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1. Impact of energy limitations on function and resilience in long-wavelength Photosystem II

Author

Stefania Viola, William Roseby, Stefano Santabarbara, Dennis Nürnberg, Ricardo Assunção, Holger Dau, Julien Sellés, Alain Boussac, Andrea Fantuzzi, and A William Rutherford

Subjects
photosystem II, cyanobacteria, photochemistry, Medicine, Science, Biology (General), QH301-705.5
Abstract

Photosystem II (PSII) uses the energy from red light to split water and reduce quinone, an energy-demanding process based on chlorophyll a (Chl-a) photochemistry. Two types of cyanobacterial PSII can use chlorophyll d (Chl-d) and chlorophyll f (Chl-f) to perform the same reactions using lower energy, far-red light. PSII from Acaryochloris marina has Chl-d replacing all but one of its 35 Chl-a, while PSII from Chroococcidiopsis thermalis, a facultative far-red species, has just 4 Chl-f and 1 Chl-d and 30 Chl-a. From bioenergetic considerations, the far-red PSII were predicted to lose photochemical efficiency and/or resilience to photodamage. Here, we compare enzyme turnover efficiency, forward electron transfer, back-reactions and photodamage in Chl-f-PSII, Chl-d-PSII, and Chl-a-PSII. We show that: (i) all types of PSII have a comparable efficiency in enzyme turnover; (ii) the modified energy gaps on the acceptor side of Chl-d-PSII favour recombination via PD1+Phe- repopulation, leading to increased singlet oxygen production and greater sensitivity to high-light damage compared to Chl-a-PSII and Chl-f-PSII; (iii) the acceptor-side energy gaps in Chl-f-PSII are tuned to avoid harmful back reactions, favouring resilience to photodamage over efficiency of light usage. The results are explained by the differences in the redox tuning of the electron transfer cofactors Phe and QA and in the number and layout of the chlorophylls that share the excitation energy with the primary electron donor. PSII has adapted to lower energy in two distinct ways, each appropriate for its specific environment but with different functional penalties.

Published
2022
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2. The Evolution and Evolvability of Photosystem II

Author

Thomas Oliver, Tom D. Kim, Joko P. Trinugroho, Violeta Cordón-Preciado, Nitara Wijayatilake, Aaryan Bhatia, A. William Rutherford, and Tanai Cardona

Subjects
photosynthesis, D1, water oxidation, Physiology, photosystem, Cell Biology, Plant Science, SDG 6 - Clean Water and Sanitation, cyanobacteria, Molecular Biology, origin of life
Abstract

Photosystem II is the water-oxidizing and O2-evolving enzyme of photosynthesis. How and when this remarkable enzyme arose are fundamental questions in the history of life that have remained difficult to answer. Here, recent advances in our understanding of the origin and evolution of photosystem II are reviewed and discussed in detail. The evolution of photosystem II indicates that water oxidation originated early in the history of life, long before the diversification of cyanobacteria and other major groups of prokaryotes, challenging and transforming current paradigms on the evolution of photosynthesis. We show that photosystem II has remained virtually unchanged for billions of years, and yet the nonstop duplication process of the D1 subunit of photosystem II, which controls photochemistry and catalysis, has enabled the enzyme to become adaptable to variable environmental conditions and even to innovate enzymatic functions beyond water oxidation. We suggest that this evolvability can be harnessed to develop novel light-powered enzymes with the capacity to carry out complex multistep oxidative transformations for sustainable biocatalysis.

Published
2023

3. Bicarbonate activation of the monomeric photosystem II-PsbS/Psb27 complex

Author

Andrea Fantuzzi, Patrycja Haniewicz, Domenica Farci, M Cecilia Loi, Keunha Park, Claudia Büchel, Matthias Bochtler, A William Rutherford, and Dario Piano

Subjects
Physiology, Genetics, Plant Science
Abstract

In thylakoid membranes, photosystem II (PSII) monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while PSII monomers (PSIIm) from granal regions lack these subunits. Here, we have isolated and characterized these 2 types of PSII complexes in tobacco (Nicotiana tabacum). PSIIm-S/27 showed enhanced fluorescence, the near absence of oxygen evolution, and limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA•− couple, which controls the charge recombination route, and this limits chlorophyll triplet-mediated 1O2 formation. These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit using a bicarbonate-mediated switch and protective mechanism.

Published
2023

5. Absorption changes in Photosystem II in the Soret band region upon the formation of the chlorophyll cation radical [PD1PD2]+

Author

Alain Boussac, Miwa Sugiura, Makoto Nakamura, A. William Rutherford, Stefania Viola, Julien Sellés, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Ehime University [Matsuyama, Japon], Department of Life Sciences, Imperial College London, Biologie du chloroplaste et perception de la lumière chez les micro-algues, Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), and ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011)

Subjects
[SDV]Life Sciences [q-bio]
Abstract

Flash-induced absorption changes in the Soret region arising from the [PD1PD2]+ state, the chlorophyll cation radical formed upon excitation of Photosystem II (PSII), were obtained using Mn-depleted PSII cores at pH 8.6. Under these conditions, TyrD is reduced before the first flash but oxidised before subsequent flashes. When TyrD• is present, an additional signal in the [PD1PD2]+-minus-[PD1PD2] difference spectrum was observed when compared to the first flash. The additional feature was W-shaped with troughs at 434 nm and 446 nm. This feature was absent when TyrD was reduced, but was present when TyrD was physically absent (and replaced by phenylalanine) or when its H-bonding histidine (D2-His190) was physically absent (replaced by a Leucine),. Thus, the simple difference spectrum without the double trough feature at 434 nm and 446 nm, required the native structural environment around the reduced TyrD and its H bonding partners to be present. A range of PSII variants were surveyed, and we found no evidence of involvement of PD1, ChlD1, PheD1, PheD2, TyrZ, and the Cytb559 heme in difference spectrum. Direct data ruling out the participation of PD2 is lacking. It seems possible that the specific H-bonding environment of around reduced TyrD allows a more homogenous electrostatic environment for [PD1PD2]+. A role for PD2 in the double-trough Soret signal may be tested using mutants of PD2 axial His ligand D2-His197.

Published
2022

6. Limitations to photosynthesis by proton motive force-induced photosystem II photodamage

Author

Geoffry A Davis, Atsuko Kanazawa, Mark Aurel Schöttler, Kaori Kohzuma, John E Froehlich, A William Rutherford, Mio Satoh-Cruz, Deepika Minhas, Stefanie Tietz, Amit Dhingra, and David M Kramer

Subjects
photosynthesis, proton motive force, photoinhibition, Medicine, Science, Biology (General), QH301-705.5
Abstract

The thylakoid proton motive force (pmf) generated during photosynthesis is the essential driving force for ATP production; it is also a central regulator of light capture and electron transfer. We investigated the effects of elevated pmf on photosynthesis in a library of Arabidopsis thaliana mutants with altered rates of thylakoid lumen proton efflux, leading to a range of steady-state pmf extents. We observed the expected pmf-dependent alterations in photosynthetic regulation, but also strong effects on the rate of photosystem II (PSII) photodamage. Detailed analyses indicate this effect is related to an elevated electric field (Δψ) component of the pmf, rather than lumen acidification, which in vivo increased PSII charge recombination rates, producing singlet oxygen and subsequent photodamage. The effects are seen even in wild type plants, especially under fluctuating illumination, suggesting that Δψ-induced photodamage represents a previously unrecognized limiting factor for plant productivity under dynamic environmental conditions seen in the field.

Published
2016
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7. Bicarbonate Activation of Monomeric Photosystem II-PsbS/Psb27 Complex

Author

Andrea Fantuzzi, Patrycja Haniewicz, Domenica Farci, M. Cecilia Loi, Keunha Park, Claudia Büchel, Matthias Bochtler, A. William Rutherford, and Dario Piano

Abstract

In thylakoid membranes, Photosystem II monomers from the stromal lamellae contain the subunits PsbS and Psb27 (PSIIm-S/27), while Photosystem II monomers from granal regions (PSIIm) lack these subunits. Here, we have isolated and characterised these two types of Photosystem II complexes. The PSIIm-S/27 showed enhanced fluorescence, the near-absence of oxygen evolution, as well as limited and slow electron transfer from QA to QB compared to the near-normal activities in the granal PSIIm. However, when bicarbonate was added to the PSIIm-S/27, water splitting and QA to QB electron transfer rates were comparable to those in granal PSIIm. The findings suggest that the binding of PsbS and/or Psb27 inhibits forward electron transfer and lowers the binding affinity for the bicarbonate. This can be rationalized in terms of the recently discovered photoprotection role played by bicarbonate binding via the redox tuning of the QA/QA•− couple, which controls the charge recombination route, and this limits chlorophyll triplet mediated 1O2 formation (Brinkert K et al. (2016) Proc Natl Acad Sci U S A. 113(43):12144-12149). These findings suggest that PSIIm-S/27 is an intermediate in the assembly of PSII in which PsbS and/or Psb27 restrict PSII activity while in transit, by using a bicarbonate-mediated switch and protective mechanism.One sentence summaryA photosystem II monomer with PsbS and Psb27 as additional subunits, is inactive as isolated but activated by bicarbonate, and is attributed to be a late-stage intermediate in photoassembly.

Published
2022

8. Bicarbonate-controlled reduction of oxygen by the QA semiquinone in Photosystem II in membranes

Author

Andrea Fantuzzi, Friederike Allgöwer, Holly Baker, Gemma McGuire, Wee Kii Teh, Ana P. Gamiz-Hernandez, Ville R. I. Kaila, A. William Rutherford, The Royal Society, and Biotechnology and Biological Sciences Research Council (BBSRC)

Subjects
Multidisciplinary
Abstract

Significance In Photosystem II (PSII), O 2 reduction by Q A • − is often discussed but has not been demonstrated. Here, we show in PSII membranes that Q A • − can reduce O 2 to superoxide, but only when bicarbonate is absent from its binding site on the nonheme Fe 2+ . Bicarbonate’s role in PSII was recently shown to involve a regulatory/protective redox-tuning mechanism linking PSII function to CO 2 concentration. A key aspect is the presence of stable Q A • − causing release of bicarbonate from its site on Fe 2+ . Here, we show that under these conditions, O 2 binds to the empty site on the Fe 2+ and is reduced by Q A • − . This unexpected reaction may be a further indication of cross-talk between the regulation of PSII and CO 2 fixation.

Published
2021

9. Bicarbonate-controlled reduction of oxygen by the Q

Author

Andrea, Fantuzzi, Friederike, Allgöwer, Holly, Baker, Gemma, McGuire, Wee Kii, Teh, Ana P, Gamiz-Hernandez, Ville R I, Kaila, and A William, Rutherford

Subjects
Chlorophyll, Electron Transport, Oxygen, Bicarbonates, Formates, Spinacia oleracea, Quinones, Photosystem II Protein Complex, Oxidation-Reduction, Chlamydomonas reinhardtii
Abstract

Photosystem II (PSII), the water/plastoquinone photo-oxidoreductase, plays a key energy input role in the biosphere. [Formula: see text], the reduced semiquinone form of the nonexchangeable quinone, is often considered capable of a side reaction with O

Published
2021

12. Photosynthetic constraints on fuel from microbes

Author

Charles A. R. Cotton, Jeffrey Stephen Douglass, Sven eDe Causmaecker, Katharina eBrinkert, Tanai eCardona, Andrea eFantuzzi, Alfred William Rutherford, and James William Murray

Subjects
Chlorophyll, Photosynthesis, biofuel, Rubisco, light harvesting, photosynthetic efficiency, Biotechnology, TP248.13-248.65
Published
2015
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14. The energetics of the two types of far-red Photosystem II

Author

Stefania Viola, William Roseby, Stefano Santabarabara, Dennis Nürnberg, Ricardo Assunção, Holger Dau, Julien Sellés, Alain Boussac, Andrea Fantuzzi, and A. William Rutherford

Subjects
Biophysics, Cell Biology, Biochemistry
Published
2022

15. Photosystem II in a State of Disassembly

Author

A. William Rutherford, James W. Murray, Peter J. Nixon, and Biotechnology and Biological Sciences Research Council (BBSRC)

Subjects
Photosystem II, food and beverages, chemistry.chemical_element, macromolecular substances, 02 engineering and technology, Manganese, 010402 general chemistry, 021001 nanoscience & nanotechnology, Photosynthesis, 01 natural sciences, Oxygen, 0104 chemical sciences, General Energy, chemistry, Cluster (physics), Biophysics, 0210 nano-technology, Biogenesis
Abstract

The light-driven oxidation of water to oxygen characteristic of oxygenic photosynthesis is catalyzed by a redox-active manganese/calcium cluster embedded in the Photosystem II (PSII) complex. How the cluster is assembled during the biogenesis and repair of PSII is unclear. Cryo-electron microscopy data have now provided new insights into the structure of a PSII complex lacking the cluster and have identified features that might be important for delivery and stabilization of Mn during assembly.

Published
2020

16. Energetics of the exchangeable quinone, Q B , in Photosystem II

Author

Wolfgang Nitschke, Jeffrey S. Douglass, A. William Rutherford, Sven De Causmaecker, Andrea Fantuzzi, Department of Life Sciences, Imperial College London, Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), The Royal Society, and Biotechnology and Biological Sciences Research Council (BBSRC)

Subjects
0106 biological sciences, Photosynthetic reaction centre, THERMOSYNECHOCOCCUS-ELONGATUS, proton-coupled electron transfer, Photoinhibition, Semiquinone, Photosystem II, [SDV]Life Sciences [q-bio], Analytical chemistry, Plastoquinone, SECONDARY QUINONE, EPR SIGNALS, 01 natural sciences, redox potential, law.invention, 03 medical and health sciences, Electron transfer, chemistry.chemical_compound, law, ELECTRON-PARAMAGNETIC-RES, MESH: photoinhibition, photosynthesis, Electron paramagnetic resonance, 030304 developmental biology, 0303 health sciences, Science & Technology, Multidisciplinary, photoinhibition, PHOTOSYNTHETIC REACTION CENTERS, Chemistry, REDOX STATE, PLASTOQUINONE POOL, WATER OXIDATION, Multidisciplinary Sciences, ACCEPTOR QUINONE, THERMO-LUMINESCENCE, Science & Technology - Other Topics, Proton-coupled electron transfer, 010606 plant biology & botany
Abstract

Significance The E m values of the 2 Q B redox couples, E m (Q B /Q B •− ) and E m (Q B •− /Q B H 2 ), in Photosystem II are fundamental for understanding the enzyme function, but they remain vague. Using EPR signals arising from the semiquinone, Q B •− , we have now measured both redox couples. The semiquinone is thermodynamically stable and has a relatively high potential. This minimizes back-reactions and electrons leaking onto O 2 , explaining the remarkable stability of Q B •− . The release of Q B H 2 is thermodynamically favorable, and the binding of the substrate PQ is greatly favored over the product PQH 2 : This optimizes PSII function in the presence of a largely reduced plastoquinone pool. This work corrects and explains a recent report of anomalous values for the Q B redox couples.

Published
2019

17. Femtosecond infrared spectroscopy of chlorophyll f-containing photosystem I

Author

Noura Zamzam, Jasper J. van Thor, Marius Kaucikas, Dennis J. Nürnberg, A. William Rutherford, and The Leverhulme Trust

Subjects
DYNAMICS, Chlorophyll, Chlorophyll a, Spectrophotometry, Infrared, Photosystem II, Infrared Rays, Chlorophyll f, Analytical chemistry, General Physics and Astronomy, 02 engineering and technology, Physics, Atomic, Molecular & Chemical, Cyanobacteria, 010402 general chemistry, Photosystem I, PRIMARY ELECTRON-TRANSFER, 01 natural sciences, CHLAMYDOMONAS-REINHARDTII, chemistry.chemical_compound, EXCITATION, PUMP-PROBE, REACTION CENTERS, Physical and Theoretical Chemistry, ULTRAFAST TRANSIENT ABSORPTION, Synechococcus, Science & Technology, 02 Physical Sciences, Chemical Physics, P700, Photosystem I Protein Complex, Chemistry, Physical, Physics, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Chemistry, Kinetics, Energy Transfer, chemistry, Absorption band, Excited state, Physical Sciences, CORE ANTENNA, ENERGY-TRANSFER, 03 Chemical Sciences, 0210 nano-technology, CHARGE SEPARATION
Abstract

The recent discovery of extremely red-shifted chlorophyll f pigments in both photosystem I (PSI) and photosystem II has led to the conclusion that chlorophyll f plays a role not only in the energy transfer, but also in the charge separation processes [Nürnberg et al., Science, 2018, 360, 1210–1213]. We have employed ultrafast transient infrared absorption spectroscopy to study the contribution of far-red light absorbing chlorophyll f to energy transfer and charge separation processes in far-red light-grown PSI (FRL-PSI) from the cyanobacterium Chroococcidiopsis thermalis PCC 7203. We compare the kinetics and spectra of FRL-grown PSI excited at 670 nm and 740 nm wavelengths to those of white light-grown PSI (WL-PSI) obtained at 675 nm excitation. We report a fast decay of excited state features of chlorophyll a and complete energy transfer from chlorophyll a to chlorophyll f in FRL-PSI upon 670 nm excitation, as indicated by a frequency shift in a carbonyl absorption band occurring within a 1 ps timescale. While the WL-PSI measurements support the assignment of initial charge separation to A−1+˙A0−˙ [Di Donato et al., Biochemistry, 2011, 50, 480–490] from the kinetics of a distinct cation feature at 1710 cm−1, in the case of FRL-PSI, small features at 1715 cm−1 from the chlorophyll cation are present from sub-ps delays instead, supporting the replacement of the A−1 pigment with chlorophyll f. Comparisons of nanosecond spectra show that charge separation proceeds with 740 nm excitation, which selectively excites chlorophyll f, and modifications in specific carbonyl absorption bands assigned to P700+˙ minus P700 and A1−˙ minus A1 indicate dielectric differences of FRL-PSI compared to WL-PSI in one or both of the two electron transfer branches of FRL-PSI.

Published
2019

18. Femtosecond visible transient absorption spectroscopy of chlorophyll- f -containing photosystem II

Author

Noura Zamzam, Rafal Rakowski, Marius Kaucikas, Gabriel Dorlhiac, Sefania Viola, Dennis J. Nürnberg, Andrea Fantuzzi, A. William Rutherford, Jasper J. van Thor, Imperial College London, and The Leverhulme Trust

Subjects
0301 basic medicine, 03 medical and health sciences, photosynthesis, 030104 developmental biology, Multidisciplinary, 030102 biochemistry & molecular biology, [SDV]Life Sciences [q-bio], photosystem II, chlorophyll-f
Abstract

The recently discovered, chlorophyll-f-containing, far-red photosystem II (FR-PSII) supports far-red light photosynthesis. Participation and kinetics of spectrally shifted far-red pigments are directly observable and separated from that of bulk chlorophyll-a. We present an ultrafast transient absorption study of FR-PSII, investigating energy transfer and charge separation processes. Results show a rapid subpicosecond energy transfer from chlorophyll-a to the long-wavelength chlorophylls-f/d. The data demonstrate the decay of an ∼720-nm negative feature on the picosecond-to-nanosecond timescales, coinciding with charge separation, secondary electron transfer, and stimulated emission decay. An ∼675-nm bleach attributed to the loss of chl-a absorption due to the formation of a cation radical, PD1+•, is only fully developed in the nanosecond spectra, indicating an unusually delayed formation. A major spectral feature on the nanosecond timescale at 725 nm is attributed to an electrochromic blue shift of a FR-chlorophyll among the reaction center pigments. These time-resolved observations provide direct experimental support for the model of Nürnberg et al. [D. J. Nürnberg et al., Science 360, 1210–1213 (2018)], in which the primary electron donor is a FR-chlorophyll and the secondary donor is chlorophyll-a (PD1 of the central chlorophyll pair). Efficient charge separation also occurs using selective excitation of long-wavelength chlorophylls-f/d, and the localization of the excited state on P720* points to a smaller (entropic) energy loss compared to conventional PSII, where the excited state is shared over all of the chlorin pigments. This has important repercussions on understanding the overall energetics of excitation energy transfer and charge separation reactions in FR-PSII.

Published
2020

19. The primary donor of far-red Photosystem II: ChlD1 or PD2?

Author

Robin Purchase, Dennis J. Nürnberg, Jennifer Morton, A. William Rutherford, Martyna Judd, Andrea Fantuzzi, Nicholas Cox, and Elmars Krausz

Subjects
0301 basic medicine, Photosynthetic reaction centre, Circular dichroism, Photosystem II, Magnetic circular dichroism, Biophysics, Electron donor, Cell Biology, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, Fluorescence, 0104 chemical sciences, Crystallography, 03 medical and health sciences, chemistry.chemical_compound, 030104 developmental biology, chemistry, Electrochromism, Spectroscopy
Abstract

Far-red light (FRL) Photosystem II (PSII) isolated from Chroococcidiopsis thermalis is studied using parallel analyses of low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies in conjunction with fluorescence measurements. This extends earlier studies (Nurnberg et al 2018 Science 360 (2018) 1210-1213). We confirm that the chlorophyll absorbing at 726 nm is the primary electron donor. At 1.8 K efficient photochemistry occurs when exciting at 726 nm and shorter wavelengths; but not at wavelengths longer than 726 nm. The 726 nm absorption peak exhibits a 21 ± 4 cm−1 electrochromic shift due to formation of the semiquinone anion, QA•-. Modelling indicates that no other FRL pigment is located among the 6 central reaction center chlorins: PD1, PD2 ChlD1, ChlD2, PheoD1 and PheoD2. Two of these chlorins, ChlD1 and PD2, are located at a distance and orientation relative to QA•- so as to account for the observed electrochromic shift. Previously, ChlD1 was taken as the most likely candidate for the primary donor based on spectroscopy, sequence analysis and mechanistic arguments. Here, a more detailed comparison of the spectroscopic data with exciton modelling of the electrochromic pattern indicates that PD2 is at least as likely as ChlD1 to be responsible for the 726 nm absorption. The correspondence in sign and magnitude of the CD observed at 726 nm with that predicted from modelling favors PD2 as the primary donor. The pros and cons of PD2 vs ChlD1 as the location of the FRL-primary donor are discussed.TOC GraphicHighlightsPrimary Donor confirmed at 726 nmDetermination of far-red chl pigment Qy excitation positions, widths, CD and MCD amplitudesQuantification of electrochromic shifts and QA•- photoconversion yieldElectrochromic shift consistent with primary donor at either ChlD1 or PD2The CD amplitude favors the primary donor at PD2

Published
2020
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20. The primary donor of far-red photosystem II: Chl

Author

Martyna, Judd, Jennifer, Morton, Dennis, Nürnberg, Andrea, Fantuzzi, A William, Rutherford, Robin, Purchase, Nicholas, Cox, and Elmars, Krausz

Subjects
Electron Transport, Photosynthetic Reaction Center Complex Proteins, Phycocyanin, Photosystem II Protein Complex, Cyanobacteria
Abstract

Far-red light (FRL) Photosystem II (PSII) isolated from Chroococcidiopsis thermalis is studied using parallel analyses of low-temperature absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies in conjunction with fluorescence measurements. This extends earlier studies (Nurnberg et al 2018 Science 360 (2018) 1210-1213). We confirm that the chlorophyll absorbing at 726 nm is the primary electron donor. At 1.8 K efficient photochemistry occurs when exciting at 726 nm and shorter wavelengths; but not at wavelengths longer than 726 nm. The 726 nm absorption peak exhibits a 21 ± 4 cm

Published
2020

21. Energetics of the exchangeable quinone, Q

Author

Sven, De Causmaecker, Jeffrey S, Douglass, Andrea, Fantuzzi, Wolfgang, Nitschke, and A William, Rutherford

Subjects
Light, Plastoquinone, Electron Spin Resonance Spectroscopy, Quinones, Thermosynechococcus, Photosystem II Protein Complex, Water, Biological Sciences, Cyanobacteria, Electron Transport, Kinetics, Benzoquinones, Thermodynamics, Photosynthesis, Oxidation-Reduction
Abstract

Photosystem II (PSII), the light-driven water/plastoquinone photooxidoreductase, is of central importance in the planetary energy cycle. The product of the reaction, plastohydroquinone (PQH(2)), is released into the membrane from the Q(B) site, where it is formed. A plastoquinone (PQ) from the membrane pool then binds into the Q(B) site. Despite their functional importance, the thermodynamic properties of the PQ in the Q(B) site, Q(B), in its different redox forms have received relatively little attention. Here we report the midpoint potentials (E(m)) of Q(B) in PSII from Thermosynechococcus elongatus using electron paramagnetic resonance (EPR) spectroscopy: E(m) Q(B)/Q(B)(•−) ≈ 90 mV, and E(m) Q(B)(•−)/Q(B)H(2) ≈ 40 mV. These data allow the following conclusions: 1) The semiquinone, Q(B)(•−), is stabilized thermodynamically; 2) the resulting E(m) Q(B)/Q(B)H(2) (∼65 mV) is lower than the E(m) PQ/PQH(2) (∼117 mV), and the difference (ΔE ≈ 50 meV) represents the driving force for Q(B)H(2) release into the pool; 3) PQ is ∼50× more tightly bound than PQH(2); and 4) the difference between the E(m) Q(B)/Q(B)(•−) measured here and the E(m) Q(A)/Q(A)(•−) from the literature is ∼234 meV, in principle corresponding to the driving force for electron transfer from Q(A)(•−) to Q(B). The pH dependence of the thermoluminescence associated with Q(B)(•−) provided a functional estimate for this energy gap and gave a similar value (≥180 meV). These estimates are larger than the generally accepted value (∼70 meV), and this is discussed. The energetics of Q(B) in PSII are comparable to those in the homologous purple bacterial reaction center.

Published
2020

22. Time-resolved comparative molecular evolution of oxygenic photosynthesis

Author

Tom H. Oliver, Tanai Cardona, Anthony W. D. Larkum, A. William Rutherford, Patricia Sánchez-Baracaldo, Leverhulme Trust, Biotechnology and Biological Sciences Research Council (BBSRC), and UKRI

Subjects
0106 biological sciences, Cyanobacteria, dD0, duplication leading to the origin of D1 and D2 subunits of photosystem II, Water oxidation, Photosystem II, Bioenergetics, 0601 Biochemistry and Cell Biology, dAB, duplication leading to the origin of Alpha and Beta subunits of ATP synthase, Biochemistry, 01 natural sciences, Ribosome, Reaction centre, Gene duplication, LUCA, the last universal common ancestor or the most recent common ancestor of life, Ga, Giga-annum, billion years, MSV, Margulisbacteria, Sericytochromatia and Vampirovibrionia, PSII, photosystem II, Photosynthesis, Molecular clock, δ Ga−1, Amino acid substitutions per site per billion years, Phylogeny, Photosystem, 0306 Physical Chemistry (incl. Structural), 0303 health sciences, MRCA, most recent common ancestor, Phototroph, ATP synthase, biology, Chemistry, Evolution of photosynthesis, Oxidation-Reduction, RC, reaction centre, Biophysics, Origin of photosynthesis, 010603 evolutionary biology, Article, ν, Rate of amino acid substitutions per site, Evolution, Molecular, 03 medical and health sciences, ROS, reactive oxygen species, Bacterial Proteins, Molecular evolution, Abiogenesis, Origin of life, Botany, GOE, Great Oxidation Event, ΔT, the span of time between dD0, dCP, dAB, or the LUCA, and the MRCA of Cyanobacteria, 030304 developmental biology, Ma, Mega-annum, million years, Photosystem II Protein Complex, Cell Biology, biology.organism_classification, Anoxygenic photosynthesis, dCP, duplication leading to the origin of CP43 and CP47 subunits of photosystem II, Oxygen, Evolutionary biology, PSI, photosystem I, biology.protein, CBP, chlorophyll-binding protein
Abstract

Oxygenic photosynthesis starts with the oxidation of water to O2, a light-driven reaction catalysed by photosystem II. Cyanobacteria are the only prokaryotes capable of water oxidation and therefore, it is assumed that the origin of oxygenic photosynthesis is a late innovation relative to the origin of life and bioenergetics. However, when exactly water oxidation originated remains an unanswered question. Here we use phylogenetic analysis to study a gene duplication event that is unique to photosystem II: the duplication that led to the evolution of the core antenna subunits CP43 and CP47. We compare the changes in the rates of evolution of this duplication with those of some of the oldest well-described events in the history of life: namely, the duplication leading to the Alpha and Beta subunits of the catalytic head of ATP synthase, and the divergence of archaeal and bacterial RNA polymerases and ribosomes. We also compare it with more recent events such as the duplication of Cyanobacteria-specific FtsH metalloprotease subunits and the radiation leading to Margulisbacteria, Sericytochromatia, Vampirovibrionia, and other clades containing anoxygenic phototrophs. We demonstrate that the ancestral core duplication of photosystem II exhibits patterns in the rates of protein evolution through geological time that are nearly identical to those of the ATP synthase, RNA polymerase, or the ribosome. Furthermore, we use ancestral sequence reconstruction in combination with comparative structural biology of photosystem subunits, to provide additional evidence supporting the premise that water oxidation had originated before the ancestral core duplications. Our work suggests that photosynthetic water oxidation originated closer to the origin of life and bioenergetics than can be documented based on phylogenetic or phylogenomic species trees alone., Highlights • The origin of oxygenic photosynthesis cannot be located on a species tree. • Vampirovibrionia, Sericytochromatia and Margulisbacteria lost photosynthesis. • Photosystem II can be as old as the oldest enzymes. • Earliest type II reaction centres were structurally like water-splitting Photosystem II. • Anoxygenic photosynthesis is not more primitive than oxygenic photosynthesis.

Published
2020

23. Bicarbonate-Mediated CO2 Formation on Both Sides of Photosystem II

Author

Johannes Messinger, Andrea Fantuzzi, A. William Rutherford, Dmitriy Shevela, and Hoang-Nguyen Do

Subjects
Photosystem II, Bicarbonate, Biochemistry and Molecular Biology, Biophysics, Photosystem II Protein Complex, Carbon Dioxide, Thylakoids, Biochemistry, Article, Biofysik, Electron Transport, Bicarbonates, chemistry.chemical_compound, chemistry, Spinacia oleracea, Oxidation-Reduction, Biokemi och molekylärbiologi
Abstract

The effect of bicarbonate (HCO3-) on photosystem II (PSII) activity was discovered in the 1950s, but only recently have its molecular mechanisms begun to be clarified. Two chemical mechanisms have been proposed. One is for the electron-donor side, in which mobile HCO3- enhances and possibly regulates water oxidation by acting as proton acceptor, after which it dissociates into CO2 and H2O. The other is for the electron-acceptor side, in which (i) reduction of the Q(A) quinone leads to the release of HCO(3)(- )from its binding site on the non-heme iron and (ii) the E-m potential of the Q(A)/Q(.-) couple increases when HCO3- dissociates. This suggested a protective/regulatory role of HCO3- as it is known that increasing the E-m of Q(A) decreases the extent of back-reaction-associated photodamage. Here we demonstrate, using plant thylakoids, that time-resolved membrane-inlet mass spectrometry together with C-13 isotope labeling of HCO3- allows donor- and acceptor-side formation of CO2 by PSII to be demonstrated and distinguished, which opens the door for future studies of the importance of both mechanisms under in vivo conditions.

Published
2020

24. Proton-coupled electron transfer from an interfacial phenol monolayer

Author

Robert J. Forster, Johan Hjelm, Martin Sjödin, and A. William Rutherford

Subjects
Proton, Kinetic models, General Chemical Engineering, Kinetics, chemistry.chemical_element, Phenol-terminated alkane-thiol monolayer, Proton-coupled electron transfer, Photochemistry, Analytical Chemistry, chemistry.chemical_compound, Reaction rate constant, chemistry, Monolayer, Electrochemistry, Phenol, Titration, pH dependence, Platinum
Abstract

A tert-butyl protected phenol is covalently link to a mixed self-assembled monolayer on a gold or platinum electrode. The phenol reduction potential shows the expected pH-dependence of a one-electron, one-proton couple with a decrease in formal reduction potential of 59 ± 5 mV per pH. A titration of the phenol is observed with a pKa of 13.2 and the reduction potential for the phenolate is 0.11 V. NHE. The kinetic behavior of the proton-coupled oxidation deviates substantially from the commonly used models for a step-wise reaction that assume the proton transfer reactions are in equilibrium throughout the reaction. A novel model for step-wise proton-coupled reactions is presented that fully accounts for the pH dependent kinetics and allows the formal rate constants and the transfer coefficients to be determined.

Published
2020

25. Glycolate Induces Redox Tuning Of Photosystem II in Vivo: Study of a Photorespiration Mutant

Author

Andrea Fantuzzi, Hermann Bauwe, A. William Rutherford, Wolfram Weckwerth, Anja Krieger-Liszkay, Marine Messant, Stefan Timm, Department of Life Sciences, Imperial College London, Molecular Systems Biology, universite de Vienne, Système membranaires, photobiologie, stress et détoxication (SMPSD), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Mécanismes régulateurs chez les organismes photosynthétiques (MROP), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
0106 biological sciences, 0301 basic medicine, Photosystem II, Physiology, [SDV]Life Sciences [q-bio], Bicarbonate, Arabidopsis, Plant Science, Photosynthesis, Photosystem I, Thylakoids, 01 natural sciences, Fluorescence, Electron Transport, 03 medical and health sciences, chemistry.chemical_compound, Spinacia oleracea, Genetics, Chlorophyll fluorescence, Singlet Oxygen, Arabidopsis Proteins, Chemistry, Photosystem II Protein Complex, food and beverages, Articles, Electron transport chain, Glycolates, Plant Leaves, Bicarbonates, 030104 developmental biology, Thylakoid, Luminescent Measurements, Mutation, Biophysics, Photorespiration, MROP, B3S, 010606 plant biology & botany
Abstract

Bicarbonate removal from the nonheme iron at the acceptor side of photosystem II (PSII) was shown recently to shift the midpoint potential of the primary quinone acceptor Q(A) to a more positive potential and lowers the yield of singlet oxygen ((1)O(2)) production. The presence of Q(A)(−) results in weaker binding of bicarbonate, suggesting a redox-based regulatory and protective mechanism where loss of bicarbonate or exchange of bicarbonate by other small carboxylic acids may protect PSII against (1)O(2) in vivo under photorespiratory conditions. Here, we compared the properties of Q(A) in the Arabidopsis (Arabidopsis thaliana) photorespiration mutant deficient in peroxisomal HYDROXYPYRUVATE REDUCTASE1 (hpr1-1), which accumulates glycolate in leaves, with the wild type. Photosynthetic electron transport was affected in the mutant, and chlorophyll fluorescence showed slower electron transport between Q(A) and Q(B) in the mutant. Glycolate induced an increase in the temperature maximum of thermoluminescence emission, indicating a shift of the midpoint potential of Q(A) to a more positive value. The yield of (1)O(2) production was lowered in thylakoid membranes isolated from hpr1-1 compared with the wild type, consistent with a higher potential of Q(A)/Q(A)(−). In addition, electron donation to photosystem I was affected in hpr1-1 at higher light intensities, consistent with diminished electron transfer out of PSII. This study indicates that replacement of bicarbonate at the nonheme iron by a small carboxylate anion occurs in plants in vivo. These findings suggested that replacement of the bicarbonate on the nonheme iron by glycolate may represent a regulatory mechanism that protects PSII against photooxidative stress under low-CO(2) conditions.

Published
2018

26. The low spin - high spin equilibrium in the S2-state of the water oxidizing enzyme

Author

Antoine Marion, Ilke Ugur, A. William Rutherford, Alain Boussac, Miwa Sugiura, Ville R. I. Kaila, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Photosystème II (PS2), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), FRAMATOME, Proteo-Science Research Center, Ehime University [Matsuyama, Japon], Department of Life Sciences, Imperial College London, Ehime University, Bunkyo-cho, Matsuyama,Ehime 790-8577, Japan, The Royal Society, Biotechnology and Biological Sciences Research Council (BBSRC), and Biotechnology and Biological Sciences Research Cou

Subjects
Absorption spectroscopy, Photosystem II, Spin states, [SDV]Life Sciences [q-bio], Biophysics, 02 engineering and technology, Electronic structure, 010402 general chemistry, DFT, 01 natural sciences, Biochemistry, law.invention, Mn(4)CaO(5) cluster, law, Electron paramagnetic resonance, 02 Physical Sciences, biology, Chemistry, Oxygen evolution, Active site, Cell Biology, 06 Biological Sciences, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Spin state, biology.protein, Physical chemistry, Density functional theory, EPR, 0210 nano-technology
Abstract

In Photosystem II (PSII), the Mn4CaO5-cluster of the active site advances through five sequential oxidation states (S0 to S4) before water is oxidized and O2 is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S2 using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S2LS and S2HS is pH dependent, with a pKa ≈ 8.3 (n ≈ 4) for the native Mn4CaO5 and pKa ≈ 7.5 (n ≈ 1) for Mn4SrO5. The DFT results suggest that exchanging Ca with Sr modifies the electronic structure of several titratable groups within the active site, including groups that are not direct ligands to Ca/Sr, e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of the pKa upon the Ca/Sr exchange. EPR also showed that NH3 addition reversed the effect of high pH, NH3-S2LS being present at all pH values studied. Absorption spectroscopy indicates that NH3 is no longer bound in the S3TyrZ state, consistent with EPR data showing minor or no NH3-induced modification of S3 and S0. In both Ca-PSII and Sr-PSII, S2HS was capable of advancing to S3 at low temperature (198 K). This is an experimental demonstration that the S2LS is formed first and advances to S3 via the S2HS state without detectable intermediates. We discuss the nature of the changes occurring in the S2LS to S2HS transition which allow the S2HS to S3 transition to occur below 200 K. This work also provides a protocol for generating S3 in concentrated samples without the need for saturating flashes.

Published
2018

27. The First State in the Catalytic Cycle of the Water-Oxidizing Enzyme: Identification of a Water-Derived μ-Hydroxo Bridge

Author

Dimitrios A. Pantazis, Frank Neese, A. William Rutherford, Thomas Lohmiller, Vera Krewald, Nicholas Cox, Arezki Sedoud, and Wolfgang Lubitz

Subjects
biology, 010405 organic chemistry, Chemistry, Stereochemistry, Substrate (chemistry), General Chemistry, 010402 general chemistry, 01 natural sciences, Biochemistry, Catalysis, Cofactor, 0104 chemical sciences, law.invention, Crystallography, Colloid and Surface Chemistry, Catalytic cycle, law, Oxidation state, Oxidizing agent, biology.protein, Molecule, Electron paramagnetic resonance
Abstract

Nature’s water-splitting catalyst, an oxygen-bridged tetramanganese calcium (Mn4O5Ca) complex, sequentially activates two substrate water molecules generating molecular O2. Its reaction cycle is composed of five intermediate (Si) states, where the index i indicates the number of oxidizing equivalents stored by the cofactor. After formation of the S4 state, the product dioxygen is released and the cofactor returns to its lowest oxidation state, S0. Membrane-inlet mass spectrometry measurements suggest that at least one substrate is bound throughout the catalytic cycle, as the rate of 18O-labeled water incorporation into the product O2 is slow, on a millisecond to second time scale depending on the S state. Here, we demonstrate that the Mn4O5Ca complex poised in the S0 state contains an exchangeable hydroxo bridge. On the basis of a combination of magnetic multiresonance (EPR) spectroscopies, comparison to biochemical models and theoretical calculations we assign this bridge to O5, the same bridge identifie...

Published
2017

29. New insights on ChlD1 function in Photosystem II from site-directed mutants of D1/T179 in $Thermosynechococcus\ elongatus$

Author

Alain Boussac, Yuki Takegawa, Shin Nakamura, Miwa Sugiura, Makoto Nakamura, Julien Sellés, Takumi Noguchi, A. William Rutherford, Ehime University [Matsuyama, Japon], Nagoya City University [Nagoya, Japan], Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Department of Life Sciences, Imperial College London, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Proteo-Science Research Center, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), The Leverhulme Trust, and Biotechnology and Biological Sciences Research Council (BBSRC)

Subjects
0106 biological sciences, 0301 basic medicine, Photosynthetic reaction centre, Pheophytin, Chl(D1), Photoinhibition, Photosystem II, [SDV]Life Sciences [q-bio], Biophysics, Electron donor, Photochemistry, 01 natural sciences, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Reaction center, 030102 biochemistry & molecular biology, Chemistry, Singlet oxygen, 0601 Biochemistry And Cell Biology, Cell Biology, Fluorescence, ChlD1P680, P(680), Chlorophyll, Electrochromic shifts, 010606 plant biology & botany
Abstract

International audience; The monomeric chlorophyll, Chl D1 , which is located between the P$_{D1}$ P$_{D2}$ chlorophyll pair and the pheophytin, Pheo D1, is the longest wavelength chlorophyll in the heart of Photosystem II and is thought to be the primary electron donor. Its central Mg$^{2+}$ is liganded to a water molecule that is H-bonded to D1/T179. Here, two sitedirected mutants, D1/T179H and D1/T179V, were made in the thermophilic cyanobacterium, Thermosynechococcus elongatus, and characterized by a range of biophysical techniques. The Mn 4 CaO 5 cluster in the water-splitting site is fully active in both mutants. Changes in thermoluminescence indicate that i) radiative recombination occurs via the repopulation of *Chl D1 itself; ii) non-radiative charge recombination reactions appeared to be faster in the T179H-PSII; and iii) the properties of P D1 P D2 were unaffected by this mutation, and consequently iv) the immediate precursor state of the radiative excited state is the Chl D1 + Pheo D1 − radical pair. Chlorophyll bleaching due to high intensity illumination correlated with the amount of 1 O 2 generated. Comparison of the bleaching spectra with the electrochromic shifts attributed to Chl D1 upon QA$_−$ formation, indicates that in the T179H-PSII and in the WT*3-PSII, the Chl D1 itself is the chlorophyll that is first damaged by $^1$O$_2$ , whereas in the T179V-PSII a more red chlorophyll is damaged, the identity of which is discussed. Thus, Chl D1 appears to be one of the primary damage site in recombination-mediated photoinhibition. Finally, changes in the absorption of Chl D1 very likely contribute to the well-known electrochromic shifts observed at ~430 nm during the S-state cycle.

Published
2019

30. Effects of fluoxetine on functional outcomes after acute stroke (FOCUS) : a pragmatic, double-blind, randomised, controlled trial

Author

Martin Dennis, Gillian Mead, John Forbes, Catriona Graham, Maree Hackett, Graeme J Hankey, Allan House, Stephanie Lewis, Erik Lundström, Peter Sandercock, Karen Innes, Carol Williams, Jonathan Drever, Aileen Mcgrath, Ann Deary, Ruth Fraser, Rosemary Anderson, Pauli Walker, David Perry, Connor Mcgill, David Buchanan, Yvonne Chun, Lynn Dinsmore, Emma Maschauer, Amanda Barugh, Shadia Mikhail, Gordon Blair, Ingrid Hoeritzauer, Maggie Scott, Greig Fraser, Katherine Lawrence, Alison Shaw, Judith Williamson, David Burgess, Malcolm Macleod, Dan Morales, Frank Sullivan, Marian Brady, Ray French, Frederike Van Wijck, Caroline Watkins, Fiona Proudfoot, Joanna Skwarski, Diane Mcgowan, Rachael Murphy, Seona Burgess, William Rutherford, Katrina Mccormick, Ruaridh Buchan, Allan Macraild, Ruth Paulton, Adnan Fazal, Pat Taylor, Ruwan Parakramawansha, Neil Hunter, Jack Perry, John Bamford, Dean Waugh, Emelda Veraque, Caroline Bedford, Mary Kambafwile, Luis Idrovo, Linetty Makawa, Paula Smalley, Marc Randall, Tharani Thirugnana-Chandran, Ahamad Hassan, Richard Vowden, Joanne Jackson, Ajay Bhalla, Anthony Rudd, Chi Kai Tam, Jonathan Birns, Charlotte Gibbs, Leonie Lee Carbon, Elizabeth Cattermole, Katherine Marks, Angela Cape, Lisa Hurley, Sagal Kullane, Nigel Smyth, Charlotte Eglinton, Jennifer Wilson, Elio Giallombardo, Angela Frith, Paul Reidy, Matthew Pitt, Lucy Sykes, Deborah Dellafera, Victoria Croome, Lauriane Kerwood, Mirea Hancevic, Christina Narh, Carley Merritt, John Duffy, Duncan Cooke, Juliet Willson, Ali Ali, Aaizza Naqvi, Christine Kamara, Helen Bowler, Simon Bell, Tracy Jackson, Kirsty Harkness, Kathy Stocks, Suzanna Duty, Clare Doyle, Geoffrey Dunn, Keith Endean, Fiona Claydon, Emma Richards, Jo Howe, Ralf Lindert, Arshad Majid, Katy Dakin, Ahmad Maatouk, Luke Barron, Madana Meegada, Pratap Rana, Anand Nair, Christine Brighouse-Johnson, Jill Greig, Myint Kyu, Sanjeev Prasad, Matthew Robinson, Irfan Alam, Belinda Mclean, Lindsay Greenhalgh, Zenab Ahmed, Christine Roffe, Susan Brammer, Carole Beardmore, Kay Finney, Adrian Barry, Paul Hollinshead, Jeanette Grocott, Holly Maguire, Indira Natarajan, Jayan Chembala, Ranjan Sanyal, Sue Lijko, Nenette Abano, Alda Remegoso, Phillip Ferdinand, Stephanie Stevens, Resti Varquez, Chelsea Causley, Adrian Butler, Philip Whitmore, Caroline Stephen, Racquel Carpio, Joanne Hiden, Girish Muddegowda, Hayley Denic, Jane Sword, Ross Curwen, Martin James, Paul Mudd, Fiona Hall, Julie Cageao, Samantha Keenan, Caroline Roughan, Hayley Kingwell, Anthony Hemsley, Christoph Lohan, Sue Davenport, Angela Bowring, Tamika Chapter, Max Hough, David Strain, Karin Gupwell, Keniesha Miller, Anita Goff, Ellie Cusack, Shirley Todd, Rebecca Partridge, Georgiana Jennings, Kevin Thorpe, Jacquelyn Stephenson, Kelly Littlewood, Mark Barber, Fiona Brodie, Steven Marshall, Derek Esson, Irene Coburn, Caroline Mcinnes, Fiona Ross, Emma Bowie, Heather Barcroft, Victoria Withers, Laura Miller, Paul Willcoxson, Michelle Donninson, Richard Evans, Di Daniel, John Coyle, Michael Keeling, Peter Wanklyn, Mark Elliott, John Wightman, Elizabeth Iveson, Natasha Dyer, Anne-Marie Porteous, Monica Haritakis, Mandy Ward, Lucy Doughty, Lisa Carr, Mark O Neill, Cosmas Anazodo, Paul Wood, Poppy Cottrell, Cheryl Donne, Romina Rodriguez, Ruhail Mir, Jax Westmoreland, Judith Bell, Christopher Emms, Lorraine Wright, Pearl Clark Brown, Elizabeth Bamford, Andrew Stanners, Mike Carpenter, Prabal Datta, Richard Davey, Ann Needle, Marjorie Jane Eastwood, Fathima Zeena Razik, Imran Ghouri, Gavin Bateman, Judy Archer, Venkatesh Balasubramanian, Richard Bowers, Julie Ball, Louise Benton, Linda Jackson, Julie Ellam, Kate Norton, Paul Guyler, Terry Dowling, Sharon Tysoe, Paula Harman, Ashish Kundu, Ololade Omodunbi, Thayalini Loganathan, Stuart Chandler, Shanas Noor, Anwer Siddiqui, Amber Siddiqui, Swapna Kunhunny, Devesh Sinha, Martin Sheppard, Sindhu Rashmi, Elena France, Rajalakshmi Orath Prabakaran, Laura Wilson, Amiirah Ropun, Shyam Kelavkar, Kheng Xiong Ng, Lucy Kamuriwo, Sweni Shah, David Mangion, Camen Constantin, Luigi De Michele Hock, Anne Hardwick, Jayne Borley, Skarlet Markova, Kimberley Netherton, Tara Lawrence, Jo Fletcher, Rebecca Spencer, Helen Palmer, Claire Cullen, Dolores Hamill, Ramesh Durairaj, Zoe Mellor, Tanya Fluskey, Diane Wood, Alison Keeling, Victoria Hankin, Jennifer Peters, Daniela Shackcloth, Thant Hlaing, Rebecca Tangney, Jordan Ewing, Melanie Harrison, Sarah Stevenson, Victoria Sutton, Mohamed Soliman, Julia Hindle, Elizabeth Watson, Claire Hewitt, Susie Butler, Ibrahim Wahishi, Sarwat Arif, Amy Fields, Jagdish Sharma, Rose Brown, Caroline Taylor, Sarah Bell, Simon Leach, Chris Patterson, Sophia Khan, Helen Wilson, Joanne Price, Hawraman Ramadan, Stuart Maguire, Ruth Bellfield, Michaela Hooley, Umair Hamid, Waqar Gaba, Robina Ghulam, Leslie Masters, Outi Quinn, Lakshmanan Sekaran, Margaret Tate, Niaz Mohammed, Kiranjit Bharaj, Frances Justin, Rajan Pattni, Lanka Alwis, Sakthivel Sethuraman, Rianne Robinson, Lianne Eldridge, Susan Mintias, Meena Chauhan, Chi-Kai Tam, Jeremias Palmones, Clare Holmes, Lucy Belle Guthrie, Mairead Osborn, Lindsay Ball, Sarah Caine, Amy Steele, Peter Murphy, Nikki Devitt, Jayne Leonard, Ronak Patel, Ian Penwarden, Emily Dodd, Amy Holloway, Pauline Baker, Samantha Clarke, Sandra Williams, Lindsey Dow, Roland Wynn-Williams, James Kennedy, Rachel Teal, Ursula Schulz, Gary Ford, Philip Mathieson, Ian Reckless, Ana Deveciana, Paige Mccann, Gillian Cluckie, Geoffrey Howell, Jonathan Ayer, Barry Moynihan, Rita Ghatala, Brian Clarke, Geoffrey Cloud, Bhavini Patel, Usman Khan, Nia Al-Samarrai, Sarah Trippier, Neha Chopra, Temi Adedoyin, Fran Watson, Val Jones, Liqun Zhang, Lillian Choy, Rebecca Williams, Natasha Clarke, Adrian Blight, Kate Kennedy, Alice Dainty, Johann Selvarajah, Dheeraj Kalladka, Bharath Cheripelli, Wilma Smith, Fiona Moreton, Angela Welch, Xuya Huang, Elizabeth Douglas, Audrey Lush, Nicola Day, Salwa El Tawil, Karen Montgomery, Helen Hamilton, Doreen Ritchie, Sankaranarayanan Ramachandra, Kirsty Mcleish, Kamy Thavanesan, Sathyabama Loganathan, Josh Roberts, Chantel Cox, Sarah Orr, Alison Hogan, Divya Tiwari, Gail Hann, Barbara Longland, Owen David, Jo Bell, Catherine Ovington, Emily Rogers, Rachel Bower, Marketa Keltos, David Cohen, Joseph Devine, Lankantha Alwis, Lucy Southworth, Laura Burgess, Matilda Lang, Bhavna Badiani, Fenglin Guo, Anne Oshodi, Emmanuelle Owoyele, Norah Epie, Anette David, Mushiya Mpelembue, Rajaram Bathula, Mudhar Abdul-Saheb, Angela Chamberlain, Varthi Sudkeo, Khalid Rashed, Barbara Williams-Yesson, Joanne Board, Sarah De Bruijn, Clare Buckley, Sarah Board, Joanna Allison, Elizabeth Keeling, Tracey Duckett, Dave Donaldson, Carinna Vickers, Claire Barron, Linda Balian, Jodhi Wilson, Adam Edwards, Timothy England, Amanda Hedstrom, Elizabeth Bedford, Margaret Harper, Elina Melikyan, Wendy Abbott, Kashmira Subramanian, Marie Goldsworthy, Meena Srinivasan, Angela Yeomans, Denise Donaldson, Frances Hurford, Riquella Chapman, Sana Shahzad, Nicki Motherwell, Louise Tonks, Rachel Young, Usman Ghani, Indranil Mukherjee, Dipankar Dutta, Mudhar Obaid, Pauline Brown, Fiona Davis, Deborah Ward, Jennifer Turfrey, Bethan Cartwright, Bilal Topia, Judith Spurway, Kayleigh Collins, Rehana Bakawala, Chloe Hughes, Susan Oconnell, Linda Hill, Kausik Chatterjee, Tim Webster, Syed Haider, Pamela Rushworth, Fiona Macleod, Arumugam Nallasivan, Charlotte Perkins, Edel Burns, Sandra Leason, Tom Carter, Samantha Seagrave, Eman Sami, Lisa Armstrong, Syed Naseem Naqvi, Muhammad Hassan, Sharron Parkinson, Samantha Mawer, Gillian Darnbrook, Carl Booth, Brigid Hairsine, Matthew Smith, Sue Williamson, Fiona Farquhar, Bernard Esisi, Tim Cassidy, Gavin Mankin, Beverley Mcclelland, Maria Bokhari, David Sproates, Elliot Epstein, Steve Hurdowar, Ruth Blackburn, Nazran Sukhdeep, Saika Razak, Khalid Osman, Amina Hashmi, Natasha Upton, Frances Harrington, Gillian Courtauld, Christine Schofield, Linda Lucas, Katja Adie, Kirsty Bond, Abhijit Mate, Jo Skewes, Ali James, Carolyn Brodie, Matthew Johnson, Linda Allsop, Emma Driver, Karina Harris, Mark Drake, Sam Ellis, Bev Maund, Emma Thomas, Kimberley Moore, Matthew Burn, Adam Hamilton, Shageetha Mahalingam, Amulya Misra, Farrah Reid, Adrienne Benford, Derek Hilton, Lorraine Hazell, Keziah Ofori, Anne Louise Thomas, Moncy Mathew, Sonia Dayal, Iona Burn, Kenneth Fotherby, Karla Jennings-Preece, Angela Willberry, Debbie Morgan, Donna Butler, Gurminder Sahota, Kelly Kauldhar, Nasar Ahmad, Angela Stevens, Saugata Das, David Bruce, Yogish Pai, Khin Nyo, Lynsey Stephenson, Richard Nendick, Gill Rogers, Mahesh Dhakal, Sofia Dima, Ellen Brown, Susan Clayton, Penny Gamble, Muhammad Naeem, Rachel Hayman, Rachel Burnip, Philip Earnshaw, David Hargroves, Barbara Ransom, Hannah Rudenko, Ibrahim Balogun, Kirsty Griffiths, Kim Mears, Tom Webb, Linda Cowie, Tessa Hammond, Audrey Thomson, Daniela Ceccarelli, Navraj Chattha, Eva Beranova, Anna Verrion, Andrew Gillian, Natasha Schumacher, Anna Bahk, Susannah Walker, Vera Cvoro, Nicola Chapman, Susan Pound, Rebecca Cain, Sean Mcauley, Mandy Couser, Maria Simpson, Athan Tachtatzis, Khalil Ullah, Don Sims, Rachael Jones, Jonathan Smith, Rebecca Tongue, Mark Willmot, Claire Sutton, Edward Littleton, Jattinder Khaira, Susan Maiden, James Cunningham, Carole Green, Yin-May Chin, Michelle Bates, Katherine Ahlquist, Ingrid Kane, Joanna Breeds, Tenesa Sargent, Laura Latter, Alexandra Pitt Ford, Nicola Gainsborough, Tom Levett, Philip Thompson, Emma Barbon, Angela Dunne, Simon Hervey, Suzanne Ragab, Tracy Sandell, Christine Dickson, Judith Dube, Sharon Power, Nick Evans, Beverley Wadams, Savina Elitova, Beth Aubrey, Tatiana Garcia, James Mcilmoyle, Carol Jeffs, Christina Dickinson, Anis Ahmed, Sanjeev Kumar, Julie Frudd, Charlotte Armer, Andrew Potter, Stacey Donaldson, Joanne Howard, Kirsty Jones, Saikat Dhar, David Collas, Saul Sundayi, Lynn Denham, Deepali Oza, Elaine Walker, Mohit Bhandari, Sissy Ispoglou, Rachel Evans, Kamel Sharobeem, Elaine Walton, Steven Shanu, Anne Hayes, Jennifer Howard-Brown, Steven Billingham, Nic Weir, Vanessa Pressly, Emma Wood, Gabriella Howard, Holly Burton, Pam Crawford, Shuna Egerton, Sue Evans, Jasmine Hakkak, Janet Andrews, Rebecca Lampard, Christopher Allen, Ashleigh Walters, Rasha Said, James Richard Marigold, Sau-Mon Tsang, Robyn Creeden, Chloe Cox, Simon Smith, Imogen Gartrell, Fiona Smith, Colin Jenkins, Joanna Pryor, Andrew Hedges, Fiona Price, Linda Moseley, Lily Mercer, Claire Hughes, Abul Azim, Julie White, Milena Krasinska-Chavez, Shaun Chaplin, James Curtis, Deepwant Singh, Javed Imam, Anne Nicolson, Sajid Alam, Simon Whitworth, Lisa Wood, Elizabeth Warburton, Siobhan Kelly, Joanne Mcgee, Hugh Markus, Denish Chandrasena, Derek Hayden, Juliana Sesay, Helen Hayhoe, Mark Bolton, Jane Macdonald, Jenny Mitchell, Charlotte Farron, Elaine Amis, Diana Day, Ainsley Culbert, Ailene Espanol, Niamh Hannon, Dominic Handley, Sarah Finlay, Sarah Crisp, Lynne Whitehead, Jobbin Francis, Janice Oconnell, Emily Osborne, Rod Beard, Ramesh Krishnamurthy, Langanani Mokoena, Naweed Sattar, Min Myint, Michelle Edwards, Andrew Smith, Paul Corrigan, Anthony Byrne, Joanne Blackburn, Caroline Mcghee, Amanda Smart, Fiona Donaldson, Claire Copeland, Jill Wilson, Rhona Scott, Paul Fitzsimmons, Paula Lopez, Mark Wilkinson, Aravindakshan Manoj, Penelope Cox, Leona Trainor, Glyn Fletcher, Lisa Denny, Karen Kavanagh, Hannah Allsop, Hedley Emsley, Sulaiman Sultan, Alison Mcloughlin, Benjamin Walmsley, Louise Hough, Shakeel Ahmed, Donna Doyle, Bindu Gregary, Sonia Raj, Kirubananthan Nagaratnam, Neelima Mannava, Nyla Haque, Norma Shields, Kate Preston, Geraldine Mason, Kirsty Short, Gemma Lumsdale, Giulia Uitenbosch, Ugnius Sukys, Stacey Valentine, David Jarrett, Kerry Dodsworth, Mary Wands, Nisa Khan, Jane Tandy, Catrin Watkinson, Wendy Golding, Rebecca Butler, Max Williams, Yasmin Davies, Keith Yip, Claire James, Anne Suttling, Aditya Maney, Giles Edward Gamble, Adam Hague, Bethan Charles, Sujata Blane, Beatriz Duran, Caroline Lambert, Katherine Stagg, Robert Whiting, Jane E Homan, Sarah Brown, Malik Hussain, Miriam Harvey, Libby Graham, Leanne Foote, Catherine Lane, Liz (Joan) Kemp, Joy Rowe, Helen Durman, Jayne Foot, Lucy Brotherton, Nicholas Hunt, Corinne Pawley, Alison Whitcher, Patrick Sutton, Susan Mcdonald, Denys Pak, Alison Wiltshire, Jennifer Jagger, Anthony K Metcalf, Gail Louise Healey, Joyce Balami, Clare Marie Self, Melissa Crofts, Annie Chakrabarti, Chit Hmu, Garth Ravenhill, Charmaine Grimmer, Thandar Soe, Jocelyn Keshet-Price, Margaret Langley, Ian Potter, Pui-Lin Tam, Mary Joan Macleod, Patricia Cooper, Michael Christie, Janice Irvine, Faye Annison, David Christie, Celia Meneses, Amber Johnson, Anu Joyson, Sandra Nelson, Vicky Taylor, John Reid, Rebecca Clarke, Jacqueline Furnace, Heather Gow, Youssif Abousleiman, Tania Beadling, Sally Collins, Stuart Jones, Jessica Purcell, Samantha Bloom, Shelly Goshawk, Marcial Landicho, Sivatharshini Sangaralingham, Yasmin Begum, Sherree Mutton, Elangovan Munuswamy Vaiyapuri, Jane Allen, Jemma Lowe, Martin Hughes, Ivan Wiggam, Sarah Cuddy, Suzanne Tauro, Brian Wells, Azlisham Mohd Nor, Nicola Persad, Maggie Kalita, Stuart Weatherby, Claire Brown, Adrian Pace, Daniel Lashley, Mike Marner, Marie Weinling, Natasha Wilmshurst, Darren Waugh, Anna Mucha, Alex Shah, John Baker, Jacqueline Westcott, Richard Cowan, Evangelos Vasileiadis, Samira Mumani, Anthea Parry, Cathy Mason, Melinda Holden, Katerina Petrides, Tomoko Nishiyama, Hina Mehta, Manju Krishnan, Dacey Lynne, Lisa Thomas, Connor Lynda, Catherine Hughes, Clare Clements, Rhys Williams, Tal Anjum, Storton Sharon, Susan Tucker, Paul Jones, Deanne Colwill, Helen Thompson Jones, Dinesh Chadha, Mark Fairweather, Deborah Walstow, Rosanna Fong, Stuart Johnston, Christine Almadenboyle, Sarah Ross, Shona Carson, Priya Nair, Emily Tenbruck, Mairi Stirling, Aparna Pusalkar, Hannah Beadle, Kelly Chan, Puneet Dangri, Asaipillai Asokanathan, Anita Rana, Sunita Gohil, Mark Massyn, Prabhu Aruldoss, Angela Cook, Karen Crabtree, Sura Dabbagh, Toby Black, Caroline Clarke, Denise Mead, Ruth Fennelly, Alpha Anthony, Linda Nardone, Victoria Dimartino, Michele Tribbeck, David Broughton, Dinesh Tryambake, Lynn Dixon, Agnieszka Skotnicka, Jane Thompson, Sarah Whitehouse, Andrew Sigsworth, Jason Wong, Arunkumar Annamalai, Julie Pagan, Brendan Affley, Caroline Sunderland, Lynda Goldenberg, Atif Khan, Peter Wilkinson, Raad Nari, Lucy Abbott, Emma Young, Amritpal Shakhon, Sally Lock, Jack Stewart, Rita Pereira, Margaret Dsouza, Sally Dunn, Anne-Marie Mckenna, Nina Cron, Michelle Kidd, Grace Hull, Kerry Bunworth, Graham Drummond, Karim Mahawish, Nicola Hayes, Lynne Connell, Jennifer Simpson, Helen Penney, Shuja Punekar, Joanne Nevinson, William Wareing, Jacqueline Ward, Richard Greenwood, Duncan Austin, Azra Banaras, Carolin Hogan, Thomas Corbett, Nnebuife Oji, Emma Elliott, Maria Brezitski, Nathalie Passeron, Laura Howaniec, Caroline Watchurst, Krishna Patel, Renuka Erande, Rahi Shah, Nabarun Sengupta, Maria Metiu, Celia Gonzalez, Sarah Funnell, Jordi Margalef, Gillian Peters, Indra Chadbourn, Ramachandran Sivakumar, Rajesh Saksena, Jane Ketley-O'donel, Richard Needle, Elaine Chinery, Alison Wright, Sue Cook, Joseph Ngeh, Harald Proeschel, Paige Cook, Pauline Ashcroft, Simon Sharpe, Stephanie Jones, Damian Jenkinson, Deborah Kelly, Holly Bray, Gunaratnam Gunathilagan, Sally Jones, Sorrell Tilbey, Saidu Abubakar, Joseph Vassallo, Dee Leonard, Lucy Orrell, Aziz Hasan, Asif Khan, Sulmaaz Qamar, Susan Graham, Emma Hewitt, Jennifer Awolesi, Muhammad Haque, Alissa Kent, Elizabeth Bradshaw, Martin Cooper, Inez Wynter, Anoja Rajapakse, Joumana Janbieh, Abu M Nasar, Lynne Wade, Linda Otter, Steve Haigh, Jamie-Rae Burgoyne, Rebecca Boulton, Andrew Boulton, Rayessa Rayessa, Emma Clarkson, Horne Rhian, Amy Fleming, Kim Mitchelson, Vicki Lowthorpe, Ahmed Abdul-Hamid, Phil Jones, Claire Duggan, Abigail Hynes, Emma Nurse, Syed Abid Raza, Sarah Jones, Udaya Pallikona, Bleddyn Edwards, Geraint Morgan, Kirsty Dennett, Helen Tench, Ronda Loosley, Toby Trugeon-Smith, Rhian Jones, Richard Williams, Donna Robson, Sunanda Mavinamane, Sanjeevi Meenakshisundaram, Lalitha Ranga, Sharon Dealing, Andrew Hill, Margaret Hargreaves, Tom Smith, Julie Bate, Linda Harrison, Ramanathan Kirthivasan, Emma Cannon, Joanne Topliffe, Rebecca Keskeys, Sarah Williams, Fiona Mcneela, Frances Cairns, Thomas James, Amanda Lyle, Sheela Shah, George Zachariah, Lauren Fergey, Susan Smolen, Lucy Cooper, Elizabeth Bohannan, Siddiq Omer, Sageet Amlani, Nadia Hunter, Melissa Hawkes-Blackburn, Giosue Gulli, Alice Peacocke, Justine Amero, Maria Burova, Ottilia Speirs, Steph Levy, Lynda Francis, Susan Holland, Sean Brotheridge, Helen Lyon, Christine Hare, Samantha Jackson, Lorraine Stephenson, Samer Al Hussayni, James Featherstone, Agness Bwalya, Arun Singh, M N Goorah, Jamie Walford, Angela Bell, Christine Kelly, Darren Rusk, Deborah Sutton, Farzana Patel, Stephen Duberley, Kathryn Hayes, Lorraine Hunt, Ahmed El Nour, Sacha Honour, Chloe Box, Simon Dyer, Lynne Brown, Kerry Elliott, Emma Temlett, John Paterson, Rosie Furness, Shelli Young, Enoch Orugun, Chris Brewer, Sarah Thornthwaite, Hannah Crowther, Rachel Glover, Moe Sein, Kashif Haque, Elspeth Gibson, Sam Wong, Karen Rotchell, Karen Burton, Lisa Brookes, Linda Bailey, Chris Lindley, Abbi Murray, Karen Waltho, Maureen Holland, Pradeep Kumar, Purnima Harlekar, Laura Booth, Charlotte Culmsee, Jade Drew, Mohammad Khan, Nicola Mackenzie, Carmel Thomas, Jane Ritchie, James Barker, Michael Haley, Donna Cotterill, Lynne Lane, Christine Little, Dawn Simmons, Glenn Saunders, Harvey Dymond, Sarah Kidd, Rachel Warinton, Yara Neves-Silva, Branimir Nevajda, Michael Villaruel, Udayaraj Umasankar, Seema Patel, Anna Man, Natasha Christmas, Ravi Rangasamy, Richard Ladner, Georgina Butt, Wilson Alvares, Narasimha Gadi, Michael Power, Belinda Wroath, Kevin Dynan, David Wilson, Sarah Crothers, Catherine Leonard, Samantha Hagan, Geraldine Douris, Djamil Vahidassr, Alastair Thompson, Brian Gallen, Shirley Mckenna, Collette Edwards, Clare Mcgoldrick, Murdi Bhattad, Khalil Kawafi, Deborah Morse, Patricia Jacob, Lisa Turner, Narayanamoorti Saravanan, Linda Johnson, Sadie Humphrey, Robert Namushi, Ramesh Patel, Jemma Mclaughlin, Paul Omahony, Esther Osikominu, Chukwuka Orefo, Chisha Mcdonald, Esther Makanju, Tabindah Khan, Grace Appiatse, Helena Stone, Martia Augustin, Alicia Wardale, Maqsud Salehin, Duncan Bailey, Luciano Garcia-Alen, Latheef Kalathil, Sarah Tinsley, Taya Jones, Kelly Amor, Andrew Ritchings, Emma Margerum, Jane Horton, Richard Miller, Nireekshana Gautam, Julie Meir, Amaryl Jones, Janet Putteril, Mirella Lepore, Rachel Gallifent, Laura-Louise Arundell, Catherine Mcredmond, Alicia Goulding, Vivek Nadarajan, Julia Laurence, Su Fung Lo, Sabina Melander, Paul Nicholas, Elizabeth Woodford, Gillian Mckenzie, Vietland Le, Jacolene Crause, Robert Luder, Maneesh Bhargava, Girish Bhome, Venetia V Johnson, Dara Chesser, Hayley Bridger, Elodie Murali, Jon Scott, Susan Morrison, Amy Burns, Julie Graham, Madeline Duffy, Khalid Ali, Emma Pitcher, Jane Gaylard, Julie Newman, Sunil Punnoose, Sarah Besley, Kirtan Purohit, Amy Rees, Mark Davy, Osman Chohan, Muhammad Fozan Khan, Rachel Walker, Vicky Murray, Charlotte Bent, Susan Oakley, Cassilda Peixoto, Suzanne Jones, Gaybrielle Livingstone, Fiona Butler, Sally Bradfield, Laura Gordon, Jenneke Schmit, Anika Wijewardane, Tineke Edmunds, Rebecca Wills, Catherine Medcalf, Lucia Argandona, Larissa Cuenoud, Hanna Hassan, Esther Erumere, Aidan Ocallaghan, Patrick Gompertz, Ozlem Redjep, Grace Auld, Anna Song, Tillana Tarkas, Hashim Kabash, Rumbi Hungwe, University of St Andrews. Sir James Mackenzie Institute for Early Diagnosis, University of St Andrews. Population and Behavioural Science Division, and University of St Andrews. School of Medicine

Subjects
RM, medicine.medical_specialty, Neurology, Neurologi, NDAS, 030204 cardiovascular system & hematology, Placebo, Minimisation (clinical trials), Article, B700, law.invention, 03 medical and health sciences, 0302 clinical medicine, Double-Blind Method, Randomized controlled trial, Modified Rankin Scale, law, Fluoxetine, Internal medicine, Humans, Medicine, 030212 general & internal medicine, R2C, Acute stroke, business.industry, ~DC~, General Medicine, Odds ratio, RM Therapeutics. Pharmacology, Stroke, RC Internal medicine, BDC, business, Selective Serotonin Reuptake Inhibitors, RC, medicine.drug
Abstract

Background: Results of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects.Methods: FOCUS was a pragmatic, multicentre, parallel group, double-blind, randomised, placebo-controlled trial done at 103 hospitals in the UK. Patients were eligible if they were aged 18 years or older, had a clinical stroke diagnosis, were enrolled and randomly assigned between 2 days and 15 days after onset, and had focal neurological deficits. Patients were randomly allocated fluoxetine 20 mg or matching placebo orally once daily for 6 months via a web-based system by use of a minimisation algorithm. The primary outcome was functional status, measured with the modified Rankin Scale (mRS), at 6 months. Patients, carers, health-care staff, and the trial team were masked to treatment allocation. Functional status was assessed at 6 months and 12 months after randomisation. Patients were analysed according to their treatment allocation. This trial is registered with the ISRCTN registry, number ISRCTN83290762.Findings: Between Sept 10, 2012, and March 31, 2017, 3127 patients were recruited. 1564 patients were allocated fluoxetine and 1563 allocated placebo. mRS data at 6 months were available for 1553 (99·3%) patients in each treatment group. The distribution across mRS categories at 6 months was similar in the fluoxetine and placebo groups (common odds ratio adjusted for minimisation variables 0·951 [95% CI 0·839–1·079]; p=0·439). Patients allocated fluoxetine were less likely than those allocated placebo to develop new depression by 6 months (210 [13·43%] patients vs 269 [17·21%]; difference 3·78% [95% CI 1·26–6·30]; p=0·0033), but they had more bone fractures (45 [2·88%] vs 23 [1·47%]; difference 1·41% [95% CI 0·38–2·43]; p=0·0070). There were no significant differences in any other event at 6 or 12 months.Interpretation: Fluoxetine 20 mg given daily for 6 months after acute stroke does not seem to improve functional outcomes. Although the treatment reduced the occurrence of depression, it increased the frequency of bone fractures. These results do not support the routine use of fluoxetine either for the prevention of post-stroke depression or to promote recovery of function.Funding: UK Stroke Association and NIHR Health Technology Assessment Programme.

Published
2019

31. A low-potential terminal oxidase associated with the irononly nitrogenase from the nitrogen-fixing bacterium Azotobacter vinelandii

Author

Burak V. Kabasakal, Charles A. R. Cotton, Febin Varghese, Jörg Schumacher, A. William Rutherford, Andrea Fantuzzi, James W. Murray, Biotechnology and Biological Sciences Research Council (BBSRC), and Biotechnology and Biological Sciences Research Council

Subjects
0301 basic medicine, Protein Conformation, Flavodoxin, PROTEIN, Crystallography, X-Ray, Biochemistry, ELECTRON-TRANSFER, FIXATION, 11 Medical and Health Sciences, Ferredoxin, Oxidase test, Rhodobacter, biology, Chemistry, oxidase, REDOX PROPERTIES, Nitrogenase, HEME, Enzyme structure, enzyme structure, Azotobacter vinelandii, dioxygenase, Oxidoreductases, 03 Chemical Sciences, Life Sciences & Biomedicine, Oxidation-Reduction, Biochemistry & Molecular Biology, CYTOCHROME-C-OXIDASE, Nitrogen, Iron, SEQUENCE, Cofactor, 03 medical and health sciences, Bacterial Proteins, Nitrogen Fixation, Molecular Biology, Science & Technology, NITRIC-OXIDE, COMPLEX, 030102 biochemistry & molecular biology, Cell Biology, ALTERNATIVE NITROGENASE, 06 Biological Sciences, nitrogenase, biology.organism_classification, Oxygen, 030104 developmental biology, 13. Climate action, biology.protein
Abstract

The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the flavin adenine dinucleotide cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of -420 ± 10 mV and -330 ± 10 mV for the two FAD potentials and -340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.

Published
2019

32. A low-potential terminal oxidase associated with the iron-only nitrogenase from the nitrogen-fixing bacterium

Author

Febin, Varghese, Burak Veli, Kabasakal, Charles A R, Cotton, Jörg, Schumacher, A William, Rutherford, Andrea, Fantuzzi, and James W, Murray

Subjects
Azotobacter vinelandii, Nitrogen, Protein Conformation, Iron, oxidase, Crystallography, X-Ray, nitrogenase, enzyme structure, Oxygen, Bacterial Proteins, nitrogen fixation, Editors' Picks, dioxygenase, Oxidoreductases, Oxidation-Reduction
Abstract

The biological route for nitrogen gas entering the biosphere is reduction to ammonia by the nitrogenase enzyme, which is inactivated by oxygen. Three types of nitrogenase exist, the least-studied of which is the iron-only nitrogenase. The Anf3 protein in the bacterium Rhodobacter capsulatus is essential for diazotrophic (i.e. nitrogen-fixing) growth with the iron-only nitrogenase, but its enzymatic activity and function are unknown. Here, we biochemically and structurally characterize Anf3 from the model diazotrophic bacterium Azotobacter vinelandii. Determining the Anf3 crystal structure to atomic resolution, we observed that it is a dimeric flavocytochrome with an unusually close interaction between the heme and the FAD cofactors. Measuring the reduction potentials by spectroelectrochemical redox titration, we observed values of −420 ± 10 and −330 ± 10 mV for the two FAD potentials and −340 ± 1 mV for the heme. We further show that Anf3 accepts electrons from spinach ferredoxin and that Anf3 consumes oxygen without generating superoxide or hydrogen peroxide. We predict that Anf3 protects the iron-only nitrogenase from oxygen inactivation by functioning as an oxidase in respiratory protection, with flavodoxin or ferredoxin as the physiological electron donors.

Published
2018

33. Evolution of Photochemical Reaction Centres: More Twists?

Author

A. William Rutherford, Tanai Cardona, and Leverhulme Trust

Subjects
0106 biological sciences, 0301 basic medicine, Photosystem II, Plant Biology & Botany, photosystem, 0607 Plant Biology, 0703 Crop and Pasture Production, Electron donor, Plant Science, Photosynthesis, 01 natural sciences, Redox, Evolution, Molecular, 03 medical and health sciences, chemistry.chemical_compound, Molecular evolution, oxygenic, Photosystem, chemistry.chemical_classification, 0602 Ecology, biology, Chemistry, Photosystem II Protein Complex, Water, anoxygenic, Electron acceptor, biology.organism_classification, Anoxygenic photosynthesis, reaction centre, Photosynthetic water oxidation, Oxygen, Crystallography, Transmembrane domain, 030104 developmental biology, water oxidation, Heliobacteria, Oxidation-Reduction, 010606 plant biology & botany
Abstract

The earliest event recorded in the molecular evolution of photosynthesis is the structural and functional specialisation of Type I (ferredoxin-reducing) and Type II (quinone-reducing) reaction centres. Here we point out that the homodimeric Type I reaction centre of Heliobacteria has a Ca2+-binding site with a number of striking parallels to the Mn4CaO5 cluster of cyanobacterial Photosystem II. This structural parallels indicate that water oxidation chemistry originated at the divergence of Type I and Type II reaction centres. We suggests that this divergence was triggered by a structural rearrangement of a core transmembrane helix resulting in a shift of the redox potential of the electron donor side and electron acceptor side at the same time and in the same redox direction.

Published
2018

34. Reprint of 'Proton-coupled electron transfer from an interfacial phenol monolayer'

Author

A. William Rutherford, Johan Hjelm, Martin Sjödin, and Robert J. Forster

Subjects
Proton, Chemistry, General Chemical Engineering, Kinetics, chemistry.chemical_element, Photochemistry, Analytical Chemistry, chemistry.chemical_compound, Reaction rate constant, Monolayer, Electrochemistry, Phenol, Titration, Proton-coupled electron transfer, Platinum
Abstract

A tert-butyl protected phenol is covalently link to a mixed self-assembled monolayer on a gold or platinum electrode. The phenol reduction potential shows the expected pH-dependence of a one-electron, one-proton couple with a decrease in formal reduction potential of 59 ± 5 mV per pH. A titration of the phenol is observed with a pKa of 13.2 and the reduction potential for the phenolate is 0.11 V . NHE. The kinetic behavior of the proton-coupled oxidation deviates substantially from the commonly used models for a step-wise reaction that assume the proton transfer reactions are in equilibrium throughout the reaction. A novel model for step-wise proton-coupled reactions is presented that fully accounts for the pH dependent kinetics and allows the formal rate constants and the transfer coefficients to be determined.

Published
2020

35. Early Archean origin of Photosystem II

Author

Patricia Sánchez-Baracaldo, Tanai Cardona, Anthony W. D. Larkum, A. William Rutherford, Leverhulme Trust, and Biotechnology and Biological Sciences Research Council (BBSRC)

Subjects
Photosystem II Protein Complex/analysis, 0106 biological sciences, Cyanobacteria, Life Sciences & Biomedicine - Other Topics, Archean, Geochemistry & Geophysics, Photosystem II, photosystem, PROTEIN, OXIDATION, 01 natural sciences, Astrobiology, RAPID EVOLUTION, ELECTRON-TRANSFER, Geosciences, Multidisciplinary, Photosynthesis, Phylogeny, Photosystem, 0303 health sciences, biology, Chemistry, reaction center, Geology, Physical Sciences, Evolutionary history of life, Photosynthetic bacteria, Life Sciences & Biomedicine, OXYGENIC PHOTOSYNTHESIS, Photosynthetic reaction centre, Bacterial Proteins/analysis, Environmental Sciences & Ecology, DIVERGENCE TIMES, Evolution, Molecular, 03 medical and health sciences, Bacterial Proteins, evolution, Proteobacteria, 0402 Geochemistry, Biology, 030304 developmental biology, Science & Technology, COMPLEX, PHOTOSYNTHETIC REACTION CENTERS, 0602 Ecology, BETA-CAROTENE, Photosystem II Protein Complex, Cyanobacteria/genetics, Bayes Theorem, Chloroflexi, biology.organism_classification, Anoxygenic photosynthesis, CHLOROPHYLL, water oxidation, 0403 Geology, Environmental Sciences, 010606 plant biology & botany
Abstract

© 2018 The Authors. Geobiology Published by John Wiley & Sons Ltd. Photosystem II is a photochemical reaction center that catalyzes the light-driven oxidation of water to molecular oxygen. Water oxidation is the distinctive photochemical reaction that permitted the evolution of oxygenic photosynthesis and the eventual rise of eukaryotes. At what point during the history of life an ancestral photosystem evolved the capacity to oxidize water still remains unknown. Here, we study the evolution of the core reaction center proteins of Photosystem II using sequence and structural comparisons in combination with Bayesian relaxed molecular clocks. Our results indicate that a homodimeric photosystem with sufficient oxidizing power to split water had already appeared in the early Archean about a billion years before the most recent common ancestor of all described Cyanobacteria capable of oxygenic photosynthesis, and well before the diversification of some of the known groups of anoxygenic photosynthetic bacteria. Based on a structural and functional rationale, we hypothesize that this early Archean photosystem was capable of water oxidation to oxygen and had already evolved protection mechanisms against the formation of reactive oxygen species. This would place primordial forms of oxygenic photosynthesis at a very early stage in the evolutionary history of life.

Published
2018

36. New insights on Chl

Author

Yuki, Takegawa, Makoto, Nakamura, Shin, Nakamura, Takumi, Noguchi, Julien, Sellés, A William, Rutherford, Alain, Boussac, and Miwa, Sugiura

Subjects
Chlorophyll, Electron Transport, Bacterial Proteins, Light, Catalytic Domain, Mutagenesis, Site-Directed, Photosystem II Protein Complex, Cyanobacteria
Abstract

The monomeric chlorophyll, Chl

Published
2018

37. Early Archean origin of Photosystem II

Author

Tanai, Cardona, Patricia, Sánchez-Baracaldo, A William, Rutherford, and Anthony W, Larkum

Subjects
Archean, reaction center, photosystem, Photosystem II Protein Complex, Bayes Theorem, Original Articles, Chloroflexi, Cyanobacteria, Evolution, Molecular, Bacterial Proteins, water oxidation, evolution, Proteobacteria, Original Article, Photosynthesis, Phylogeny
Abstract

Photosystem II is a photochemical reaction center that catalyzes the light‐driven oxidation of water to molecular oxygen. Water oxidation is the distinctive photochemical reaction that permitted the evolution of oxygenic photosynthesis and the eventual rise of eukaryotes. At what point during the history of life an ancestral photosystem evolved the capacity to oxidize water still remains unknown. Here, we study the evolution of the core reaction center proteins of Photosystem II using sequence and structural comparisons in combination with Bayesian relaxed molecular clocks. Our results indicate that a homodimeric photosystem with sufficient oxidizing power to split water had already appeared in the early Archean about a billion years before the most recent common ancestor of all described Cyanobacteria capable of oxygenic photosynthesis, and well before the diversification of some of the known groups of anoxygenic photosynthetic bacteria. Based on a structural and functional rationale, we hypothesize that this early Archean photosystem was capable of water oxidation to oxygen and had already evolved protection mechanisms against the formation of reactive oxygen species. This would place primordial forms of oxygenic photosynthesis at a very early stage in the evolutionary history of life.

Published
2018

38. Oxygenic Photoreactivity in Photosystem II Studied by Rotating Ring Disk Electrochemistry

Author

Nikolay, Kornienko, Jenny Z, Zhang, Katarzyna P, Sokol, Sarah, Lamaison, Andrea, Fantuzzi, Rienk, van Grondelle, A William, Rutherford, and Erwin, Reisner

Subjects
Article
Abstract

Protein film photoelectrochemistry has previously been used to monitor the activity of photosystem II, the water-plastoquinone photooxidoreductase, but the mechanistic information attainable from a three-electrode setup has remained limited. Here we introduce the four-electrode rotating ring disk electrode technique for quantifying light-driven reaction kinetics and mechanistic pathways in real time at the enzyme–electrode interface. This setup allows us to study photochemical H2O oxidation in photosystem II and to gain an in-depth understanding of pathways that generate reactive oxygen species. The results show that photosystem II reacts with O2 through two main pathways that both involve a superoxide intermediate to produce H2O2. The first pathway involves the established chlorophyll triplet-mediated formation of singlet oxygen, which is followed by its reduction to superoxide at the electrode surface. The second pathway is specific for the enzyme/electrode interface: an exposed antenna chlorophyll is sufficiently close to the electrode for rapid injection of an electron to form a highly reducing chlorophyll anion, which reacts with O2 in solution to produce O2•–. Incomplete H2O oxidation does not significantly contribute to reactive oxygen formation in our conditions. The rotating ring disk electrode technique allows the chemical reactivity of photosystem II to be studied electrochemically and opens several avenues for future investigation.

Published
2018

39. Photochemistry beyond the red limit in chlorophyll f–containing photosystems

Author

Nürnberg, D.J.aEmail Author, Morton, J.b, Santabarbara, S.c, Telfer, A.a, Joliot, P.d, Antonaru, L.A.a, Ruban, A.V.e, Cardona, T.a, Krausz, E.c, Boussac, A.f, Fantuzzi, A.aEmail Author, William Rutherford, A, Department of Life Sciences, Imperial College London, Australian National University (ANU), Consiglio Nazionale delle Ricerche [Milano] (CNR), Physiologie membranaire et moléculaire du chloroplaste (PMMC), Sorbonne Université (SU)-Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Collège de France (CdF (institution)), Queen Mary University of London (QMUL), Research School of Chemistry, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Biotechnology and Biological Sciences Research Council (BBSRC)

Subjects
0106 biological sciences, 0301 basic medicine, Chlorophyll, separation, Light, Acaryochloris marina, OXIDATION, Photochemistry, 01 natural sciences, chemistry.chemical_compound, analogs and derivatives, wavelength, biophysics, Acaryochloris, pigmentation, ANGSTROM, Photosynthesis, thermoluminescence, Photosystem, photochemistry, Multidisciplinary, spectral sensitivity, biology, development and aging, unclassified drug, Multidisciplinary Sciences, thylakoid membrane protein, priority journal, visual_art, visual_art.visual_art_medium, Science & Technology - Other Topics, PRIMARY ELECTRON-ACCEPTOR, Chlorophyll a, photosystem I, radiation response, General Science & Technology, Chlorophyll f, growth, solar energy, ACARYOCHLORIS-MARINA, chlorophyll f, chemistry, Photosystem I, Cyanobacteria, Article, 03 medical and health sciences, Pigment, far red light, cyanobacterium, pigment, [CHIM]Chemical Sciences, nonhuman, Science & Technology, Photosystem I Protein Complex, Chlorophyll A, excitation, photosystem II, Photosystem II Protein Complex, biology.organism_classification, light intensity, Chroococcidiopsis thermalis, 030104 developmental biology, RESOLUTION, chemical structure, metabolism, AMBIENT, 010606 plant biology & botany
Abstract

Lower-energy photons do the work, too Plants and cyanobacteria use chlorophyll-rich photosystem complexes to convert light energy into chemical energy. Some organisms have developed adaptations to take advantage of longer-wavelength photons. Nürnberg et al. studied photosystem complexes from cyanobacteria grown in the presence of far-red light. The authors identified the primary donor chlorophyll as one of a few chlorophyll molecules in the far-red light–adapted enzymes that were chemically altered to shift their absorption spectrum. Kinetic measurements demonstrated that far-red light is capable of directly driving water oxidation, despite having less energy than the red light used by most photosynthetic organisms. Science , this issue p. 1210

Published
2018

40. Electron transfer pathways from the S2-states to the S3-states either after a Ca2+/Sr2+ or a Cl−/I− exchange in Photosystem II from Thermosynechococcus elongatus

Author

A. William Rutherford, Miwa Sugiura, Alain Boussac, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Stress Oxydants et Détoxication (LSOD), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Imperial College London, Proteo-Science Research Center, Ehime University [Matsuyama, Japon], Ehime University, Bunkyo-cho, Matsuyama,Ehime 790-8577, Japan, Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Stress Oxydants et Détoxication ( LSOD ), Département Biochimie, Biophysique et Biologie Structurale ( B3S ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), and Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 )

Subjects
Spin states, Photosystem II, [SDV]Life Sciences [q-bio], Biophysics, chemistry.chemical_element, Photochemistry, Oxygen, Biochemistry, law.invention, Electron Transport, Mn(4)CaO(5) cluster, Electron transfer, Chlorides, law, Electron paramagnetic resonance, Synechococcus, [ SDV ] Life Sciences [q-bio], Oxygen evolution, Electron Spin Resonance Spectroscopy, Temperature, Thermosynechococcus elongatus, Photosystem II Protein Complex, Cell Biology, Iodides, Crystallography, chemistry, Strontium, Spin state, Marked heterogeneity, Calcium, EPR, Mn4CaO5 cluster, Oxidation-Reduction
Abstract

International audience; The site for water oxidation in Photosystem II (PSII) goes through five sequential oxidation states (S0 to S4) before O2 is evolved. It consists of a Mn4CaO5-cluster close to a redox-active tyrosine residue (YZ). Cl- is also required for enzyme activity. By using EPR spectroscopy it has been shown that both Ca2+/Sr2+ exchange and Cl-/I- exchange perturb the proportions of centers showing high (S=5/2) and low spin (S=1/2) forms of the S2-state. The S3-state was also found to be heterogeneous with: i) a S=3 form that is detectable by EPR and not sensitive to near-infrared light; and ii) a form that is not EPR visible but in which Mn photochemistry occurs resulting in the formation of a (S2YZ)' split EPR signal upon near-infrared illumination. In Sr/Cl-PSII, the high spin (S=5/2) form of S2 shows a marked heterogeneity with a g=4.3 form generated at low temperature that converts to a relaxed form at g=4.9 at higher temperatures. The high spin g=4.9 form can then progress to the EPR detectable form of S3 at temperatures as low as 180K whereas the low spin (S=1/2) S2-state can only advance to the S3 state at temperatures≥235 K. Both of the two S2 configurations and the two S3 configurations are each shown to be in equilibrium at ≥235 K but not at 198 K. Since both S2 configurations are formed at 198 K, they likely arise from two specific populations of S1. The existence of heterogeneous populations in S1, S2 and S3 states may be related to the structural flexibility associated with the positioning of the oxygen O5 within the cluster highlighted in computational approaches and which has been linked to substrate exchange. These data are discussed in the context of recent in silico studies of the electron transfer pathways between the S2-state(s) and the S3-state(s).

Published
2015
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41. Origin and Evolution of Water Oxidation before the Last Common Ancestor of the Cyanobacteria

Author

James W. Murray, Tanai Cardona, A. William Rutherford, and Biotechnology and Biological Sciences Research Council (BBSRC)

Subjects
0106 biological sciences, Cyanobacteria, Most recent common ancestor, Gloeobacter, Light, Photosystem II, 0601 Biochemistry and Cell Biology, 01 natural sciences, cyanobacteria, EVOLVING PHOTOSYSTEM-II, Photosynthesis, GENE-EXPRESSION, Genetics & Heredity, GLOEOBACTER-VIOLACEUS PCC-7421, 0303 health sciences, biology, ELECTRON-ACCEPTOR COMPLEX, THERMOSYNECHOCOCCUS ELONGATUS, Life Sciences & Biomedicine, Oxidation-Reduction, Biochemistry & Molecular Biology, PSBA GENE, AMINO-ACID-RESIDUES, Evolution, Molecular, 03 medical and health sciences, 0603 Evolutionary Biology, Phylogenetics, Botany, Genetics, Plastid, Molecular Biology, Ecology, Evolution, Behavior and Systematics, Discoveries, 030304 developmental biology, Evolutionary Biology, 0604 Genetics, Science & Technology, PHOTOSYNTHETIC REACTION CENTERS, D1 PROTEIN, FTIR DIFFERENCE SPECTROSCOPY, Photosystem II Protein Complex, Water, biology.organism_classification, Synechococcus, Oxygen, water oxidation, PsbA, 010606 plant biology & botany, oxygenic photosynthesis
Abstract

Photosystem II, the water oxidizing enzyme, altered the course of evolution by filling the atmosphere with oxygen. Here, we reconstruct the origin and evolution of water oxidation at an unprecedented level of detail by studying the phylogeny of all D1 subunits, the main protein coordinating the water oxidizing cluster (Mn4CaO5) of Photosystem II. We show that D1 exists in several forms making well-defined clades, some of which could have evolved before the origin of water oxidation and presenting many atypical characteristics. The most ancient form is found in the genome of Gloeobacter kilaueensis JS-1 and this has a C-terminus with a higher sequence identity to D2 than to any other D1. Two other groups of early evolving D1 correspond to those expressed under prolonged far-red illumination and in darkness. These atypical D1 forms are characterized by a dramatically different Mn4CaO5 binding site and a Photosystem II containing such a site may assemble an unconventional metal cluster. The first D1 forms with a full set of ligands to the Mn4CaO5 cluster are grouped with D1 proteins expressed only under low oxygen concentrations and the latest evolving form is the dominant type of D1 found in all cyanobacteria and plastids. In addition, we show that the plastid ancestor had a D1 more similar to those in early branching Synechococcus. We suggest each one of these forms of D1 originated from transitional forms at different stages towards the innovation and optimization of water oxidation before the last common ancestor of all known cyanobacteria.

Published
2015

42. The low spin - high spin equilibrium in the S

Author

Alain, Boussac, Ilke, Ugur, Antoine, Marion, Miwa, Sugiura, Ville R I, Kaila, and A William, Rutherford

Subjects
Photosystem II Protein Complex, Water, Cyanobacteria, Oxidation-Reduction
Abstract

In Photosystem II (PSII), the Mn

Published
2017

43. Photoelectrochemistry of Photosystem II in Vitro vs in Vivo

Author

Jenny Z, Zhang, Paolo, Bombelli, Katarzyna P, Sokol, Andrea, Fantuzzi, A William, Rutherford, Christopher J, Howe, and Erwin, Reisner

Subjects
Biofilms, Communication, Synechocystis, Photosystem II Protein Complex, macromolecular substances, Electrochemical Techniques, Cyanobacteria, Photochemical Processes, Electrodes
Abstract

Factors governing the photoelectrochemical output of photosynthetic microorganisms are poorly understood, and energy loss may occur due to inefficient electron transfer (ET) processes. Here, we systematically compare the photoelectrochemistry of photosystem II (PSII) protein-films to cyanobacteria biofilms to derive: (i) the losses in light-to-charge conversion efficiencies, (ii) gains in photocatalytic longevity, and (iii) insights into the ET mechanism at the biofilm interface. This study was enabled by the use of hierarchically structured electrodes, which could be tailored for high/stable loadings of PSII core complexes and Synechocystis sp. PCC 6803 cells. The mediated photocurrent densities generated by the biofilm were 2 orders of magnitude lower than those of the protein-film. This was partly attributed to a lower photocatalyst loading as the rate of mediated electron extraction from PSII in vitro is only double that of PSII in vivo. On the other hand, the biofilm exhibited much greater longevity (>5 days) than the protein-film (

Published
2017

45. Hacking the thylakoid proton motive force for improved photosynthesis: modulating ion flux rates that control proton motive force partitioning into Δ

Author

Geoffry A. Davis, David Kramer, and A. William Rutherford

Subjects
0106 biological sciences, 0301 basic medicine, Life Sciences & Biomedicine - Other Topics, Photoinhibition, Photosystem II, FLUCTUATING LIGHT, Photosynthesis, bioenergetics, 01 natural sciences, Thylakoids, General Biochemistry, Genetics and Molecular Biology, FLAVODIIRON PROTEINS, 03 medical and health sciences, PHOTOSYSTEM-II, Botany, ELECTRON-TRANSFER, REACTION CENTERS, CHARGE RECOMBINATION, Biology, Evolutionary Biology, Science & Technology, ATP synthase, biology, photoinhibition, Chemiosmosis, Non-photochemical quenching, proton motive force, ATP SYNTHESIS, Proton-Motive Force, 11 Medical And Health Sciences, Articles, 06 Biological Sciences, Plants, WATER OXIDATION, 030104 developmental biology, Thylakoid, PLANT PHOTOSYNTHESIS, biology.protein, Biophysics, General Agricultural and Biological Sciences, K+/H+ ANTIPORTER KEA3, Flux (metabolism), Life Sciences & Biomedicine, non-photochemical quenching, 010606 plant biology & botany
Abstract

There is considerable interest in improving plant productivity by altering the dynamic responses of photosynthesis in tune with natural conditions. This is exemplified by the ‘energy-dependent' form of non-photochemical quenching ( q E ), the formation and decay of which can be considerably slower than natural light fluctuations, limiting photochemical yield. In addition, we recently reported that rapidly fluctuating light can produce field recombination-induced photodamage (FRIP), where large spikes in electric field across the thylakoid membrane (Δ ψ ) induce photosystem II recombination reactions that produce damaging singlet oxygen ( 1 O 2 ). Both q E and FRIP are directly linked to the thylakoid proton motive force ( pmf ), and in particular, the slow kinetics of partitioning pmf into its ΔpH and Δ ψ components. Using a series of computational simulations, we explored the possibility of ‘hacking' pmf partitioning as a target for improving photosynthesis. Under a range of illumination conditions, increasing the rate of counter-ion fluxes across the thylakoid membrane should lead to more rapid dissipation of Δ ψ and formation of ΔpH. This would result in increased rates for the formation and decay of q E while resulting in a more rapid decline in the amplitudes of Δ ψ -spikes and decreasing 1 O 2 production. These results suggest that ion fluxes may be a viable target for plant breeding or engineering. However, these changes also induce transient, but substantial mismatches in the ATP : NADPH output ratio as well as in the osmotic balance between the lumen and stroma, either of which may explain why evolution has not already accelerated thylakoid ion fluxes. Overall, though the model is simplified, it recapitulates many of the responses seen in vivo , while spotlighting critical aspects of the complex interactions between pmf components and photosynthetic processes. By making the programme available, we hope to enable the community of photosynthesis researchers to further explore and test specific hypotheses. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’.

Published
2017

46. Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Author

Walid Habre, Nicola Disma, Katalin Virag, Karin Becke, Tom G Hansen, Martin Jöhr, Brigitte Leva, Neil S Morton, Petronella M Vermeulen, Marzena Zielinska, Krisztina Boda, Francis Veyckemans, Walter Klimscha, Regina Konecny, Robert Luntzer, Ulrike Morawk-Wintersperger, Franz Neiger, Lydia Rustemeyer, Christian Breschan, Denise Frey, Manuela Platzer, Reinhard Germann, Joachim Oeding, Birgit Stoegermüller, Bernhard Ziegler, Philipp Brotatsch, Anton Gutmann, Gerlinde Mausser, Brigitte Messerer, Wolfgang Toller, Maria Vittinghoff, Gregor Zangl, Natascha Seidel-Ahyai, Christoph Hochhold, Ruth Kroess, Peter Paal, Steven Cnudde, Patricia Coucke, Birgit Loveniers, John Mitchell, David Kahn, Thierry Pirotte, Caroline Pregardien, Marc Coppens, Stefan De Hert, Björn Heyse, Martine Neckebroek, Aliaksandra Parashchanka, Jurgen Van Limmen, Nancy Van Den Eynde, Caroline Vanpeteghem, Piet Wyffels, Michaël Lalot, Jean-Paul Lechat, France Stevens, Sari Casaer, Françoise De Groote, Françoise De Pooter, Andrée De Villé, Marc Gerin, Natalia Magasich, Cristel Sanchez Torres, Denise Van Deenen, Johan Berghmans, Dirk Himpe, Eva Roofthooft, Ellen Joukes, Carine Smitz, Veronique Van Reeth, Christel Huygens, Julie Lauweryns, Karen De Smet, Nadia Najafi, Jan Poelaert, Anne Van de Velde, Veerle Van Mossevelde, Ivan Bekavac, Diana Butkovic, Dubravka Heli Litvic, Ivana Kerovec Soric, Hrvoje Maretic, Djurdjica Moscatello, Ljiljana Popovic, Slobodan Micici, Ivana Stuck Tus, Lada Kalagac Fabris, Tatjana Simurina, Nina Sulen, Gordana Kesic-Valpotic, Dajana Djapic, Jirí Žurek, Lucie Jureckova, Iveta Mackova, Monika Skacel, Stanislava Weinlichova, Jan Divák, Michal Frelich, René Urbanec, Vera Biskupova, Vladimir Mixa, Jana Pavlickova, Arash Afshari, Morten Bøttger, Marguerite B. Ellekvist, Mathias Johansen, Birgitte Ingeborg Madsen, Jens Christian Nilsson, Birgitte MB Schousboe, Nicola G. Clausen, Tom G. Hansen, Nick Phaff Steen, Mari-Liis Ilmoja, Virge Tonise, Juri Karjagin, Reet Kikas, Mika Isohanni, Anniina Lyly, Annika Takala, Johanna Happo, Kai Kiviluoma, Kati Martikainen, Riku Aantaa, Tuula Manner, Sanna Vilo, Catherine Amory, Hugues Ludot, Patricia Lambotte, René Busche, Olivier Jacqmarcq, Corinne Lejus, Julien Corouge, Christian Erb, Delphine Garrigue, Patricia Gillet, Anne Laffargue, Veronique Lambelin, Hélène Le Freche, Daliana Peresbota, Pierre Richart, Jerome Berton, Catherine Chapotte, Laurent Colbus, Thierry Lehousse, Jean Monrigal, Catherine Baujard, Philippe Roulleau, Giuseppe Staiti, Hélène Batoz, Maryline Bordes, Anne Didier, Yann Hamonic, Sylvaine Lagarde, Karine Nouette-Gaulain, François Semjen, Brigitte Zaghet, Jacky Dekens, Axelle Delcuze, Hervé Dupont, Aurélien Legrand, Celine Raffoflandreur, Noémie Audren, Blandine Camus, Marielle Cartal, Chantal Chazelet, Isabelle Davin, Marion Guillier, Luc Desjeux, Claire Larcher, Elodie Grein, Mickeal Leclercq, Roxana Levitchi, Lilica Rosu, Dominique Simon, Aurélien Zang, Anne Migeon, Anne-Charlotte Gagey, Nathalie Bourdaud, Anne-Charlotte Carre, Frédéric Duflo, Jean-Claude Riche, Philippe Robert, Emilie Druot, Olivier Maupain, Gilles Orliaguet, Lucie Sabau, Hanna Taright, Lynn Uhrig, Juliette Verchere-Montmayeur, Lise Debrabant, Clotaire Pilla, Alexandre Podvin, Benjamin Roth, Souhayl Dahmani, Florence Julien-Marsollier, Nada Sabourdin, Bogdan Alexandri, Gilles Brezac, Francois de la Brière, Catherine Hayem, Elizabeth Lhubat, Jean Paul Mission, Charlotte Rémond, Christophe Dadure, Maud Maniora, Anais Marie, Philippe Pirat, Anne-Charlotte Saour, Chrystelle Sola, Claude Ecoffey, Eric Wodey, Christian Adam, Thomas Standl, Ehrenfried Schindler, Tomohiro Yamamoto, Michael Brackhahn, Christoph Bernhard Eich, Holger Guericke, Petra Kindermann, Michael Laschat, Cornel Schink, Frank Wappler, Claudia Hoehne, Natalia Skordou, Johanna Ulrici, Martin Jetzek-Zader, Peter Kienbaum, Tanja Meyer-Treschan, Olaf Picker, Maximilian S. Schaefer, Golo Mielke, Sabine Baethge, Axel Ramminger, Martin Bauer, Matthias Bollinger, José Hinz, Michael Quintel, Sebastian G. Russo, Michael Bauer, Dfominik Geil, Andreas Kortgen, Niels-Peter Preussler, Ulrich Hofmann, Manfred Raber, Doris Reindl, Karin Oppenrieder, Bettina Schierlinger, Jens Roth, Wolfgang Funk, Thomas Fischer, Christian Gernoth, Christina Wiefelspütz, Hauke Volger, Nicole Zederer, Anja Diers, Matthias Huber, Clemens Schorer, Andreas Weyland, Konrad Schwarzkopf, Catharina Grau, Winfried Roth, Rolf Holy, Thomas Mader, Laura Peter, Hauke Supthut, Thomas Kuehhirt, Alexander Milde, Fritz Fiedler, Carsten Isselhorst, Ulrich Grundmann, Alexander Pattar, Jennifer Reinert, Birgit Ehm, Katrin Fritzsche, Ralf Gaebler, Patrick Meybohm, Maximilian Hein, Ines Guzman, Johanna Jokinen, Peter Kranke, Ulrich Goebel, Sarah Harris, Christoph Eisner, Miriam Ochsenreiter, Michael Schoeler, Elke Thil, Richard Ellerkmann, Andreas Hoeft, Claudia Neumann, Stefan Weber, Julia Keilhauer, Jan Kloessing, Michael Schramm, Uwe Trieschmann, Kristina Knauss, Barbara Sinner, Johannes Steinmann, Herbert Koessler, Evagelia Kalliardou, Anna Malisiova, Adelais Tsiotou, Pelagia Chloropoulou, Mpratzou Chrysi, Despoina Iordanidou, Merkourios Ntavlis, Krisztina B Boda, Christilynn Guerin, Janice Irwin, Claire Magner, Solmaz Nakhjavani, Brendan O'Hare, Deborah Galvin, Yosry Jamil, Carlos Lesmes, Yuri Barak, Haran Fisher, Ludmyla Kachko, Jacob Katz, Dmitry Kirilov, Michael Levinzon, Yair Manevich, Konstantin Nekrasov, Elia Peled, Elena Sanko, Dmitri Schmain, Olga Sheinkin, Eliahu Simhi, Alex Tarabikin, Evelina Trabkin, Irena Yagudaev, Yelena Zeitlin, Igor Zunser, Elisabetta Cerutti, Maria Maddalena Schellino, Silvia Valzan, Rosa Lucia Pinciroli, Luciano Bortone, Giorgia Cerati, Fabiana Salici, Leonardo Bussolin, Giuliana Rizzo, Francesca Rossetti, Laura Marchesini, Simonetta Tesoro, Brita De Lorenzo, Fabio Guarracino, Beate Kuppers, Marinella Astuto, Sofia Pitino, Rita Scalisi, Lucia Scordo, Sandra D'Alessandro, Luigi Dei Giudici, Ivano Farinelli, Giuseppe Lofino, Giuliano Marchetti, Sergio Giuseppe Picardo, Simone Reali, Alessandro Vittori, Francesco Antonio Idone, Maria Sammartino, Fabio Sbaraglia, Cinzia Barbera, Michela Bevilacqua, Valeria Cento, Svetlana Kotzeva, Leila Mameli, Giovanni Montobbio, Leandro Passariello, Cinzia Punzo, Rosanna Sileo, Rosanna Viacava, Claudia Volpe, Clelia Zanaboni, Edoardo Calderini, Daniele Genco, Simona Neri, Davide Ottolina, Anna Camporesi, Francesca Izzo, Ida Salvo, Andrea Wolfler, Andrea Sanna, Angela Sciascia, Paolo Stoia, Annamaria Guddo, Maria Lapi, Giorgio Ivani, Annalisa Longobardo, Valeria Mossetti, Dino Pedrotti, Maurizia Grazzini, Luisa Meneghini, Salvatore Metrangolo, Stefania Michelon, Carmelo Minardi, Costanza Tognon, Nicola Zadra, Ilaria Busi, Magda Khotcholava, Bruno Guido Locatelli, Valter Sonzogni, Giusi Starita, Nicole Almenrader, Caterina Aurilio, Pasquale Sansone, Raffaella Albarello, Paolo Bracci, Mariateresa Cecini, Maria Cristina Mondardini, Lorena Pasini, Milo Vason, Gianluca Zani, Roberto Zoppellari, Laura Pistidda, Andrea Cortegiani, Santi Maurizio Raineri, Antigona Hasani, Medita Hashimi, Agris Ancupans, Arta Barzdina, Zane Straume, Anda Zundane, Mikhail Chlopin, Dalia Gestautaite, Laura Lukosiene, Evelina Paliokaite, Ilona Razlevice, Inesa Armoniene, Aurelija Bernotiene, Vidunas Daugelavicius, Ilona Dockiene, Lina Gaidelyte, Nijole Saviciene, Jolita Krikstaponiene, Dominika Sidlovskaite-Baltake, Vladyslav Stasevski, Agne Vaitoskaite, David Gatt, Stephanie Mifsud, Simone Zammit, Celia Allison, Hamid Aslami, Susanne Eberl, Mireille F M van Stijn, Markus F Stevens, Kees Punt, Rob van Osch, Arthur Bauwman, Harm Scholten, Vesna Svircevic, Veronique Adriaens, Maaike Dirckx, Jaap Dogger, Ilse Dons-Sinke, Andreas Machotta, Xavier Moors, Mandana Rad, Lonneke Staals, Anouk van der Knijff - van Dortmont, Caroline van der Marel, Anne Sieben, Tim van der Zwan, Marianne Veldhuizen, David Alders, Wolfgang Buhre, Nicole Engel, Carine Vossen, Ravin Mahadewsing, Patrick Meijer, Volker Gerling, Roelof van der Schatte Olivier, Thea van Doorn, Kristy Vons Mark Hendriks, Sandra Lako, Gert jan Scheffer, Luc Tielens, Marieke Voet, Anthony Absalom, Margot Bergsma, Vera Spanjer, Rob Spanjersberg, Yvette van de Riet, Martin Volkers, Jurgen C. de Graaff, Geranne A.J. Hopman, Teus H. Kappen, J. Hannie, A.M. Megens, Sandra C. Numan, Anton N.J. Schouten, Nigel M. Turner, Désirée B.M. Van Der Werff, Renee T.M. Wensing, Erik Ephraim, Claire Nolte, Tore Reikvam, Ole Fredrik Lund, Lene Skaaden, Kari Marthe Ballovarre, Wenche Bakken Boerke, Guro Grindheim, Pal H H Lindenskov, Anne Beate Solas, Sjur Sponheim, Kyrre Ullensvang, Oddbjorn Viken, Inger Marie Drage, Kasper Gymoese Berthelsen, Bjørn Anders Kroken, Unni Bergland, Miroslaw Pryzmont, Mariola Talalaj, Malgorzata Wasiluk, Dorota Zalewska, Maria Damps, Jadzia Siemek-Mitela, Pawel Wieczorek, Magdalena Juzwa, Jowita Rosada-Kurasinska, Alicja Bartkowska-Sniatkowska, Maciej Cettler, Renata Kopycinska, Iwona Rudewicz, Jaek Sobczyk, Dariusz Wojciechowski, Artur Baranowski, Ewa Basiewicz, Magdalena Mierzewska-Schmidt, Wlodzimierz Retka, Piotr Sawicki, Magdalena Checinska, Magdalena Zurawska, Teresa Leal, Catia Mascarenhas, António Pedro Pina, Maria Joao Susano, Antonio Moniz, Maria Teresa Rocha, Carolina Calvao Santos, M Domingas Patuleia, Ricardo Pereira, Helena Roxo, Rosa Amaral, Isabel Guedes, Cristina Gomes, Marta Gonçalves, Helena Salgado, Maria Santos, Sara Rodrigues, Angela Sa, Elvira Machado, Sandra Pé d'Arca, Manuel Seabra, Ligia Mihaela Gheorghe, Constantin Ivascu, Lucia Moraru-Draghici, Mirela Suvejan, Remus Babici, Kovacs Eniko, Cristiana Hogea, Dubau Mihaela, Daraban Nicoleta, Danut Barbunc, Alina Maria Nistor, Violeta Stefan, Gabriela Catalina Ionescu, Irina Davidescu, Alina Teodora Nastase, Florin Dumitru Rusu, Victoria Badarau, Iulia Cindea, Melania Moscaliuc, Dana Olteanu, Luxita Petrescu, Daniela Ceuca, Irena Galinescu, Rodica Badeti, Alin Capusan, Cosmina Cucui-Cozma, Barbura Popescu, Luminita Cimpeanu, Mihaela-Petronela Birliba, Magdalena Miulescu, Stefania Balamat, Adriana Gurita, Luminita Ilie, Gabriel Mocioiu, Darina Pick, Rodica Sirghie, Radu Tabacaru, Irinel Trante, Lucian Horhota, Daniela Bandrabur, Tudor Ciobanu, Veaceslav Cuciuc, Valentin Munteanu, Valentin Olaru, Corneliu Paiu, Anca Savu, Oana Trifan, Anca Elena Malos, Anton Glazunov, Alexander Ivanov, Evgeny Poduskov, Alexander Popov, Igor 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Sanem, Alkis, Neslihan, Yildirim Guclu, Cigdem, Ceyda Meco, Basak, Hatipoglu, Zehra, Ozcengiz, Dilek, Begec, Zekine, Ilksen Toprak, Huseyin, Kendigelen, Pinar, Cigdem Tütüncü, Ayse, Karadeniz, Meltem Savran, Seyhan Ozkan, Tulay, Sivrikoz, Nuket, Kemal Arslantas, Mustafa, Hizal, Ayse, Tore Altun, Gülbin, Umuroglu, Tumay, Baris, Sibel, Kazak Bengisun, Zuleyha, Goncharenko, Galyna, Khrapak, Maksym, Klymenko, Tetyana, Pavlenko, Vitaliy, Prysiazhniuk, Dmytro, Rudio, Olga, Varyvoda, Mykhailo, Vodianytskyi, Sergii, Boryshkevych, Ivanna, Kyselova, Iryna, Trikash, Nikolay, Albokrinov, Andrew, Perova Sharonova, Valentina, Sklyar, Vitaliy, Surkov, Deni, Abdelaal, Amr, Barber, Nicola, Checuti, Sarah, Godsiff, Leisha, Johanne, Lynch, Simpson, Joanna, Underhill, Helen, Diwan, Rishi, Kelgeri, Nivedita, Masip, Nuria, Ravi, Radha, Roberts, Steve, Cillis, Annagrazia, Marcus, Ritchie, Merella, Federica, Love, David, Baraggia, Paolo, Bird, Victoria, Hussey, Joseph, Alderson, Peter, Bartholomew, Karen, Moncreiff, Michael, Davidson, Sarah, Hare, Alison, Kotecha, Ami, Lee, Corina, Liyanage, Ganga, Patel, Shivali, Samani, Amee, Abou Samra, Mark, Boyd, Matthew, Hullatt, Laurence, Levy, David, Pauling, Mark, Sharman, Sarah Jane, Smith, Natalie, Rutherford, John, Cavalier, Alison, Locke, Christie, Sage, Frederic, Bapat, Smita, Hammerschlag, Jacinda, Ioannou, Ioanni, King, Stephanie, Pegg, Rachel, Salota, Vishal, Sketcher, Jonathan, Thadsad, Muthu, Zeitlin, Deborah, Jack, Ewan, Lang, Colin, Ahmed, Samira, Ayyash, Reema, Bari, Francoise, Bell, Sarah J, Elizabeth Biercamp, Claire, Briggs, Sandra, Gabriella Elena Clement, Maria, Dalton, Mark, Ali Eissa Eid, Mohamed, Gandhi, Monica, Harmen Herrema, Idse, Khaffaf, Ranj, Jeng Min Law, Savio, Mcclintock, Joanna, Ireland, Nichola, Majid Saleem, Mohammed, Smith, Fiona, Cohen, Marc, Lee, Clover Ann, O'Donahue, Lizanne, Powell, Alex, Rawlinson, Ellen, Snoek, Aarjan, Weiss, Katja, Wellesley, Hugo, Crawford, Michael, Abdel Hafiz, Mostafa, Day, Alexandra, Rajamani, Balaji, Saha, Rita, Wright, Dave, Chee, Lew Chin, Bew, Stephanie, Homer, Rachel, Malarkkan, Nalini, Wolfe Barry, Juliet, Angadi, Pradeep, Cagney, Brid, De Melo, Eric, Dekker, Elsa, Helm, Emma, Jones, Gareth, Peiris, Kawshala, Russell, William, Slater, Patricia, Sodhi, Puja, Browning, Mike, Phillips, Trudie, Van Hecke, Rachel, Muir, Vimty, Singh, Piyush, Soskova, Tereza, Cumming, Craig, Farquharson, Pamela, Pearson, Karen, Shaw, Neil, Whiteside, Jonathan, Whyte, Emma, Byers, Gordon, Davies, Kay, Engelhardt, Thoma, Faliszewski, Isabella, Johnston, Graham, Kaufmann, Nil, Kusnirikova, Zuzana, Wilson, Graham, Carachi, Peter, Makin, Andrew, Foster, Brian, Lipczynski, Dariusz, Mawer, Rebecca, Rutherford, William, Rogerson, David, Rushman, Sarah, Taylor, Christopher, Tomlinson, William, Dix, Philippa, Woodward, Tom, Bell, Graham, Boyle, David, Cloherty, Marianne, Cullen, Julia, Cullen, Pauline, Fairgrieve, Ro, Ghent, Robert, Glasgow, Russell, Gordeeva, Elena, Harden, Alison, Hivey, Sarah, Jerome, Kevin, Mckee, Lesley, Morton, Neil, Pribul, Vicky, Sinclair, John, Steiner, Maria, Steward, Heather, Sweeney, Lorraine, Thomson, Wendy, Whiteside, Jeanette, Dalton, Andrew, Ross, Mark, Smith, Carolyn, Allen, Claire, Anders, Nicola, Barlow, Victoria, Bassett, Mike, Darwin, Leanne, Davison, Rachel, Diacono, Jacque, Hobbs, Amy, Hutchinson, Adel, Lomas, Bernadette, Lonsdale, Hannah, Nasser, Leila, Oshan, Vimmi, Patel, Pradip, Raistrick, Christopher, Scott Warren, Victoria, Talbot, Laura, Wai, Carolynn, Childs, Sophie, Dickinson, Matthew, Bloomfield, Tom, Garrioch, Sweyn, Watson, Karen, Gaynor, Jame, Harrison, Richard, Lee, Juliette, Blythe, Elizabeth, Dorman, Teresa, Eissa, Ayman, Ellwood, Jame, Gooch, Ingrid, Hearn, Robert, Hodgetts, Vanessa, John, Robert, Kirton, Christine, Ladak, Nadia, Morgan, Judith, Plant, Nina, Shepherd, Elizabeth, Short, Judith, Stack, Charle, Steel, Simon, Taylor, Matthew, Thomas, Deborah, Wilson, Catharine, Wilson Smith, Elaine, Bradbury, Carol L., Hussain, Nageena, Mayell, Antonia, Mesbah, Ahmed, Qureshi, Arif, Vaidyanath, Chandra, Geary, Tim, Hawksworth, Chri, Parasuraman, Tamaraichelvi, Perry, Nicole, Banerjee, Indrani, Barr, Katharine, Butler, Patrick, Davies, Jack, Flewin, Lisa, Gande, Richard, Montague, Jame, Plumb, Jame, Pratt, Thoma, Sutherland, Paul, Vail, Hannah, Wilkins, Andrew, Hunter, Catherine, Russell, Susan, Thomas, Alun, Mifsud, Mark, Strachan, Dominic, Plichon, Benoit, Harlet, Pierre, Child and Adolescent Psychiatry / Psychology, Morawk-Wintersperger, Ulrike, Seidel-Ahyai, Natascha, Lechat, Jean-Paul, Kesic-Valpotic, Gordana, Ilmoja, Mari-Lii, Nouette-Gaulain, Karine, Gagey, Anne-Charlotte, Carre, Anne-Charlotte, Riche, Jean-Claude, Verchere-Montmayeur, Juliette, Julien-Marsollier, Florence, Saour, Anne-Charlotte, Jetzek-Zader, Martin, Meyer-Treschan, Tanja, Preussler, Niels-Peter, Sidlovskaite-Baltake, Dominika, van Stijn, Mireille F M, Dons-Sinke, Ilse, van der Knijff - van Dortmont, Anouk, Hopman, Geranne A.J., Megens, A.M., Schouten, Anton N.J., Van Der Werff, Désirée B.M., Wensing, Renee T.M., Lindenskov, Pal H H, Siemek-Mitela, Jadzia, Rosada-Kurasinska, Jowita, Bartkowska-Sniatkowska, Alicja, Mierzewska-Schmidt, Magdalena, Moraru-Draghici, Lucia, Cucui-Cozma, Cosmina, Birliba, Mihaela-Petronela, Perez- Ferrer, Antonio, Spahr-Schopfer, Isabelle, Cakar Turhan, K.Sanem, Perova-Sharonova, Valentina, Abou-Samra, Mark, Sharman, Sarah-Jane, McClintock, Joanna, Lee, Clover-Ann, Abdel-Hafiz, Mostafa, Chee, Lew-Chin, McKee, Lesley, Scott-Warren, Victoria, and Wilson-Smith, Elaine

Subjects
Male, Pediatrics, Health Status, OPERATING-ROOM, Respiratory Tract Diseases, CHILDREN, 0302 clinical medicine, REGIONAL ANESTHESIA, Postoperative Complications, 030202 anesthesiology, CARDIAC-ARREST, Medicine, General anaesthesia, Prospective Studies, Prospective cohort study, Child, Intraoperative Complications, COMPLICATIONS, ddc:617, Incidence (epidemiology), Mortality rate, musculoskeletal, neural, and ocular physiology, Incidence, Age Factors, Hospitals, Europe, Cardiovascular Diseases, Child, Preschool, RESPIRATORY ADVERSE EVENTS, Female, Clinical Competence, Cohort study, Pulmonary and Respiratory Medicine, medicine.medical_specialty, Adolescent, macromolecular substances, Anesthesia, General, Drug Hypersensitivity, 03 medical and health sciences, MORBIDITY, Journal Article, Humans, business.industry, Infant, Newborn, Infant, 030208 emergency & critical care medicine, Perioperative, nervous system, PROSPECTIVE COHORT, Relative risk, RISK-FACTORS, Observational study, Human medicine, Nervous System Diseases, business, SYSTEM
Abstract

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31 127 anaesthetic procedures in 30 874 children with a mean age of 6·35 years (SD 4·50) were included. The incidence of perioperative severe critical events was 5·2% (95% CI 5·0–5·5) with an incidence of respiratory critical events of 3·1% (2·9–3·3). Cardiovascular instability occurred in 1·9% (1·7–2·1), with an immediate poor outcome in 5·4% (3·7–7·5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10 000. This was independent of type of anaesthesia. Age (relative risk 0·88, 95% CI 0·86–0·90; p

Published
2017

47. Description of DeltaPsi.py code from Hacking the thylakoid proton motive force (pmf) for improved photosynthesis: modulating ion flux rates that control pmf partitioning into Δψ and ΔpH

Author

Geoffry A. Davis, A. William Rutherford, and Kramer, David M.

Abstract

The rationale and initial constants used to simulate photosynthesis used in the DeltaPsi.py code are presented. An example of running the DeltaPsi.py code is also presented. The text for the DeltaPsi.py code follows.

Published
2017
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48. Modification of the pheophytin redox potential in Thermosynechococcus elongatus Photosystem II with PsbA3 as D1

Author

Miwa Sugiura, A. William Rutherford, Chizuko Azami, Alain Boussac, Kazumi Koyama, and Fabrice Rappaport

Subjects
Pheophytin, Light, Photosystem II, Glutamine, Biophysics, Glutamic Acid, macromolecular substances, Cyanobacteria, Photochemistry, Biochemistry, Redox, Electron Transport, chemistry.chemical_compound, Photosensitivity, Thermosynechococcus elongatus, chemistry.chemical_classification, Singlet oxygen, Chemistry, Pheophytins, Photosystem II Protein Complex, food and beverages, DCMU, Cell Biology, Electron acceptor, Crystallography, PsbA, Yield (chemistry), Mutation, Cyanobacterium, Oxidation-Reduction, Recombination
Abstract

In Photosystem II (PSII) of the cyanobacterium Thermosynechococcus elongatus, glutamate 130 in the high-light variant of the D1-subunit (PsbA3) was changed to glutamine in a strain lacking the two other genes for D1, psbA1 and psbA2. The resulting PSII (PsbA3/Glu130Gln) was compared with those from the "native" high-light (PsbA3-PSII) and low-light (PsbA1-PSII) variants, which differ by 21 amino acid including Glu130Gln. H-bonding from D1-Glu130Gln to the primary electron acceptor, PheophytinD1 (PheoD1), is known to affect the Em of the PheoD1/PheoD1(-) couple. The Gln130 mutation here had little effect on water splitting, charge accumulation and photosensitivity but did slow down S2QA(-) charge recombination and up-shift the thermoluminescence while increasing its yield. These changes were consistent with a ≈-30mV shift of the PheoD1/PheoD1(-)Em, similar to earlier single site-mutation results from other species and double the ≈-17mV shift seen for PsbA1-PSII versus PsbA3-PSII. This is attributed to the influence of the other 20 amino-acids that differ in PsbA3. A computational model for simulating S2QA(-) recombination matched the experimental trend: the S2QA(-) recombination rate in PsbA1-PSII differed only slightly from that in PsbA3-PSII, while in Glu130-PsbA3-PSII there was a more pronounced slowdown of the radical pair decay. The simulation predicted a major effect of the PheoD1/PheoD1(-) potential on (1)O2 yield (~60% in PsbA1-PSII, ~20% in PsbA3-PSII and ~7% in Gln130-PsbA3-PSII), reflecting differential sensitivities to high light.

Published
2014

49. Covalent Immobilization of Oriented Photosystem II on a Nanostructured Electrode for Solar Water Oxidation

Author

Tanai Cardona, Erwin Reisner, A. William Rutherford, and Masaru Kato

Subjects
Models, Molecular, Photosystem II, Static Electricity, 02 engineering and technology, Cyanobacteria, 010402 general chemistry, Photochemistry, 7. Clean energy, 01 natural sciences, Biochemistry, Catalysis, chemistry.chemical_compound, Electron transfer, Colloid and Surface Chemistry, Monolayer, Solar Energy, Carboxylate, Electrodes, chemistry.chemical_classification, Communication, Photosystem II Protein Complex, Tin Compounds, Water, General Chemistry, Electron acceptor, Enzymes, Immobilized, 021001 nanoscience & nanotechnology, Nanostructures, 0104 chemical sciences, Indium tin oxide, chemistry, Covalent bond, Electrode, 0210 nano-technology, Oxidation-Reduction
Abstract

Photosystem II (PSII) offers a biological and sustainable route of photochemical water oxidation to O2 and can provide protons and electrons for the generation of solar fuels, such as H2. We present a rational strategy to electrostatically improve the orientation of PSII from a thermophilic cyanobacterium, Thermosynechococcus elongatus , on a nanostructured indium tin oxide (ITO) electrode and to covalently immobilize PSII on the electrode. The ITO electrode was modified with a self-assembled monolayer (SAM) of phosphonic acid ITO linkers with a dangling carboxylate moiety. The negatively charged carboxylate attracts the positive dipole on the electron acceptor side of PSII via Coulomb interactions. Covalent attachment of PSII in its electrostatically improved orientation to the SAM-modified ITO electrode was accomplished via an amide bond to further enhance red-light-driven, direct electron transfer and stability of the PSII hybrid photoelectrode.

Published
2013

50. Bicarbonate-induced redox tuning in Photosystem II for regulation and protection

Author

Katharina Brinkert, Andrea Fantuzzi, Anja Krieger-Liszkay, A. William Rutherford, Sven De Causmaecker, Department of Life Sciences, Imperial College London, London, United Kingdom, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Imperial College London, The Royal Society, and Biotechnology and Biological Sciences Research Council (BBSRC)

Subjects
0106 biological sciences, 0301 basic medicine, Photosystem II, Light, Bicarbonate, [SDV]Life Sciences [q-bio], Analytical chemistry, Nanotechnology, 01 natural sciences, Redox, Carbon Cycle, Electron Transport, 03 medical and health sciences, chemistry.chemical_compound, Electron transfer, Spinacia oleracea, Superoxides, Plant Proteins, CO2 fixation, Multidisciplinary, photosynthesis, [ SDV ] Life Sciences [q-bio], photoinhibition, Cell Membrane, Quinones, Photosystem II Protein Complex, Carbon Dioxide, Hydrogen-Ion Concentration, Biological Sciences, Acceptor, Quinone, Oxygen, Plant Leaves, Bicarbonates, Kinetics, 030104 developmental biology, chemistry, water oxidation, Chlorophyll, Thermodynamics, photoassembly, Titration, Oxidation-Reduction, 010606 plant biology & botany
Abstract

Significance The E m of Q A / Q A − • is the reference pegging the thermodynamics of Photosystem II (PSII) to an absolute value. This work resolves a long-standing discrepancy in the literature, removing this ambiguity at the heart of PSII bioenergetics. The discrepancy in the literature reflects the loss of bicarbonate in some titrations. This surprising finding shows that bicarbonate binding controls the E m of Q A / Q A − • , and thus the redox state of Q A influences the binding of bicarbonate. This process constitutes a previously unrecognized regulation of PSII: Q A − • triggers bicarbonate release, slowing electron transfer and tuning the potential of Q A to protect PSII against photodamage. This work provides an explanation for the existence of bicarbonate in PSII, a mystery for more than half a century.

Published
2016

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