Structures of the intermediates of Kok’s photosynthetic water oxidation clock

Ever since the discovery that Mn was required for oxygen evolution in plants by Pirson in 1937 and the period-four oscillation in flash-induced oxygen evolution by Joliot and Kok in the 1970s, understanding of this process has advanced enormously using state-of-the-art methods. The most recent in this series of innovative techniques was the introduction of X-ray free-electron lasers (XFELs) a decade ago, which led to another quantum leap in the understanding in this field, by enabling operando X-ray structural and X-ray spectroscopy studies at room temperature. This review summarizes the current understanding of the structure of Photosystem II (PS II) and its catalytic centre, the Mn4 CaO5 complex, in the intermediate Si (i= 0-4)-states of the Kok cycle, obtained using XFELs.

Form the Series Editor, Preface A personal tribute to an eminent photosynthesis researcher, Martin D. Kamen (1913-2002) Biographies of the Editors Colour Plates Part I Editorials: Celebrating the Millennium - Historical Highlights of Photosynthesis Research, Part 1.- Part 2.- Part 3 .- Part II Overviews and Timelines: History of the word photosynthesis and evolution of its definition.- In one era and out the other.- Timeline of discoveries: Anoxygenic photosynthesis.- Discoveries in oxygenic photosynthesis (1727-2002): a perspective.- Part III Tributes: 'And whose bright presence'-an appreciation of Robert Hill and his reaction.- The Contributions of James Franck to photosynthetic research: a tribute.- Hydrogen metabolism of green algae: discovery and early research-a tribute to Hans Gaffron and his coworkers.- Samuel Ruben's contributions to research on photosynthesis and bacterial metabolism with radioactive carbon. Contributions of Henrik Lundegardh.-Part IV Excitation Energy Transfer: Photosynthetic exciton theory in the 1960s.- Excitation energy trapping in anoxygenic photosynthetic bacteria.- Fluorescence lifetime, yield, energy transfer and spectrum in photosynthesis, 1950-1960.- Visualization of excitation energy transfer processes in plants and algae.-Plastoquinone redox control of chloroplast thylakoid protein phosphorylation and distribution of excitation energy between photosystems: discovery, background, implications.- Excitation transfer between photosynthetic units: the 1964 experiment.-Part V Reaction Centers: Research on photosynthetic reaction centers from 1932 to 1987.- Chlorophyll chemistry before and after crystals of photosynthetic reaction centers.- Electron donors and acceptors in the initial steps of photosynthesis in purple bacteria: a personal account.- My daily constitutional in Martinsried.- The two-electron gate in photosynthetic bacteria.- Steps on the way to building-blocks, 3-D crystalsand X-ray structural analysis of photosystem I and II of water-oxidizing photosynthesis. a personal account.- The identification of the Photosystem II reaction center: a personal story.- The isolated Photosystem II reaction center: first attempts to directly measure the kinetics of primary charge separation.- Discovery of pheophytin function in the photosynthetic energy conversion as the primary electron acceptor of Photosystem II.- Engine of life and big bang of evolution.- Role of bicarbonate at the acceptor side of Photosystem II.- Unraveling the PS I reaction center: a long history or the sum of many efforts.- Photosystem I reaction center: past and future.- P 430: a retrospective, 1971-2001.- Part VI Oxygen Evolution: Apparatus and mechanism of photosynthetic oxygen evolution: a personal perspective.- Period-four oscillations of the flash-induced oxygen formation in photosynthesis.- Period four oscillations in chlorophyll a fluorescence- Chloride and calcium in Photosystem II: from effects to enigma.- The bicarbonate effect, oxygen evolution, and the shadow of Otto Warburg.- Early indications for manganese oxidation state changes during photosynthetic oxygen production.-Part VII Light-harvesting and Pigment-protein Complexes: Purple bacterial light-harvesting complexes: From dreams to structures.- The Fenna-Matthews-Olson protein.- Physical separation of chlorophyll-protein complexes.-How the chlorophyll-proteins got their names.- Phycobiliproteins and phycobilisomes: the early observations.- Part VIII Electron Transport and ATP Synthesis: Discovery and characterization of electron transfer proteins in the photosynthetic bacteria.- Membrane-anchored cytochrome c as an electron carrier in photosynthesis and respiration: past, present and future of an unexpected discovery.- The Q-cycle:a personal perspective.- The isolation of functional cytochrome b6-f complex: From lucky encounter to rewardingexperience.- Ironies in photosynthetic electron transport

The light-induced oxidation of water by Photosystem II (PS II) of higher plants, algae and cyanobacteria is the main source of atmospheric oxygen. The discovery of the flash-induced period-four oscillations in the O2 evolution [Joliot P, Barbieri G and Chabaud R (1969) Photochem Photobiol 10: 309–329] has had a lasting impact on current photosynthesis research. Such oscillations were explained by introducing the cycle of flash-induced transitions of states of an oxygen evolving complex [Kok B, Forbush B and McGloin M (1970) Photochem Photobiol 11: 467–475]. In order to describe dampening of the oscillations in the O2 evolution the Kok model introduces misses, which characterize the failure to advance the S-states, and double hits, which characterize the two-step advancement of S-states. While the Kok model has been successfully used for over 30 years for interpretation of experimental data in photosynthesis, until recently there was no simple analytical solution for it. A much overdue analytical solution is presented here. Correlation of S-states transitions at the donor side of the PS II and QB transitions at the acceptor side leads to the recognition of two different reaction sequence cycles of PS II, so called cycles V and W [Shinkarev VP and Wraight CA (1993a) Proc Natl Acad Sci USA 90:1834–1838]. In each of these cycles the quantitative description of binary oscillations of the QB semiquinone can be obtained from the analytical solution for individual S-states. Standard application of the Kok model consists in finding misses and double hits from measured sequence of flash-induced O2 evolution. In alternative approach known kinetic and thermodynamic data are used to reconstruct period-four oscillations of O2 evolution. This general kinetic model allows calculation of all transition probabilities in the Kok model from first principles. The model predicts that misses in the V and W cycles are different. The general character of the model allows simultaneous consideration of different flash-induced oscillation phenomena at the donor and acceptor sides of PS II, without limitations on the number of states needed to be considered.

Response to Kinetic Models of Photosystem II Should Incorporate a Role for Q B-Nonreducing Reaction Centers

The functional role of cytochrome (cyt) b(559) in photosystem II (PSII) was investigated in H22K alpha and Y18S alpha cyt b(559) mutants of the cyanobacterium Synechocystis sp. PCC6803. H22K alpha and Y18S alpha cyt b(559) mutant carries one amino acid substitution on and near one of heme axial ligands of cyt b(559) in PSII, respectively. Both mutants grew photoautotrophically, assembled stable PSII, and exhibited the normal period-four oscillation in oxygen yield. However, both mutants showed several distinct chlorophyll a fluorescence properties and were more susceptible to photoinhibition than wild type. EPR results indicated the displacement of one of the two axial ligands to the heme of cyt b(559) in H22K alpha mutant reaction centers, at least in isolated reaction centers. The maximum absorption of cyt b(559) in Y18S alpha mutant PSII core complexes was shifted to 561 nm. Y18S alpha and H22K alpha mutant PSII core complexes contained predominately the low potential form of cyt b(559). The findings lend support to the concept that the redox properties of cyt b(559) are strongly influenced by the hydrophobicity and ligation environment of the heme. When the cyt b(559) mutations placed in a D1-D170A genetic background that prevents assembly of the manganese cluster, accumulation of PSII is almost completely abolished. Overall, our data support a functional role of cyt b(559) in protection of PSII under photoinhibition conditions in vivo.

Molecular oxygen evolution from water is a universal signature of oxygenic photosynthesis. Detection of the presence, speed and efficiency of the enzymatic machinery that catalyzes this process in vivo has been limited. We describe a laser-based fast repetition rate fluorometer (FRRF) that allows highly accurate and rapid measurements of these properties via the kinetics of Chl-a variable fluorescence yield (Fv) in living cells and leaves at repetition rates up to 10 kHz. Application to the detection of quenching of Fv is described and compared to flash-induced O2 yield data. Period-four oscillations in both Fv and O2, caused by stimulation of primary charge recombination by the O2 evolving complex (WOC) within Photosystem II (PS II), are directly compared. The first quantitative calculations of the enzymatic parameters of the Kok model (alpha - miss; beta - double hit; S-state populations) are reported from Fv data over a 5 kHz range of flash frequencies that is 100-fold wider than previously examined. Comparison of a few examples of cyanobacteria, green algae and spinach reveals that Arthrospira m., a cyanobacterium that thrives in alkaline carbonate lakes, exhibits the fastest water-splitting rates ever observed thus farin vivo. In all oxygenic phototrophs examined thus far, an unprecedented large increase in the Kok alpha and beta parameters occur at both high and low flash frequencies, which together with their strong correlation, indicates that PS II-WOC centers split water at remarkably lower efficiencies and possibly by different mechanisms at these extreme flash frequencies. Revisions to the classic Kok model are anticipated.

The effect of redox-active amines NH2R (R = OH or NH2) on the period-four oscillation pattern of oxygen evolution has been analyzed in isolated spinach thylakoids as a function of the redox state Si (i = 0, ..., 3) of the water oxidase. The following results were obtained: (a) In dark-adapted samples with a highly populated S1 state, NH2R leads via a dark reaction sequence to the formal redox state "S-1"; (b) the reaction mechanism is different between the NH2R species; NH2OH acts as a one-electron donor, whereas NH2NH2 mainly functions as a two-electron donor, regardless of the interacting redox state Si (i = 0, ..., 3). For NH2NH2, the modified oxygen oscillation patterns strictly depend upon the initial ratio [S0(0)]/[S1(0)] before the addition of the reductant; while due to kinetic reasons, for NH2OH this dependence largely disappears after a short transient period. (c) The existence of the recently postulated formal redox state "S-2" is confirmed not only in the presence of NH2NH2 [Renger, G., Messinger, J., & Hanssum, B. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. 1, pp 845-848, Kluwer, Dordrecht] but also in the presence of NH2OH. (d) Activation energies, EA, of 50 kJ/mol were determined for the NH2R-induced reduction processes that alter the oxygen oscillation pattern from dark-adapted thylakoids. (e) Although marked differences exist between NH2OH and NH2NH2 in terms of the reduction mechanism and efficiency (which is about 20-fold in favor of NH2OH), both NH2R species exhibit the same order of rate constants as a function of the redox state Si in the nonperturbed water oxidase: kNH2R(S0) greater than kNH2R(S1) much less than kNH2R(S2) much greater than kNH2R(S3) The large difference between S2 and S3 in their reactivity toward NH2R is interpreted to indicate that a significant change in the electronic configuration and nuclear geometry occurs during the S2----S3 transition that makes the S3 state much less susceptible to NH2R. The implications of these findings are discussed with special emphasis on the possibility of complexed peroxide formation in redox state S3 postulated previously on the basis of theoretical considerations [Renger, G. (1978) in Photosynthetic Water Oxidation (Metzner, H., Ed.) pp 229-248, Academic Press, London].

Flash-Induced Oxygen Evolution in Photosynthesis: Simple Solution for the Extended S-State Model that includes Misses, Double-Hits, Inactivation, and Backward-Transitions

In photosynthetic water oxidation performed in the water oxidizing center (WOC) of photosystem II (PSII), two water molecules are converted into one oxygen molecule and four protons through a light-driven cycle of intermediates called S states (S(0)-S(4)). To understand the molecular mechanism of water oxidation and the chemical nature of substrate intermediates, it is essential to determine the stoichiometry of proton release from substrate water at individual S-state transitions. In this study, we have monitored proton release during water oxidation by means of isotope-edited Fourier transform infrared (FTIR) spectroscopy. FTIR difference spectra upon successive flash illumination were measured using PSII core complexes from a thermophilic cyanobacterium Thermosynechococcus elongatus, which were suspended in a high concentration (200 mM) Mes buffer at pH 6.0. The spectra involved, in addition to protein bands, the bands of the Mes buffer that trapped virtually all protons from the WOC. Mes-only signals were extracted by subtracting the spectra measured in deuterated-Mes (Mes-d(12)). The flash-number dependence of the intensity increase of the isotope-edited Mes signal showed a clear period-four oscillation. By simulating the oscillation with different assumptions about miss factors, the proton release pattern was estimated to be 0.8-1.0:0.2-0.3:0.9-1.2:1.5-1.6 for the S(0)-->S(1)-->S(2)-->S(3)-->S(0) transitions. The effect of H/D exchange on the COOH region of proteins in FTIR difference spectra of the S-state cycle showed that protonation/deprotonation of carboxylic groups contributed little to the observed proton release pattern. Together with the present and previous FTIR results suggesting no involvement of also His and Cys side groups, it was concluded that proton release from substrate water takes place with a 1:0:1:2 stoichiometry, which is perturbed by partial protonation/deprotonation of side groups probably of Arg, Lys, or Tyr located nearby the WOC.

Correlation between flash-induced oxygen evolution and fluorescence yield kinetics in the 0 to 16 μs range in Chlorella pyrenoidosa during incubation with hydroxylamine

Many weed-controlling agents act by inhibiting the process of photosynthesis. Their mode of action is a displacement of the secondary quinone electron acceptor of photosystem II from its proteinaceous binding environment. This results in a blocking of the electron transport. Consequently plants are no longer able to produce ATP and to reduce NADP +, which eventually leads to starvation and death.In this thesis an attempt is made to characterize the interactions of herbicides with their binding environment. Several herbicides were used to measure binding kinetics in a wildtype and a triazine -resistant biotype of Common Lambsquarters, Chenopodium album L.In Chapter 1 a general introduction is given on photosynthesis, chloroplast structure and function, the chloroplast genome, and herbicide action and resistances.Chapter 2 describes the methods used to isolate chloroplasts and to measure oxygen production. Also the fluorescence induction measurements are described, which can be used as a rapid method to discriminate between herbicide-sensitive and -resistant plants. Procedures for the simultaneous isolation of total plant nucleic acids, for the separation of DNA and RNA and for the respective sequence analyses are outlined.In Chapter 3 a model is described which can be used to simulate flash- induced oxygen production by isolated thylakoids. In the literature a model was described before, which explains the observed 4-step oscillation in flash-induced oxygen evolution by assuming the existence of 4 different S-states of the oxygen evolving complex. This model is refined here by assuming the values of the miss parameters αto be dependent on both the redox state of the quinone acceptor complex (Q A .Fe.Q B ) of photosystem II and on the S-state transition involved. The best fit between theoretical and experimental oxygen evolution patterns is obtained when 4 different miss parameters are distinguished, corresponding with the 4 S-state transitions of the oxygen evolving complex. The values of two of these αparameters, notably those for the 2 ndand 4 thflash, are found to approach 0, while the other two have values of about 0.2 and 0.4. The value for the double hit parameter βis found to be about 0.06. The oxygen evolving complexes of thoroughly dark adapted chloroplasts are found to be for almost 100 % in the most stable single oxidized state S 1 . A fraction of 30 % of the reaction centers is assumed to be connected to a oneelectron donor D, which is able to reduce the S 2 - or S 3 -state of the oxygen evolving complex with a half time of 2-3 seconds.In Chapter 4 the derived model is used to determine the exchange ,parameters of herbicides with the secondary acceptor Q B . These exchanges, which influence the oxygen evolution patterns, can be determined by comparing experimental and theoretical patterns for various herbicide concentrations and flash frequencies. The I 50 -values derived from the measurements are in agreement with values measured with other methods. The resistance to triazine compounds proved to be caused by an increase in the herbicide release rather than by a lower binding rate. Configuration around a chiral carbon atom, present in two pairs of isomers of cyanoacrylate inhibitors, has a strong influence on inhibition properties. The differences are largely due to alterations in the release kinetics. This observation suggests that herbicide binding is determined mainly by physical properties, like e.g. hydrophobicity. A stationary binding, resulting in a significant electron transport inhibition, requires a strict molecular shape.In Chapter 5 partial sequence analyses from DNA and mRNA isolated from a Chenopodium album wildtype and a triazine -resistant biotype are presented. The only difference found was an adenine to guanine point mutation resulting in a serine to glycine alteration at position 264 in the D1 (herbicide binding) protein.The thesis is concluded with Chapter 6, the general discussion, in which an alternative explanation for redox reactions at the photosystem II acceptor side is presented. In this model the non-heme iron, located between the primary and the secondary acceptor of photosystem II, is proposed to play an active role in in vivo electron transport.

Oxygen evolution by photosynthetic water oxidation has shaped life on planet Earth. This unique biological reaction may provide important clues for developing artificial devices (‘artificial leaves’) for splitting water into O2 and H2 by visible light. In this chapter, we summarize the latest structural and mechanistic information on photosystem II, its water-oxidizing complex (WOC), and O2 formation from water at its Mn4CaO5 cluster. Structural information on the WOC derived by X-ray crystallography, X-ray spectroscopy, EPR/ENDOR, and theoretical approaches are compared and discussed in detail. After a description of the basic mechanism of water-oxidation (Kok model), our knowledge about the storage of oxidizing equivalents, substrate water-binding, the release of the products O2 and H+, and the energetics of water-oxidation is described. The near electroneutrality of the water-splitting process via coupling of electron and proton transfer steps and the stabilization of the initial light-driven charge separation by spatial separation and heat dissipation are emphasized as important design principles for coupling fast photochemistry with comparatively slow chemical reactions. The chapter concludes with a discussion of recent DFT-based mechanistic proposals for photosynthetic water oxidation, and with an attempt to identify lessons for the design of artificial systems.

Thermodynamically accurate modeling of the catalytic cycle of photosynthetic oxygen evolution: A mathematical solution to asymmetric Markov chains

Photosynthetic water oxidation, where water is oxidized to dioxygen, is a fundamental chemical reaction that sustains the biosphere. This reaction is catalyzed by a Mn4Ca complex in the photosystem II (PS II) oxygen-evolving complex (OEC): a multiprotein assembly embedded in the thylakoid membranes of green plants, cyanobacteria, and algae. The mechanism of photosynthetic water oxidation by the Mn4Ca cluster in photosystem II is the subject of much debate, although lacking structural characterization of the catalytic intermediates. Biosynthetically exchanged Ca/Sr-PS II preparations and x-ray spectroscopy, including extended x-ray absorption fine structure (EXAFS), allowed us to monitor Mn-Mn and Ca(Sr)-Mn distances in the four intermediate S states, S0 through S3, of the catalytic cycle that couples the one-electron photochemistry occurring at the PS II reaction center with the four-electron water-oxidation chemistry taking place at the Mn4Ca(Sr) cluster. We have detected significant changes in the structure of the complex, especially in the Mn-Mn and Ca(Sr)-Mn distances, on the S2-to-S3 and S3-to-S0 transitions. These results implicate the involvement of at least one common bridging oxygen atom between the Mn-Mn and Mn-Ca(Sr) atoms in the O-O bond formation. Because PS II cannot advance beyond the S2 state in preparations that lack Ca(Sr), these results show that Ca(Sr) is one of the critical components in the mechanism of the enzyme. The results also show that Ca is not just a spectator atom involved in providing a structural framework, but is actively involved in the mechanism of water oxidation and represents a rare example of a catalytically active Ca cofactor.

Probing S-state advancements and recombination pathways in photosystem II with a global fit program for flash-induced oxygen evolution pattern