chromatography combines a highly reactive surface per bed volume with extremely high elution flow rates. We present an overview of this chromatographic method and show the rapid isolation of reaction centres from plant Photosystems I and II and photosynthetic purple bacteria, as well as the fractionation of the chlorophyll a/b-binding proteins of Photosystem I (LHC I). The isolation times have been drastically reduced compared to earlier approaches. The pronounced reduction in time for separation of photosynthetic complexes is convenient and permits purification of proteins in a more native state, including the maintainance of ligands and the possibility to isolate proteins trapped in intermediate metabolic or structural states.Abbreviations Chl chlorophyll - LDAO N,N dimethyldodecylamine-N-oxide - LHC light-harvesting complex - PS photosystem - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis