Natural Variants of Photosystem II Subunit D1 Tune Photochemical Fitness to Solar Intensity

Photosystem II (PSII) is composed of six core polypeptides that make up the minimal unit capable of performing the primary photochemistry of light-driven charge separation and water oxidation in all oxygenic phototrophs. The D1 subunit of this complex contains most of the ligating amino acid residues for the Mn₄CaO₅ core of the water-oxidizing complex (WOC). Most cyanobacteria have 3–5 copies of the psbA gene coding for at least two isoforms of D1, whereas algae and plants have only one isoform. Synechococcus elongatus PCC 7942 contains two D1 isoforms; D1:1 is expressed under low light conditions, and D1:2 is up-regulated in high light or stress conditions. Using a heterologous psbA expression system in the green alga Chlamydomonas reinhardtii, we have measured growth rate, WOC cycle efficiency, and O₂ yield as a function of D1:1, D1:2, or the native algal D1 isoform. D1:1-PSII cells outcompete D1:2-PSII cells and accumulate more biomass in light-limiting conditions. However, D1:2-PSII cells easily outcompete D1:1-PSII cells at high light intensities. The native C. reinhardtii-PSII WOC cycles less efficiently at all light intensities and produces less O₂ than either cyanobacterial D1 isoform. D1:2-PSII makes more O₂ per saturating flash than D1:1-PSII, but it exhibits lower WOC cycling efficiency at low light intensities due to a 40% faster charge recombination rate in the S₃ state. These functional advantages of D1:1-PSII and D1:2-PSII at low and high light regimes, respectively, can be explained by differences in predicted redox potentials of PSII electron acceptors that control kinetic performance.     

How chloride functions to enable proton conduction in photosynthetic water oxidation: Time-resolved kinetics of intermediates (S-states) in vivo and bromide substitution

Chloride (Cl⁻) is essential for O₂ evolution during photosynthetic water oxidation. Two chlorides near the water-oxidizing complex (WOC) in Photosystem II (PSII) structures from Thermosynechococcus elongatus (and T. vulcanus) have been postulated to transfer protons generated from water oxidation. We monitored four criteria: primary charge separation flash yield (P* → P⁺Q_A⁻), rates of water oxidation steps (S-states), rate of proton evolution, and flash O₂ yield oscillations by measuring chlorophyll variable fluorescence (P* quenching), pH-sensitive dye changes, and oximetry. Br-substitution slows and destabilizes cellular growth, resulting from lower light-saturated O₂ evolution rate (−20 %) and proton release (−36 % ΔpH gradient). The latter implies less ATP production. In Br- cultures, protonogenic S-state transitions (S₂ → S₃ → S₀’) slow with increasing light intensity and during O₂/water exchange (S₀’ → S₀ → S₁), while the non-protonogenic S₁ → S₂ transition is kinetically unaffected. As flash rate increases in Cl⁻ cultures, both rate and extent of acidification of the lumen increase, while charge recombination is suppressed relative to Br⁻. The Cl⁻ advantage in rapid proton escape from the WOC to lumen is attributed to correlated ion-pair movement of H₃O⁺Cl⁻ in dry water channels vs. separated Br⁻ and H⁺ ion movement through different regions (>200-fold difference in Bronsted acidities). By contrast, at low flash rates a previously unreported reversal occurs that favors Br⁻ cultures for both proton evolution and less PSII charge recombination. In Br⁻ cultures, slower proton transfer rate is attributed to stronger ion-pairing of Br⁻ with AA residues lining the water channels. Both anions charge-neutralize protons and shepherd them to the lumen using dry aqueous channels.     

Molecular origin of the pH dependence of tyrosine D oxidation kinetics and radical stability in photosystem II

A role for redox-active tyrosines has been demonstrated in many important biological processes, including water oxidation carried out by photosystem II (PSII) of oxygenic photosynthesis. The rates of tyrosine oxidation and reduction and the Tyr/Tyr reduction potential are undoubtedly controlled by the immediate environment of the tyrosine, with the coupling of electron and proton transfer, a critical component of the kinetic and redox behavior. It has been demonstrated by Faller et al. that the rate of oxidation of tyrosine D (Tyr_D) at room temperature and the extent of Tyr_D oxidation at cryogenic temperatures, following flash excitation, dramatically increase as a function of pH with a pK_a of ≈ 7.6 [Faller et al. 2001 Proc. Natl. Acad. Sci. USA 98, 14368-14373; Faller et al. 2001 Biochemistry 41, 12914-12920]. In this work, we investigated, using FTIR difference spectroscopy, the mechanistic reasons behind this large pH dependence. These studies were carried out on Mn-depleted PSII core complexes isolated from Synechocystis sp. PCC 6803, WT unlabeled and labeled with ¹³C₆-, or ¹³C₁(4)-labeled tyrosine, as well as on the D2-Gln164Glu mutant. The main conclusions of this work are that the pH-induced changes involve the reduced Tyr_D state and not the oxidized Tyr_D state and that Tyr_D does not exist in the tyrosinate form between pH 6 and 10. We can also exclude a change in the protonation state of D2-His189 as being responsible for the large pH dependence of Tyr_D oxidation. Indeed, our data are consistent with D2-His189 being neutral both in the Tyr_D and Tyr_D states in the whole pH6-10 range. We show that the interactions between reduced Tyr_D and D2-His189 are modulated by the pH. At pH greater than 7.5, the ν(CO) mode frequency of Tyr_D indicates that Tyr_D is involved in a strong hydrogen bond, as a hydrogen bond donor only, in a fraction of the PSII centers. At pH below 7.5, the hydrogen-bonding interaction formed by Tyr_D is weaker and Tyr_D could be also involved as a hydrogen bond acceptor, according to calculations performed by Takahashi and Noguchi [J. Phys. Chem. B 2007 111, 13833-13844]. The involvement of Tyr_D in this strong hydrogen-bonding interaction correlates with the ability to oxidize Tyr_D at cryogenic temperatures and rapidly at room temperature. A strong hydrogen-bonding interaction is also observed at pH 6 in the D2-Gln164Glu mutant, showing that the residue at position D2-164 regulates the properties of Tyr_D. The IR data point to the role of a protonatable group(s) (with a pK_a of ≈ 7) other than D2-His189 and Tyr_D, in modifying the characteristics of the Tyr_D hydrogen-bonding interactions, and hence its oxidation properties. It remains to be determined whether the strong hydrogen-bonding interaction involves D2-His189 and if Tyr_D oxidation involves the same proton transfer route at low and at high pH.     

Action spectra of photosystems II and I and quantum yield of photosynthesis in leaves in State 1

The spectral global quantum yield (Y_II, electrons/photons absorbed) of photosystem II (PSII) was measured in sunflower leaves in State 1 using monochromatic light. The global quantum yield of PSI (Y_I) was measured using low-intensity monochromatic light flashes and the associated transmittance change at 810 nm. The 810-nm signal change was calibrated based on the number of electrons generated by PSII during the flash (4 · O₂ evolution) which arrived at the PSI donor side after a delay of 2 ms. The intrinsic quantum yield of PSI (y_I, electrons per photon absorbed by PSI) was measured at 712 nm, where photon absorption by PSII was small. The results were used to resolve the individual spectra of the excitation partitioning coefficients between PSI (a_I) and PSII (a_II) in leaves. For comparison, pigment–protein complexes for PSII and PSI were isolated, separated by sucrose density ultracentrifugation, and their optical density was measured. A good correlation was obtained for the spectral excitation partitioning coefficients measured by these different methods. The intrinsic yield of PSI was high (y_I = 0.88), but it absorbed only about 1/3 of quanta; consequently, about 2/3 of quanta were absorbed by PSII, but processed with the low intrinsic yield y_II = 0.63. In PSII, the quantum yield of charge separation was 0.89 as detected by variable fluorescence F_v/F_m, but 29% of separated charges recombined (Laisk A, Eichelmann H and Oja V, Photosynth. Res. 113, 145–155). At wavelengths less than 580 nm about 30% of excitation is absorbed by pigments poorly connected to either photosystem, most likely carotenoids bound in pigment–protein complexes.     

Perturbations at the chloride site during the photosynthetic oxygen-evolving cycle

Photosystem II (PSII) catalyzes the oxidation of water to O₂ at the manganese-containing, oxygen-evolving complex (OEC). Photoexcitation of PSII results in the oxidation of the OEC; four sequential oxidation reactions are required for the generation and release of molecular oxygen. Therefore, with flash illumination, the OEC cycles among five S _n states. Chloride depletion inhibits O₂ evolution. However, the binding site of chloride in the OEC is not known, and the role of chloride in oxygen evolution has not as yet been elucidated. We have employed reaction-induced FT-IR spectroscopy and selective flash excitation, which cycles PSII samples through the S state transitions. On the time scale employed, these FT-IR difference spectra reflect long-lived structural changes in the OEC. Bromide substitution supports oxygen evolution and was used to identify vibrational bands arising from structural changes at the chloride-binding site. Contributions to the vibrational spectrum from bromide-sensitive bands were observed on each flash. Sulfate treatment led to an elimination of oxygen evolution activity and of the FT-IR spectra assigned to the S₃ to S₀ (third flash) and S₀ to S₁ transitions (fourth flash). However, sulfate treatment changed, but did not eliminate, the FT-IR spectra obtained with the first and second flashes. Solvent isotope exchange in chloride-exchanged samples suggests flash-dependent structural changes, which alter protein dynamics during the S state cycle. Supported by NSF MCB 03-55421.     

Cationic state distribution over the chlorophyll d-containing PD1/PD2 pair in photosystem II

Most of the chlorophyll (Chl) cofactors in photosystem II (PSII) from Acaryochloris marina are Chld, although a few Chla molecules are also present. To evaluate the possibility that Chla may participate in the P_D1/P_D2 Chl pair in PSII from A. marina, the P_D1^?+/P_D2^?+ charge ratio was investigated using the PSII crystal structure analyzed at 1.9-Å resolution, while considering all possibilities for the Chld-containing P_D1/P_D2 pair, i.e., Chld/Chld, Chla/Chld, and Chld/Chla pairs. Chld/Chld and Chla/Chld pairs resulted in a large P_D1^?+ population relative to P_D2^?+, as identified in Chla/Chla homodimer pairs in PSII from other species, e.g., Thermosynechococcus elongatus PSII. However, the Chld/Chla pair possessed a P_D1^?+/P_D2^?+ ratio of approximately 50/50, which is in contrast to previous spectroscopic studies on A. marina PSII. The present results strongly exclude the possibility that the Chld/Chla pair serves as P_D1/P_D2 in A. marina PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

D1-S169A substitution of photosystem II reveals a novel S2-state structure

In photosystem II (PSII), photosynthetic water oxidation occurs at the O₂-evolving complex (OEC), a tetramanganese-calcium cluster that cycles through light-induced redox intermediates (S₀–S₄) to produce oxygen from two substrate water molecules. The OEC is surrounded by a hydrogen-bonded network of amino-acid residues that plays a crucial role in proton transfer and substrate water delivery. Previously, we found that D1-S169 was crucial for water oxidation and its mutation to alanine perturbed the hydrogen-bonding network. In this study, we demonstrate that the activation energy for the S₂ to S₁ transition of D1-S169A PSII is higher than wild-type PSII with a ~1.7–2.7× slower rate of charge recombination with Q_A⁻ relative to wild-type PSII. Arrhenius analysis of the decay kinetics shows an E_a of 5.87 ± 1.15 kcal mol⁻¹ for decay back to the S₁ state, compared to 0.80 ± 0.13 kcal mol⁻¹ for the wild-type S₂ state. In addition, we find that ammonia does not affect the S₂-state EPR signal, indicating that ammonia does not bind to the Mn cluster in D1-S169A PSII. Finally, a QM/MM analysis indicates that an additional water molecule binds to the Mn4 ion in place of an oxo ligand O5 in the S₂ state of D1-S169A PSII. The altered S₂ state of D1-S169A PSII provides insight into the S₂➔S₃ state transition.     

Lipids in photosystem II: Interactions with protein and cofactors

Photosystem II (PSII) is a homodimeric protein-cofactor complex embedded in the thylakoid membrane that catalyses light-driven charge separation accompanied by the oxidation of water during oxygenic photosynthesis. Biochemical analysis of the lipid content of PSII indicates a number of integral lipids, their composition being similar to the average lipid composition of the thylakoid membrane. The crystal structure of PSII at 3.0 Å resolution allowed for the first time the assignment of 14 integral lipids within the protein scaffold, all of them being located at the interface of different protein subunits. The reaction centre subunits D1 and D2 are encircled by a belt of 11 lipids providing a flexible environment for the exchange of D1. Three lipids are located in the dimerization interface and mediate interactions between the PSII monomers. Several lipids are located close to the binding pocket of the mobile plastoquinone Q_B, forming part of a postulated diffusion pathway for plastoquinone. Furthermore two lipids were found, each ligating one antenna chlorophyll a. A detailed analysis of lipid-protein and lipid-cofactor interactions allows to derive some general principles of lipid binding pockets in PSII and to suggest possible functional properties of the various identified lipid molecules.     

Isolation and characterization of oxygen-evolving thylakoid membranes and Photosystem II particles from a marine diatom Chaetoceros gracilis

Thylakoid membranes retaining high oxygen-evolving activity (about 250 μmol O₂/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 μmol O₂/mg Chl/h in the absence and presence of CaCl₂, respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein.     

Using site-directed mutagenesis to probe the role of the D2 carotenoid in the secondary electron-transfer pathway of photosystem II

Secondary electron transfer in photosystem II (PSII), which occurs when water oxidation is inhibited, involves redox-active carotenoids (Car), as well as chlorophylls (Chl), and cytochrome b ₅₅₉ (Cyt b ₅₅₉), and is believed to play a role in photoprotection. Car_D2 may be the initial point of secondary electron transfer because it is the closest cofactor to both P₆₈₀, the initial oxidant, and to Cyt b ₅₅₉, the terminal secondary electron donor within PSII. In order to characterize the role of Car_D2 and to determine the effects of perturbing Car_D2 on both the electron-transfer events and on the identity of the redox-active cofactors, it is necessary to vary the properties of Car_D2 selectively without affecting the ten other Car per PSII. To this end, site-directed mutations around the binding pocket of Car_D2 (D2-G47W, D2-G47F, and D2-T50F) have been generated in Synechocystis sp. PCC 6803. Characterization by near-IR and EPR spectroscopy provides the first experimental evidence that Car_D2 is one of the redox-active carotenoids in PSII. There is a specific perturbation of the Car^?+ near-IR spectrum in all three mutated PSII samples, allowing the assignment of the spectral signature of Car _D2 ^?+ ; Car _D2 ^?+ exhibits a near-IR peak at 980 nm and is the predominant secondary donor oxidized in a charge separation at low temperature in ferricyanide-treated wild-type PSII. The yield of secondary donor radicals is substantially decreased in PSII complexes isolated from each mutant. In addition, the kinetics of radical formation are altered in the mutated PSII samples. These results are consistent with oxidation of Car_D2 being the initial step in secondary electron transfer. Furthermore, normal light levels during mutant cell growth perturb the shape of the Chl^?+ near-IR absorption peak and generate a dark-stable radical observable in the EPR spectra, indicating a higher susceptibility to photodamage further linking the secondary electron-transfer pathway to photoprotection.     

Evidence for the Involvement of Loosely Bound Plastosemiquinones in Superoxide Anion Radical Production in Photosystem II

Recent evidence has indicated the presence of novel plastoquinone-binding sites, Q_C and Q_D, in photosystem II (PSII). Here, we investigated the potential involvement of loosely bound plastosemiquinones in superoxide anion radical (O₂^•−) formation in spinach PSII membranes using electron paramagnetic resonance (EPR) spin-trapping spectroscopy. Illumination of PSII membranes in the presence of the spin trap EMPO (5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide) resulted in the formation of O₂^•−, which was monitored by the appearance of EMPO-OOH adduct EPR signal. Addition of exogenous short-chain plastoquinone to PSII membranes markedly enhanced the EMPO-OOH adduct EPR signal. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, the EMPO-OOH adduct EPR signal was suppressed by 50% when the urea-type herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) was bound at the Q_B site. However, the EMPO-OOH adduct EPR signal was enhanced by binding of the phenolic-type herbicide dinoseb (2,4-dinitro-6-sec-butylphenol) at the Q_D site. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, DCMU and dinoseb inhibited photoreduction of the high-potential form of cytochrome b₅₅₉ (cyt b₅₅₉). Based on these results, we propose that O₂^•− is formed via the reduction of molecular oxygen by plastosemiquinones formed through one-electron reduction of plastoquinone at the Q_B site and one-electron oxidation of plastoquinol by cyt b₅₅₉ at the Q_C site. On the contrary, the involvement of a plastosemiquinone formed via the one-electron oxidation of plastoquinol by cyt b₅₅₉ at the Q_D site seems to be ambiguous. In spite of the fact that the existence of Q_C and Q_D sites is not generally accepted yet, the present study provided more spectroscopic data on the potential functional role of these new plastoquinone-binding sites.     

What can we still learn from the electrochromic band-shifts in Photosystem II?

Electrochromic band-shifts have been investigated in Photosystem II (PSII) from Thermosynechoccocus elongatus. Firstly, by using Mn-depleted PsbA1-PSII and PsbA3-PSII in which the Q_X absorption of Phe_D1 differs, a band-shift in the Q_X region of Phe_D2 centered at ~ 544 nm has been identified upon the oxidation, at pH 8.6, of Tyr_D. In contrast, a band-shift due to the formation of either Q_A^•- or Tyr_Z^• is observed in PsbA3-PSII at ~ 546 nm, as expected with E130 H-bonded to Phe_D1 and at ~ 544 nm as expected with Q130 H-bonded to Phe_D1. Secondly, electrochromic band-shifts in the Chla Soret region have been measured in O₂-evolving PSII in PsbA3-PSII, in the PsbA3/H198Q mutant in which the Soret band of P_D1 is blue shifted and in the PsbA3/T179H mutant. Upon Tyr_Z^•Q_A^•- formation the Soret band of P_D1 is red shifted and the Soret band of Chl_D1 is blue shifted. In contrast, only P_D1 undergoes a detectable S-state dependent electrochromism. Thirdly, the time resolved S-state dependent electrochromism attributed to P_D1 is biphasic for all the S-state transitions except for S₁ to S₂, and shows that: i) the proton release in S₀ to S₁ occurs after the electron transfer and ii) the proton release and the electron transfer kinetics in S₂ to S₃, in T. elongatus, are significantly faster than often considered. The nature of S₂Tyr_Z^• is discussed in view of the models in the literature involving intermediate states in the S₂ to S₃ transition.     

Photon- and carbon-use efficiency in<Emphasis Type="Italic"> Ulva rigida</Emphasis> at different CO<Subscript>2</Subscript> and N levels

The seaweed Ulva rigida C. Agardh (Chlorophyta) was cultured under two CO₂ conditions supplied through the air bubbling system: non-manipulated air and 1% CO₂-enriched aeration. These were also combined with N sufficiency and N limitation, using nitrate as the only N source. High CO₂ in U. rigida led to higher growth rates without increasing the C fixed through photosynthesis under N sufficiency. Quantum yields for charge separation at photosystem II (PSII) reaction centres (_PSII) and for oxygen evolution (_O2) decreased at high CO₂ even in N-sufficient thalli. Cyclic electron flow around PSII as part of a photoprotection strategy accompanied by decreased antennae size was suspected. The new re-arrangement of the photosynthetic energy at high CO₂ included reduced investment in processes other than C fixation, as well as in carbon diverted to respiration. As a result, quantum yield for new biomass-C production (^growth) increased. The calculation of the individual quantum yields for the different processes involved allowed the completion of the energy flow scheme through the cell from incident light to biomass production for each of the CO₂ and N-supply conditions studied.Abbreviations A total thallus absorptance - A_pig absorptance due to pigments - A_str Absorptance due to non-pigmented structures - a* spectrally averaged in vivo absorption cross-section of chlorophyll a - CCM carbon-concentrating mechanism - Chl chlorophyll - DOC dissolved organic carbon - ETR electron transport rate - F_v/F_m optimum quantum yield for PSII charge separation - GP gross O₂ evolution rate - k_pig specific light absorption coefficient for pigments - k_str specific light absorption coefficient for non-pigmented structures - OP optimum O₂ evolution rate - PFR photon fluence rate - POC particulate organic carbon - PS photosystem - q_N non-photochemical quenching - q_P photochemical quenching - ^growth quantum yield for new biomass-C production - _O2 quantum yield for gross O₂ evolution - _PSII quantum yield for PSII charge separation     

A seconds range component of the reoxidation of the primary Photosystem II acceptor,Q. Effects of bicarbonate depletion in chloroplasts

Broken chloroplasts depleted of bicarbonate (HCO^?₃) show 30–50% inhibition of the Hill reaction in low-intensity light. Also, photoreactions excited by repetitive flashes measured by oxygen evolution, ESR signal IIvf, and absorption changes at 680 and 334 nm show inhibition of 30–50%. An effect of HCO^?₃ was sought to explain these phenomena. The decay of chlorophyll a fluorescence yield in the millisecond and seconds range, following a single flash, was observed to be multiphasic with a very slow component of 1–2 s half-time. In HCO^?₃ -depleted samples this component is enhanced 2- or 3-fold. Since this occurs even after one flash, it is suggested that HCO^?₃ affects the Q^? B → QB^? reaction. In this work it is shown that 40% inhibition of oxygen flash yield is relieved to a great extent if the excitation flash rate is decreased from 2 to 0.33 Hz. A measurement of 520 nm absorption change in the presence of ferricyanide, which is proportional to Photosystem II charge separation, shows a similar inhibition that is dependent on flash rate. The maximum amplitude of variable fluorescence yield and 520 nm absorption change after a single flash are unaffected by HCO^?₃ depletion. The dark distribution of oxygen-evolution S-states is found to be shifted to a more reduced configuration in depleted samples. It is concluded that normal charge separation occurs in HCO^?₃ -depleted Photosystem II reaction centers but that a large fraction of Q^? decays so slowly that not all Q^? is reoxidized between flashes given at a rate of 1 or 2 Hz. Thus, a portion of the Photosystem II centers would be closed to photochemistry. There is a reversible effect of HCO^?₃ depletion on the oxygen-evolution system that is observed as a shift in the dark distribution of S-states.     

Probing the turnover efficiency of photosystem II membrane fragments with different electron acceptors

In this study we employ isotope ratio membrane-inlet mass spectrometry to probe the turnover efficiency of photosystem II (PSII) membrane fragments isolated from spinach at flash frequencies between 1Hz and 50Hz in the presence of the commonly used exogenous electron acceptors potassium ferricyanide(III) (FeCy), 2,5-dichloro-p-benzoquinone (DCBQ), and 2-phenyl-p-benzoquinone (PPBQ). The data obtained clearly indicate that among the tested acceptors PPBQ is the best at high flash frequencies. If present at high enough concentration, the PSII turnover efficiency is unaffected by flash frequency of up to 30Hz, and at 40Hz and 50Hz only a slight decrease by about 5-7% is observed. In contrast, drastic reductions of the O(2) yields by about 40% and 65% were found at 50Hz for DCBQ and FeCy, respectively. Comparison with literature data reveals that PPBQ accepts electrons from Q(A)(-) in PSII membrane fragments with similar efficiency as plastoquinone in intact cells. Our data also confirm that at high flashing rates O(2) evolution is limited by the reactions on the electron-acceptor side of PSII. The relevance of these data to the evolutionary development of the water-splitting complex in PSII and with regard to the potential of artificial water-splitting catalysts is discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Function of two β-carotenes near the D1 and D2 proteins in photosystem II dimers

The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 Å resolution), in which 11 β-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (E_m) of one-electron oxidation for all Car (E_m(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (Car_D1) in van der Waals contact with the chlorin ring of Chl_Z(D1). Each Car_D1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between Car_D1 and plastoquinone/Q_A may imply a direct charge recombination of Car⁺Q_A⁻. The calculated E_m(Car_D1) and E_m(Chl_Z(D1)) are, respectively, 83 and 126 mV higher than E_m(Car_D2) and E_m(Chl_Z(D2)), which could explain why Car_D2⁺ and Chl_Z(D2)⁺ are observed rather than the corresponding Car_D1⁺ and Chl_Z(D1)⁺.     

Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase

Cytochrome c oxidase is a multisubunit membrane-bound enzyme, which catalyzes oxidation of four molecules of cytochrome c²⁺ and reduction of molecular oxygen to water. The electrons are taken from one side of the membrane while the protons are taken from the other side. This topographical arrangement results in a charge separation that is equivalent to moving one positive charge across the membrane for each electron transferred to O₂. In this reaction part of the free energy available from O₂ reduction is conserved in the form of an electrochemical proton gradient. In addition, part of the free energy is used to pump on average one proton across the membrane per electron transferred to O₂. Our understanding of the molecular design of the machinery that couples O₂ reduction to proton pumping in oxidases has greatly benefited from studies of so called “uncoupled” structural variants of the oxidases. In these uncoupled oxidases the catalytic O₂-reduction reaction may display the same rates as in the wild-type CytcO, yet the electron/proton transfer to O₂ is not linked to proton pumping. One striking feature of all uncoupled variants studied to date is that the (apparent) pK_a of a Glu residue, located deeply within a proton pathway, is either increased or decreased (from 9.4 in the wild-type oxidase). The altered pK_a presumably reflects changes in the local structural environment of the residue and because the Glu residue is found near the catalytic site as well as near a putative exit pathway for pumped protons these changes are presumably important for controlling the rates and trajectories of the proton transfer. In this paper we summarize data obtained from studies of uncoupled structural oxidase variants and present a hypothesis that in quantitative terms offers a link between structural changes, modulation of the apparent pK_a and uncoupling of proton pumping from O₂ reduction.     

Formation of tyrosine radicals in photosystem II under far-red illumination

Photosystem II (PS II) contains two redox-active tyrosine residues on the donor side at symmetrical positions to the primary donor, P₆₈₀. Tyr_Z, part of the water-oxidizing complex, is a preferential fast electron donor while Tyr_D is a slow auxiliary donor to P₆₈₀ ⁺. We used PS II membranes from spinach which were depleted of the water oxidation complex (Mn-depleted PS II) to study electron donation from both tyrosines by time-resolved EPR spectroscopy under visible and far-red continuous light and laser flash illumination. Our results show that under both illumination regimes, oxidation of Tyr_D occurs via equilibrium with Tyr_Z ^? at pH 4.7 and 6.3. At pH 8.5 direct Tyr_D oxidation by P₆₈₀ ⁺ occurs in the majority of the PS II centers. Under continuous far-red light illumination these reactions were less effective but still possible. Different photochemical steps were considered to explain the far-red light-induced electron donation from tyrosines and localization of the primary electron hole (P₆₈₀ ⁺) on the Chl_D1 in Mn-depleted PS II after the far-red light-induced charge separation at room temperature is suggested.     

The involvement of lipids in light-induced charge separation in the reaction center of photosystem II

Extraction of PS II particles with 50 mM cholate and 1 M NaCl releases several proteins (33-, 23-, 17- and 13 kDa) and lipids from the thylakoid membrane which are essential for O₂ evolution, dichlorophenolindophenol (DCIP) reduction and for stable charge separation between P680⁺ and Q_A ^-. This work correlates the results on the loss of steady-state rates for O₂ evolution and PS II mediated DCIP photo-reduction with flash absorption changes directly monitoring the reaction center charge separation at 830 nm due to P680⁺, the chlorophyll a donor. Reconstitution of the extracted lipids to the depleted membrane restores the ability to photo-oxidize P680 reversibly and to reduce DCIP, while stimulating O₂ evolution minimally. Addition of the extracted proteins of masses 33-, 23- and 17- kDa produces no further stimulation of DCIP reduction in the presence of an exogenous donor like DPC, but does enhance this rate in the absence of exogenous donors while also stimulating O₂ evolution. The proteins alone in the absence of lipids have little influence on charge separation in the reaction center. Thus lipids are essential for stable charge separation within the reaction center, involving formation of P680⁺ and Q_A ^-.Abbreviations A₈₃₀ Absorption change at 830 nm - Chl Chlorophyll - D₁ primary electron donor to P680 - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - MOPS 3-(N-morpholino)propanesulfonic acid - P680 reaction center chlorophyll a molecule of photosystem II - PPBQ Phenyl-p-benzoquinone - PS II Photosystem II - Q_A, Q_B first and second quinone acceptors in PS II - V-DCIP rate of DCIP reduction - V-O₂ rate of oxygen evolution - Y water-oxidizing enzyme system - CHAPS 3-Cyclohexylamino-propanesulfonic acid