23 research outputs found

    Dynamics of Proton Transfer to Internal Water during the Photosynthetic Oxygen-Evolving Cycle

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    In photosynthesis, the light-driven oxidation of water is a sustainable process, which converts solar to chemical energy and produces protons and oxygen. To enable biomimetic strategies, the mechanism of photosynthetic oxygen evolution must be elucidated. Here, we provide information concerning a critical step in the oxygen-evolving, or S-state, cycle. During this S<sub>3</sub>-to-S<sub>0</sub> transition, oxygen is produced, and substrate water binds to the manganese–calcium catalytic site. Our spectroscopic and H<sub>2</sub><sup>18</sup>O labeling experiments show that this S<sub>3</sub>-to-S<sub>0</sub> step is associated with the protonation of an internal water cluster in a hydrogen-bonding network, which contains calcium. When compared to the protonated water cluster, formed during a preceding step, the S<sub>1</sub>-to-S<sub>2</sub> transition, the S<sub>3</sub>-to-S<sub>0</sub> hydronium ion is likely to be coordinated by additional water molecules. This evidence shows that internal water and the hydrogen bonding network act as a transient proton acceptor at multiple points in the oxygen-evolving cycle

    Calcium and the Hydrogen-Bonded Water Network in the Photosynthetic Oxygen-Evolving Complex

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    In photosynthesis, photosystem II evolves oxygen from water at a Mn<sub>4</sub>CaO<sub>5</sub> cluster (OEC). Calcium is required for biological oxygen evolution. In the OEC, a water network, extending from the calcium to four peptide carbonyl groups, has recently been predicted by a high-resolution crystal structure. Here, we use carbonyl vibrational frequencies as reporters of electrostatic changes to test the presence of this water network. A single flash, oxidizing Mn­(III) to Mn­(IV) (the S<sub>1</sub> to S<sub>2</sub> transition), upshifted the frequencies of peptide CO bands. The spectral change was attributable to a decrease in CO hydrogen bonding. Strontium, which supports a lower level of steady state activity, also led to an oxidation-induced shift in CO frequencies, but treatment with barium and magnesium, which do not support activity, did not. This work provides evidence that calcium maintains an electrostatically responsive water network in the OEC and shows that OEC peptide carbonyl groups can be used as solvatochromic markers

    Redox-Induced Conformational Switching in Photosystem-II-Inspired Biomimetic Peptides: A UV Resonance Raman Study

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    Long-distance electron transfer (ET) plays a critical role in solar energy conversion, DNA synthesis, and mitochondrial respiration. Tyrosine (Y) side chains can function as intermediates in these reactions. The oxidized form of tyrosine deprotonates to form a neutral tyrosyl radical, Y<sup>•</sup>, a powerful oxidant. In photosystem II (PSII) and ribonucleotide reductase, redox-active tyrosines are involved in the proton-coupled electron transfer (PCET) reactions, which are key in catalysis. In these proteins, redox-linked structural dynamics may play a role in controlling the radical’s extraordinary oxidizing power. To define these dynamics in a structurally tractable system, we have constructed biomimetic peptide maquettes, which are inspired by PSII. UV resonance Raman studies were conducted of ET and PCET reactions in these β-hairpins, which contain a single tyrosine residue. At pH 11, UV photolysis induces ET from the deprotonated phenolate side chain to solvent. At pH 8.5, interstrand proton transfer to a π-stacked histidine accompanies the Y oxidation reaction. The UV resonance Raman difference spectrum, associated with Y oxidation, was obtained from the peptide maquettes in D<sub>2</sub>O buffers. The difference spectra exhibited bands at 1441 and 1472 cm<sup>–1</sup>, which are assigned to the amide II′ (CN) vibration of the β-hairpin. This amide II′ spectral change was attributed to substantial alterations in amide hydrogen bonding, which are coupled with the Y/Y<sup>•</sup> redox reaction and are reversible. These experiments show that ET and PCET reactions can create new minima in the protein conformational landscape. This work suggests that charge-coupled conformational changes can occur in complex proteins that contain redox-active tyrosines. These redox-linked dynamics could play an important role in control of PCET in biological oxygen evolution, respiration, and DNA synthesis

    Calcium, Strontium, and Protein Dynamics during the S<sub>2</sub> to S<sub>3</sub> Transition in the Photosynthetic Oxygen-Evolving Cycle

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    Photosystem II (PSII) catalyzes the oxidation of water at a Mn<sub>4</sub>CaO<sub>5</sub> cluster. The mechanism of water oxidation requires four sequential photooxidation events and cycles the OEC through the S<sub>0–4</sub> states. Oxygen is released during a thermal transition from S<sub>4</sub> to S<sub>0</sub>, and S<sub>1</sub> is the dark stable state. Calcium is required for activity, and, of substituted cations, only strontium supports activity but at a lower steady-state rate. The S<sub>1</sub> to S<sub>2</sub> transition corresponds to a Mn oxidation reaction. Previously, we used divalent ion substitution to provide evidence that calcium activates water and that an internal water cluster (W<sub>5</sub><sup>+</sup>) is protonated during the S<sub>1</sub> to S<sub>2</sub> transition. For the next transition, S<sub>2</sub> to S<sub>3</sub>, either a Mn or a ligand oxidation event has been proposed. Here, we use strontium reconstitution and reaction-induced FT-IR spectroscopy to study this transition. We show that strontium substitution has a dramatic effect on the infrared spectrum of the S<sub>2</sub> to S<sub>3</sub> transition, reducing the intensity of all spectral bands in the mid-infrared region (1600–1200 cm<sup>–1</sup>). However, the S<sub>3</sub> to S<sub>0</sub> and S<sub>0</sub> to S<sub>1</sub> spectra and the flash dependence of W<sub>5</sub><sup>+</sup> decay are not significantly altered in strontium PSII. The observed decrease in mid-infrared intensity is consistent with inhibition of a protein reorganization event, which may be associated with a strontium-induced change in S<sub>3</sub> charge distribution. These data provide evidence that strontium replacement alters the S<sub>2</sub> to S<sub>3</sub> conformational landscape

    Tracking Reactive Water and Hydrogen-Bonding Networks in Photosynthetic Oxygen Evolution

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    ConspectusIn oxygenic photosynthesis, photosystem II (PSII) converts water to molecular oxygen through four photodriven oxidation events at a Mn<sub>4</sub>CaO<sub>5</sub> cluster. A tyrosine, YZ (Y161 in the D1 polypeptide), transfers oxidizing equivalents from an oxidized, primary chlorophyll donor to the metal center. Calcium or its analogue, strontium, is required for activity. The Mn<sub>4</sub>CaO<sub>5</sub> cluster and YZ are predicted to be hydrogen bonded in a water-containing network, which involves amide carbonyl groups, amino acid side chains, and water. This hydrogen-bonded network includes amino acid residues in intrinsic and extrinsic subunits. One of the extrinsic subunits, PsbO, is intrinsically disordered. This extensive (35 Å) network may be essential in facilitating proton release from substrate water. While it is known that some proteins employ internal water molecules to catalyze reactions, there are relatively few methods that can be used to study the role of water. In this Account, we review spectroscopic evidence from our group supporting the conclusion that the PSII hydrogen-bonding network is dynamic and that water in the network plays a direct role in catalysis. Two approaches, transient electron paramagnetic resonance (EPR) and reaction-induced FT-IR (RIFT-IR) spectroscopies, were used. The EPR experiments focused on the decay kinetics of YZ• via recombination at 190 K and the solvent isotope, pH, and calcium dependence of these kinetics. The RIFT-IR experiments focused on shifts in amide carbonyl frequencies, induced by photo-oxidation of the metal cluster, and on the isotope-based assignment of bands to internal, small protonated water clusters at 190, 263, and 283 K. To conduct these experiments, PSII was prepared in selected steps along the catalytic pathway, the S<sub><i>n</i></sub> state cycle (<i>n</i> = 0–4). This cycle ultimately generates oxygen. In the EPR studies, S-state dependent changes were observed in the YZ• lifetime and in its solvent isotope effect. The YZ• lifetime depended on the presence of calcium at pH 7.5, but not at pH 6.0, suggesting a two-donor model for PCET. At pH 6.0 or 7.5, barium and ammonia both slowed the rate of YZ• recombination, consistent with disruption of the hydrogen-bonding network. In the RIFT-IR studies of the S state transitions, infrared bands associated with the transient protonation and deprotonation of internal waters were identified by D<sub>2</sub>O and H<sub>2</sub><sup>18</sup>O labeling. The infrared bands of these protonated water clusters, W<sub><i>n</i></sub><sup>+</sup> (or <i>n</i>H<sub>2</sub>O­(H<sub>3</sub>O)<sup>+</sup>, <i>n</i> = 5–6), exhibited flash dependence and were produced during the S<sub>1</sub> to S<sub>2</sub> and S<sub>3</sub> to S<sub>0</sub> transitions. Calcium dependence was observed at pH 7.5, but not at pH 6.0. S-state induced shifts were observed in amide CO frequencies during the S<sub>1</sub> to S<sub>2</sub> transition and attributed to alterations in hydrogen bonding, based on ammonia sensitivity. In addition, isotope editing of the extrinsic subunit, PsbO, established that amide vibrational bands of this lumenal subunit respond to the S state transitions and that PsbO is a structural template for the reaction center. Taken together, these spectroscopic results support the hypothesis that proton transfer networks, extending from YZ to PsbO, play a functional and dynamic role in photosynthetic oxygen evolution

    Light-Induced Oxidative Stress, <em>N</em>-Formylkynurenine, and Oxygenic Photosynthesis

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    <div><p>Light stress in plants results in damage to the water oxidizing reaction center, photosystem II (PSII). Redox signaling, through oxidative modification of amino acid side chains, has been proposed to participate in this process, but the oxidative signals have not yet been identified. Previously, we described an oxidative modification, <em>N</em>-formylkynurenine (NFK), of W365 in the CP43 subunit. The yield of this modification increases under light stress conditions, in parallel with the decrease in oxygen evolving activity. In this work, we show that this modification, NFK365-CP43, is present in thylakoid membranes and may be formed by reactive oxygen species produced at the Mn<sub>4</sub>CaO<sub>5</sub> cluster in the oxygen-evolving complex. NFK accumulation correlates with the extent of photoinhibition in PSII and thylakoid membranes. A modest increase in ionic strength inhibits NFK365-CP43 formation, and leads to accumulation of a new, light-induced NFK modification (NFK317) in the D1 polypeptide. Western analysis shows that D1 degradation and oligomerization occur under both sets of conditions. The NFK modifications in CP43 and D1 are found 17 and 14 Angstrom from the Mn<sub>4</sub>CaO<sub>5</sub> cluster, respectively. Based on these results, we propose that NFK is an oxidative modification that signals for damage and repair in PSII. The data suggest a two pathway model for light stress responses. These pathways involve differential, specific, oxidative modification of the CP43 or D1 polypeptides.</p> </div

    Optical absorption of NFK-containing PSII peptides (A) and the model compounds (B), tryptophan, NFK, and kynurenine.

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    <p>(A) shows absorption spectra of NFK-containing peptide fractions A–C. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#pone.0042220.s002" target="_blank">Table S1</a>, for average retention times from 350 nm chromatograms. Fraction A is displayed as a solid line, fraction B as a dashed line, and fraction C as a dotted line. In (B), absorption spectra of 40 µM tryptophan (solid line), 40 µM NFK (dotted line), and 40 µM kynurenine (dashed line) are shown in water. Absorption spectra in A were derived from the HPLC chromatogram and are on an arbitrary y-scale (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#s4" target="_blank">Materials and Methods</a>). The spectra in B were measured on a Hitachi spectrophotometer (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#s4" target="_blank">Materials and Methods</a>).</p

    Representative 350 nm HPLC chromatograms of oxygen-evolving PSII with and without 2 mM NaCl or TMA.

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    <p>In (A), (C), and (E), samples were incubated in the dark at room temperature for two hours (controls). In (B), (D), and (F), samples were illuminated with ∼7,000 µmol photons m<sup>−2</sup> s<sup>−1</sup> of white light for two hours at 25°C. In (C) and (D), 2 mM NaCl was added just prior to the dark or light incubation. In (E) and (F), 2 mM TMA was added just prior to the dark or light incubation. Fraction A is filled with horizontal stripes, fraction B has solid fill, and fraction C is filled with dots. The chromatograms are displaced on the y-axis for presentation purposes. The tick increments are 0.020 A.U. See Supporting Information for average retention times and summary of light-induced changes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#pone.0042220.s002" target="_blank">Table S1</a>). Fraction C corresponds to fraction 1 in ref <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#pone.0042220-Dreaden1" target="_blank">[19]</a>.</p

    Steady state rates of oxygen evolution of PSII membranes (A) and TM (B) during high light illumination and in the dark.

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    <p>In A, PSII membranes were kept in the dark at 25°C for two hours (blue). PSII membranes were exposed to a white light intensity of 500 (red) and 7,000 (green) µmol photons m<sup>−2</sup> s<sup>−1</sup> for two hours at 25°C. In B, TM were kept in the dark (black and red) or exposed to a white light intensity of 7,000 µmol photons m<sup>−2</sup> s<sup>−1</sup> at chlorophyll concentrations of 1.0 (blue) or 0.1 mg/ml (green). Oxygen evolution was assayed every 30 minutes and normalized to time zero. The data shown are an average of three to six experiments. The error bars are plus and minus one standard deviation. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042220#s4" target="_blank">Materials and Methods</a>, Photoinhibition, for experimental conditions.</p
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