Chlorophyll fluorescence emission by leaves of <i>Arabidopsis thaliana</i> at room temperature.

<p>Two days of light 1 treatment (before monitoring the room temperature variable chlorophyll fluorescence) are assiciated with an apparent state transition minus phenotype in CSK mutants. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026372#pone-0026372-g001" target="_blank">Figure 1a</a> shows the time-course of variable chlorophyll fluorescence emission from leaves of <i>Arabidopsis thaliana</i>, which were grown in light 1. Illumination with light 2, which is absorbed primarily by photosystem II, initially increases chlorophyll fluorescence emission. Fluorescence then decreases, and one component of the decrease is the removal of light-harvesting capacity from photosystem II during the transition to state 2. Addition of light 1, absorbed primarily by photosystem I, causes an initial decrease in fluorescence and then a slow rise, as the light-harvesting capacity of photosystem II increases during the transition to state 1. The slow components attributable to the state 2 and state 1 transitions are seen in the wild type, but are absent from the CSK mutant. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026372#pone-0026372-g001" target="_blank">Figure 1b</a> shows fluorescence emission from white light grown plants. Fm 1 and Fm 2 are maximal fluorescence at state 1 and state 2 respectively.</p

CSK mutants show normal LHC II phosphorylation.

<p>(a) Autoradiographs of <i>Arabidopsis</i> thylakoid phosphoproteins separated by SDS-PAGE. The positions of molecular weight markers are indicated on the left. Thylakoid samples from the wild-type (WT) and the CSK mutant are loaded in each lane and labelled accordingly at the top. The experimental conditions for each pair of samples are labelled at the bottom. (b) Coomassie-stained gel as protein loading control. The gel from which the autoradiograph was developed is stained with Coomassie brilliant blue to show that results presented in (a) results from ³²P-labelling and not from unequal protein loading.</p

Interactions of photosynthetic electron carriers with redox-signalling components of photosystem stoichiometry adjustment and state transitions.

<p>Light reactions of photosynthesis are represented as electron transport from H₂O to NADP⁺ via two photosystems connected by a cytochrome <i>b</i>₆<i>f</i> complex which oxidizes plastiquinol (PQH₂) to plastoquinone (PQ). CSK senses the redox state of the plastoquinone pool directly by becoming autophosphorylated and activated by PQ. CSK phosphorylation and dephosphorylation initiate transcription of PS II reaction centre (<i>psbA,D</i>) and PS I reaction centre (<i>psaA,B</i>) genes, respectively, selectively controlling expression of reaction centre genes in chloroplast DNA. The LHC II kinase Stn7 responds to PQH₂ and initiates the state 2 transition, while the phospho-LHC II phosphatase, TAP38/PPH1, is redox-independent and predominates, inducing the state 1 transition, when PQ is oxidized. Even though they are both controlled by plastoquinone redox state, CSK exerts its transcriptional effect on photosystem stoichiometry independently of the effect of Stn7 in state transitions.</p

Fluorescence emission spectra of isolated thylakoids at 77 K.

<p>77 K fluorescence emission spectra from wild type and CSK null mutant thylakoids as measured by the Perkin-Elmer LS 55 luminescence spectrometer. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026372#pone-0026372-g002" target="_blank">Figure 2a and 2c</a> shows fluorescence emission spectra of thylakoids isolated from light 1 adapted plants. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026372#pone-0026372-g002" target="_blank">Figure 2b and 2d</a> show fluorescence emission spectra of thylakoids isolated from white light grown plants. The excitation wavelength was 435 nm (5 nm slit width) and emission was detected from 650 to 800 nm (2.5 nm slit width). All spectra were normalized at 685 nm.</p

Iron Binding Properties of Recombinant Class A Protein Disulfide Isomerase from <i>Arabidopsis thaliana</i>

The protein disulfide isomerase (PDI) family comprises a wide set of enzymes mainly involved in thiol–disulfide exchange reactions in the endoplasmic reticulum. Class A PDIs (PDI-A) constitute the smallest members of the family, consisting of a single thioredoxin (TRX) module without any additional domains. To date, their catalytic activity and cellular function are still poorly understood. To gain insight into the role of higher-plant class A PDIs, the biochemical properties of r<i>At</i>PDI-A, the recombinant form of <i>Arabidopsis thaliana</i> PDI-A, have been investigated. As expressed, r<i>At</i>PDI-A has only little oxidoreductase activity, but it appears to be capable of binding an iron–sulfur (Fe–S) cluster, most likely a [2Fe-2S] center, at the interface between two protein monomers. A mutational survey of all cysteine residues of r<i>At</i>PDI-A indicates that only the second and third cysteines of the CXX­X­C­KHC stretch, containing the putative catalytic site CKHC, are primarily involved in cluster coordination. A key role is also played by the lysine residue. Its substitution with glycine, which restores the canonical PDI active site CGHC, does not influence the oxidoreductase activity of the protein, which remains marginal, but strongly affects the binding of the cluster. It is therefore proposed that the unexpected ability of r<i>At</i>PDI-A to accommodate an Fe–S cluster is due to its very unique CKHC motif, which is conserved in all higher-plant class A PDIs, differentiating them from all other members of the PDI family

Structure-Based Exciton Hamiltonian and Dynamics for the Reconstituted Wild-type CP29 Protein Antenna Complex of the Photosystem II

We provide an analysis of the pigment composition of reconstituted wild type CP29 complexes. The obtained stoichiometry of 9 ± 0.6 Chls <i>a</i> and 3 ± 0.6 Chls <i>b</i> per complex, with some possible heterogeneity in the carotenoid binding, is in agreement with 9 Chls <i>a</i> and 3.5 Chls <i>b</i> revealed by the modeling of low-temperature optical spectra. We find that ∼50% of Chl <i>b</i>614 is lost during the reconstitution/purification procedure, whereas Chls <i>a</i> are almost fully retained. The excitonic structure and the nature of the low-energy (low-E) state(s) are addressed via simulations (using Redfield theory) of 5 K absorption and fluorescence/nonresonant hole-burned (NRHB) spectra obtained at different excitation/burning conditions. We show that, depending on laser excitation frequency, reconstituted complexes display two (independent) low-E states (i.e., the A and B traps) with different NRHB and emission spectra. The red-shifted state A near 682.4 nm is assigned to a minor (∼10%) subpopulation (sub. II) that most likely originates from an imperfect local folding occurring during protein reconstitution. Its lowest energy state A (localized on Chl <i>a</i>604) is easily burned with λ_B = 488.0 nm and has a red-shifted fluorescence origin band near 683.7 nm that is not observed in native (isolated) complexes. Prolonged burning by 488.0 nm light reveals a second low-E trap at 680.2 nm (state B) with a fluorescence origin band at ∼681 nm, which is also observed when using a direct low-fluence excitation near 650 nm. The latter state is mostly delocalized over the <i>a</i>611, <i>a</i>612, <i>a</i>615 Chl trimer and corresponds to the lowest energy state of the major (∼90%) subpopulation (sub. I) that exhibits a lower hole-burning quantum yield. Thus, we suggest that major sub. I correspond to the native folding of CP29, whereas the red shift of the Chl <i>a</i>604 site energy observed in the minor sub. II occurs only in reconstituted complexes

Trapping Dynamics in Photosystem I‑Light Harvesting Complex I of Higher Plants Is Governed by the Competition Between Excited State Diffusion from Low Energy States and Photochemical Charge Separation

The dynamics of excited state equilibration and primary photochemical trapping have been investigated in the photosystem I-light harvesting complex I isolated from spinach, by the complementary time-resolved fluorescence and transient absorption approaches. The combined analysis of the experimental data indicates that the excited state decay is described by lifetimes in the ranges of 12–16 ps, 32–36 ps, and 64–77 ps, for both detection methods, whereas faster components, having lifetimes of 550–780 fs and 4.2–5.2 ps, are resolved only by transient absorption. A unified model capable of describing both the fluorescence and the absorption dynamics has been developed. From this model it appears that the majority of excited state equilibration between the bulk of the antenna pigments and the reaction center occurs in less than 2 ps, that the primary charge separated state is populated in ∼4 ps, and that the charge stabilization by electron transfer is completed in ∼70 ps. Energy equilibration dynamics associated with the long wavelength absorbing/emitting forms harbored by the PSI external antenna are also characterized by a time mean lifetime of ∼75 ps, thus overlapping with radical pair charge stabilization reactions. Even in the presence of a kinetic bottleneck for energy equilibration, the excited state dynamics are shown to be principally trap-limited. However, direct excitation of the low energy chlorophyll forms is predicted to lengthen significantly (∼2-folds) the average trapping time

Exploring the Electron Transfer Pathways in Photosystem I by High-Time-Resolution Electron Paramagnetic Resonance: Observation of the B-Side Radical Pair P₇₀₀⁺A_1B^– in Whole Cells of the Deuterated Green Alga Chlamydomonas reinhardtii at Cryogenic Temperatures

Crystallographic models of photosystem I (PS I) highlight a symmetrical arrangement of the electron transfer cofactors which are organized in two parallel branches (A, B) relative to a pseudo-<i>C</i>₂ symmetry axis that is perpendicular to the membrane plane. Here, we explore the electron transfer pathways of PS I in whole cells of the deuterated green alga Chlamydomonas reinhardtii using high-time-resolution electron paramagnetic resonance (EPR) at cryogenic temperatures. Particular emphasis is given to quantum oscillations detectable in the tertiary radical pairs P₇₀₀⁺A_1A^– and P₇₀₀⁺A_1B^– of the electron transfer chain. Results are presented first for the deuterated site-directed mutant PsaA-M684H in which electron transfer beyond the primary electron acceptor A_0A on the PsaA branch of electron transfer is impaired. Analysis of the quantum oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the geometry of the B-side radical pair. The orientation of the <b>g</b> tensor of P₇₀₀⁺ in an external reference system is adapted from a time-resolved multifrequency EPR study of deuterated and ¹⁵N-substituted cyanobacteria (Link, G.; Berthold, T.; Bechtold, M.; Weidner, J.-U.; Ohmes, E.; Tang, J.; Poluektov, O.; Utschig, L.; Schlesselman, S. L.; Thurnauer, M. C.; Kothe, G. <i>J. Am. Chem. Soc.</i> <b>2001</b>, <i>123</i>, 4211–4222). Thus, we obtain the three-dimensional structure of the B-side radical pair following photoexcitation of PS I in its native membrane. The new structure describes the position and orientation of the reduced B-side quinone A_1B^– on a nanosecond time scale after light-induced charge separation. Furthermore, we present results for deuterated wild-type cells of C. reinhardtii demonstrating that both radical pairs P₇₀₀⁺A_1A^– and P₇₀₀⁺A_1B^– participate in the electron transfer process according to a mole ratio of 0.71/0.29 in favor of P₇₀₀⁺A_1A^–. A detailed comparison reveals different orientations of A_1A^– and A_1B^– in their respective binding sites such that formation of a strong hydrogen bond from A₁^– to the protein backbone is possible only in the case of A_1A^–. We suggest that this is relevant to the rates of forward electron transfer from A_1A^– or A_1B^– to the iron–sulfur center F_X, which differ by a factor of 10. Thus, the present study sheds new light on the orientation of the phylloquinone acceptors in their binding pockets in PS I and the effect this has on function