Desiccation tolerant lichens facilitate in vivo H/D isotope effect measurements in oxygenic photosynthesis

© 2018 Elsevier B.V. We have used the desiccation-tolerant lichen Flavoparmelia caperata, containing the green algal photobiont Trebouxia gelatinosa, to examine H/D isotope effects in Photosystem II in vivo. Artifact-free H/D isotope effects on both PSII primary charge separation and water oxidation yields were determined as a function of flash rate from chlorophyll-a variable fluorescence yields. Intact lichens could be reversibly dehydrated/re-hydrated with H2O/D2O repeatedly without loss of O2 evolution, unlike all isolated PSII preparations. Above a threshold flash rate, PSII charge separation decreases sharply in both D2O and H2O, reflecting loss of excitation migration and capture by PSII. Changes in H/D coordinates further slow charge separation in D2O (−23% at 120 Hz), attributed to reoxidation of the primary acceptor QA−. At intermediate flash rates (5–50 Hz) D2O decreases water oxidation efficiency (O2 evolution) by −2–5%. No significant isotopic difference is observed at slow flash rates (\u3c5 \u3eHz) where charge recombination dominates. Slower D2O diffusion, changes in hydrogen bonding networks, and shifts in the pKa\u27s of ionizable residues may all contribute to these systematic variations of H/D isotope effects. Lichens’ reversible desiccation tolerance allows highly reproducible H/D exchange kinetics in PSII reactions to be studied in vivo for the first time

Oxidized quinones signal onset of darkness directly to the cyanobacterial circadian oscillator

Synchronization of the circadian clock in cyanobacteria with the day/night cycle proceeds without an obvious photoreceptor, leaving open the question of its specific mechanism. The circadian oscillator can be reconstituted in vitro,where the activities of two of its proteins, KaiA and KaiC, are affected by metabolites that reflect photosynthetic activity: KaiC phosphorylation is directly influenced by the ATP/ADP ratio, and KaiA stimulation of KaiC phosphorylation is blocked by oxidized, but not reduced, quinones. Manipulation of the ATP/ADP ratio can reset the timing of KaiC phosphorylation peaks in the reconstituted in vitro oscillator. Here, we show that pulses of oxidized quinones reset the cyanobacterial circadian clock both in vitro and in vivo. Onset of darkness causes an abrupt oxidation of the plastoquinone pool in vivo, which is in contrast to a gradual decrease in the ATP/ADP ratio that falls over the course of hours until the onset of light. Thus, these twometabolicmeasures of photosynthetic activity act in concert to signal both the onset and duration of darkness to the cyanobacterial clock

Natural isoforms of the Photosystem II D1 subunit differ in photoassembly efficiency of the water-oxidizing complex

© 2015 Springer Science+Business Media Dordrecht. Oxygenic photosynthesis efficiency at increasing solar flux is limited by light-induced damage (photoinhibition) of Photosystem II (PSII), primarily targeting the D1 reaction center subunit. Some cyanobacteria contain two natural isoforms of D1 that function better under low light (D1:1) or high light (D1:2). Herein, rates and yields of photoassembly of the Mn4CaO5 water-oxidizing complex (WOC) from the free inorganic cofactors (Mn2+, Ca2+, water, electron acceptor) and apo-WOC-PSII are shown to differ significantly: D1:1 apo-WOC-PSII exhibits a 2.3-fold faster rate-limiting step of photoassembly and up to seven-fold faster rate to the first light-stable Mn3+ intermediate, IM1, but with a much higher rate of photoinhibition than D1:2. Conversely, D1:2 apo-WOC-PSII assembles slower but has up to seven-fold higher yield, achieved by a higher quantum yield of charge separation and slower photoinhibition rate. These results confirm and extend previous observations of the two holoenzymes: D1:2-PSII has a greater quantum yield of primary charge separation, faster [P680+ Q A- ] charge recombination and less photoinhibition that results in a slower rate and higher yield of photoassembly of its apo-WOC-PSII complex. In contrast, D1:1-PSII has a lower quantum yield of primary charge separation, a slower [P680+ Q A- ] charge recombination rate, and faster photoinhibition that together result in higher rate but lower yield of photoassembly at higher light intensities. Cyanobacterial PSII reaction centers that contain the high- and low-light D1 isoforms can tailor performance to optimize photosynthesis at varying light conditions, with similar consequences on their photoassembly kinetics and yield. These different efficiencies of photoassembly versus photoinhibition impose differential costs for biosynthesis as a function of light intensity

Natural variants of photosystem II subunit D1 tune photochemical fitness to solar intensity

Background: Cyanobacteria use multiple PSII-D1 isoforms to adapt to environmental conditions. Results: D1:2 achieves higher quantum efficiency of water oxidation and biomass accumulation rate at high light versus D1:1; the latter is more efficient at low light due to less charge recombination. Conclusion: A functional advantage for D1:1 is revealed for the first time. Significance: Improved photochemical efficiency at low light suggests an evolutionary advantage to retain D1:1. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc

Heterologous Expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] Hydrogenases in Synechococcus elongatus

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H2 evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria

Competing charge transfer pathways at the photosystem II-electrode interface.

The integration of the water-oxidation enzyme photosystem II (PSII) into electrodes allows the electrons extracted from water oxidation to be harnessed for enzyme characterization and to drive novel endergonic reactions. However, PSII continues to underperform in integrated photoelectrochemical systems despite extensive optimization efforts. Here we carried out protein-film photoelectrochemistry using spinach and Thermosynechococcus elongatus PSII, and we identified a competing charge transfer pathway at the enzyme-electrode interface that short-circuits the known water-oxidation pathway. This undesirable pathway occurs as a result of photo-induced O2 reduction occurring at the chlorophyll pigments and is promoted by the embedment of PSII in an electron-conducting fullerene matrix, a common strategy for enzyme immobilization. Anaerobicity helps to recover the PSII photoresponse and unmasks the onset potentials relating to the QA/QB charge transfer process. These findings impart a fuller understanding of the charge transfer pathways within PSII and at photosystem-electrode interfaces, which will lead to more rational design of pigment-containing photoelectrodes in general.This work was supported by the U.K. Engineering and Physical Sciences Research Council (EP/H00338X/2 to E. Reisner), the U.K. Biology and Biotechnological Sciences Research Council (BB/K010220/1 to E. Reisner), a Marie Curie International Incoming Fellowship (PIIF-GA-2012-328085 RPSII to J.J.Z.). N.P. was supported by the Winton Fund for the Physics of Sustainability. E. Romero. and R.v.G. were supported by the VU University Amsterdam, the Laserlab-Europe Consortium, the TOP grant (700.58.305) from the Foundation of Chemical Sciences part of NWO, the Advanced Investigator grant (267333, PHOTPROT) from the European Research Council, and the EU FP7 project PAPETS (GA 323901). R.v.G. gratefully acknowledges his `Academy Professor' grant from the Royal Netherlands Academy of Arts and Sciences (KNAW). We would also like to thank Miss Katharina Brinkert and Prof A. William Rutherford for a sample of T. elongatus PSII, and H. v. Roon for preparation of the spinach PSII samples

Electrode kinetics in the system SmBaCo2O6–δ | Ce0.8Sm0.2O1.9 | SmBaCo2O6–δ

The polarization resistance dependences for the SmBaCo2O6-δ electrode in contact with the Ce0.8Sm0.2O1.9 electrolyte in the temperature range 600–700 °C and oxygen pressures of 2.3-520 kPa were obtained. Using the isotopic exchange and impedance spectroscopy data it was established that in the investigated ranges of T and pO2 the electrode process in the system is determined by three stages: oxygen exchange and diffusion in SmBaCo2O6-δ and oxygen diffusion over the gas phase.Получены зависимости поляризационного сопротивления для электрода SmBaCo2O6–δ в контакте с Ce0.8Sm0.2O1.9 электролите в интервале температур 600–700 °С и давлений кислорода 2,3–520 кПа. С помощью данных изотопного обмена и импедансной спектроскопии установлено, что в указанном интервале температур и рО2 электродный процесс в исследуемой системе определяется тремя стадиями: обмен и диффузия кислорода в SmBaCo2O6–δ и диффузия кислорода по газовой фазе