Kok Model Research Articles

Binary oscillations in the Kok model of oxygen evolution in oxygenic photosynthesis

The flash-induced kinetics of various characteristics of Photosystem II (PS II) in the thylakoids of oxygenic plants are modulated by a period of two, due to the function of a two-electron gate in the electron acceptor side, and by a period of four, due to the changes in the state of the oxygen-evolving complex. In the absence of inhibitors of PS II, the assignment of measured signal to the oxygen-evolving complex or to quinone acceptor side has frequently been done on the basis of the periodicity of its flash-induced oscillations, i.e. four or two. However, in some circumstances, the period four oscillatory processes of the donor side of PS II can generate period two oscillations. It is shown here that in the Kok model of oxygen evolution (equal misses and equal double hits), the sum of the concentrations of the S 0 and S 2 states (as well as the sum of concentrations of S 1 and S 3 states) oscillates with period of two: S 0+S 2→S 1+S 3→S 0+S 2→S 1+S 3. Moreover, in the generalized Kok model (with specific miss factors and double hits for each S-state) there always exist such ε0, ε1, ε2, ε3 that the sum ε0[S0] + ε1[S1] + ε2[S2] + ε3[S3] oscillates with period of two as a function of flash number. Any other coefficients which are linearly connected with these coefficients, % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGak0dh9WrFfpC0xh9vqqj-hEeeu0xXdbba9frFj0-OqFf% ea0dXdd9vqaq-JfrVkFHe9pgea0dXdar-Jb9hs0dXdbPYxe9vr0-vr% 0-vqpWqaaeaabiGaciaacaqabeaadaqaaqaaaOqaaiqbew7aLzaaja% aaaa!3917!\[\hat \varepsilon \]i = c1εi + c2, also generate binary oscillations of this sum. Therefore, the decomposition of the flash-induced oscillations of some measured parameters into binary oscillations, depending only on the acceptor side of PS II, and quaternary oscillations, depending only on the donor side of PS II, becomes practically impossible when measured with techniques (such as fluorescence of chlorophyll a, delayed fluorescence, electrochromic shift, transmembrane electrical potential, changes of pH and others) that could not spectrally distinguish the donor and acceptor sides. This property of the Kok cycle puts limits on the simultaneous analysis of the donor and acceptor sides of the RC of PS II in vivo and suggests that binary oscillations are no longer a certain indicator of the origin of a signal in the acceptor side of PS II.

Oxidation states of the manganese cluster during the flash-induced S-state cycle of the photosynthetic oxygen-evolving complex.

The Mn K-edge x-ray absorption spectra for the pure S states of the tetranuclear Mn cluster of the oxygen-evolving complex of photosystem II during flash-induced S-state cycling have been determined. The relative S-state populations in samples given 0, 1, 2, 3, 4, or 5 flashes were determined from fitting the flash-induced electron paramagnetic resonance (EPR) multiline signal oscillation pattern to the Kok model. The edge spectra of samples given 0, 1, 2, or 3 flashes were combined with EPR information to calculate the pure S-state edge spectra. The edge positions (defined as the zero-crossing of the second derivatives) are 6550.1, 6551.7, 6553.5, and 6553.8 eV for S0, S1, S2, and S3, respectively. In addition to the shift in edge position, the S0--> S1 and S1--> S2 transitions are accompanied by characteristic changes in the shape of the edge, both indicative of Mn oxidation. The edge position shifts very little (0.3 eV) for the S2--> S3 transition, and the edge shape shows only subtle changes. We conclude that probably no direct Mn oxidation is involved in this transition. The proposed Mn oxidation state assignments are as follows: S0 (II, III, IV, IV) or (III, III, III, IV), S1 (III, III, IV, IV), S2 (III, IV, IV, IV), S3 (III, IV, IV, IV).

Analyses of pH-Induced Modifications of the Period Four Oscillation of Flash-Induced Oxygen Evolution Reveal Distinct Structural Changes of the Photosystem II Donor Side at Characteristic pH Values

This study presents a thorough analysis of the reaction pattern of flash-induced oxygen evolution in spinach thylakoids as a function of pH (4.5 < or = pH < or = 9) and the redox state of tyrosine YD in polypeptide D2. Evaluation of the experimental data within the conventional Kok model [Kok, B., Forbush, B., & McGloin, M. (1970) Photochem. Photobiol. 11, 457-475] led to the following results: (1) the probability of the miss factor is strongly pH dependent (with a pronounced minimum near neutral pH) while the double hit factor is less affected; (2) a marked increase of the apparent S0 population arises at alkaline pH in dark-adapted samples where most of the YD is reduced, but this effect is absent if the percentage of PS II containing the oxidized form YDox is high; and (3) the lifetimes of S2 and S3 exhibit a characteristic pH dependence that is indicative of conformational changes of functional relevance within the water-oxidizing complex and its environment; (4) the kinetic interaction of redox states S2 and S3 with YD is characterized by a change of its behavior at a threshold pH of 6.5-7.0; and (5) at acidic pH values the extent of S2 and S3 reduction by YD decreases concomitant with the occurrence of a very fast decay kinetics. On the basis of a detailed discussion of these results and data from the literature, the water oxidase is inferred to undergo structural changes at pH values of 5-5.5 and 6.5-7.0. These transitions are almost independent of the redox state Si and modify the reaction coordinates of the water oxidase toward endogenous reductants.

A Study on the Life Time of the S3-State in the Filamentous Cyanobacterium Oscillatoria chalybea

In the filamentous cyanobacterium Oscillatoria chalybea deactivation of the S-states starting from steady-state conditions in which S0 = S1 = S2 = S3 = 25% reveals that S3 deactivates to a finite level of approx. 10%. This level is reached under normal conditions between 10-15 seconds. This quasi metastable S3 meets all requirements for S3 in that one flash eliminates this redox conditions to give S4 and therewith molecular oxygen. An analysis of the cyanobacterial S-state system in the 5-state Kok model shows that the S-state population in the dark adapted sample contains no contribution from S-1 or a more reduced condition which under normal conditions is the case for Chlorella or higher plant chloroplasts. Hence under standard conditions, the Oscillatoria condition is a pure Kok-4-condition in which S0 is the most reduced state. Under these conditions S2 seems to deactivate to S1 and S3 to S2 and to a smaller extent to S0. In the presence of the ADRY-reagent Ant-2-p (2-(3-chloro-4-trifluoromethyl)- anilino-3,5-dinitrothiophene) introduced by Renger (Biochim. Biophys. Acta 256,428,1972), which is supposed to specifically act on the S3-state (and thereby on S2), not only the deactivation kinetic of S3 (and S2) is accelerated (hence the life time of the S3-state is shortened), but also the level of metastable S3 becomes practically zero. An analysis of the deactivation pattern shows that the agent changes the mode of deactivation of the entire system. Thus, it is seen that after deactivation of a sample in presence of this agent the dark population of S-states contains the more reduced redox condition S-1 It looks as if in this condition S2 deactivates not only to S1 but also to an appreciable extent by two steps to S-1 Another agent ABDAC (alkyl-benzyl-dimethyl-ammoniumchloride) seems to lengthen the lifetime of the S2 and S3 condition in this cyanobacterium by apparently acting on the membrane condition.

Earlier researches on the mechanism of oxygen evolution: A personal account

A brief overview is given of the research which led to the discovery of the period-4 oscillations of the flash-induced oxygen production and which is the basis of the generally accepted Kok's model for water splitting and oxygen evolution.In this paper I discuss the earlier work of the groups of Robert Emerson, James Franck, C. P. Whittingham, and myself in relation to the development of new techniques for the detection of photosynthetic oxygen evolution. Also discussed are various hypotheses and speculations related to the concept of a priming photoreaction which is required for oxygen evolution. Finally, I discuss my long scientific collaboration with Bessel Kok which led to the elaboration by Kok of the classical model in which the formation of oxygen requires the sequential accumulation of four positive charges on the same photochemical center.

Absorbance difference spectra of the S-state transitions in Photosystem II core particles

Redox changes of the oxygen evolving complex in PS II core particles were investigated by absorbance difference spectroscopy in the UV-region. The oscillation of the absorbance changes induced by a series of saturating flashes could not be explained by the minimal Kok model (Kok et al. 1970) consisting of a 4-step redox cycle, S0 → S1 → S2 → S3 → S0, although the values of most of the relevant parameters had been determined experimentally. Additional assumptions which allow a consistent fit of all data are a slow equilibration of the S3 state with an inactive state, perhaps related to Ca(2+)-release, and a low quantum efficiency for the first turnover after dark-adaptation. Difference spectra of the successive S-state transitions were determined. At wavelengths above 370 nm, they were very different due to the different contribution of a Chl bandshift in each spectrum. At shorter wavelengths, the S1 → S2 transition showed a difference spectrum similar to that reported by Dekker et al. 1984b and attributed to an Mn(III) to Mn(IV) oxidation. The spectrum of absorbance changes associated with the S2 → S3 transition was similar to that reported by Lavergne 1991 for PS II membranes. The S0 → S1 transition was associated with a smaller but still substantial absorbance increase in the UV. Differences with the spectra reported by Lavergne 1991 are attributed to electrostatic effects on electron transfer at the acceptor side associated with the S-state dependence of proton release in PS II membranes.

Improvement of four sigma analysis for the investigation of oxygen evolution by Photosystem II

We present here an improvement to the analysis of oxygen evolution with four sigma coefficients (4-S) by computing z, the sum of the S-state probabilities, which was introduced earlier (Delrieu and Rosengard 1987, Biochim Biophys Acta 892: 163-171). We demonstrate that z is equal to the ratio of two consecutive Mean Y (the estimation of the steady state oxygen production based on local properties) found by three sigma analysis. The quantity z is useful for computing double-hits, and for showing the inactivation/activation processes of PS II complexes. Three sigma analysis assumes z=1 exactly; since this is not verified, it is argued that four sigma analysis is closer to the real workings of the water oxidizing complex. Oxygen evolution can then be interpreted in the frame of a modified Kok's model where the sum of the probabilities equals z. We therefore suggest that the closer fitting of four sigma analysis to oxygen production data is not simply due to an extra, unnecessary variable, but to the fact that PS II complexes can be inactivated and reactivated under flashing light. Finally, in order to facilitate the use of four sigma analysis, a computer program is made available upon request.

Oxygen release time in leaf discs and thylakoids of peas and Photosystem II membrane fragments of spinach

The half-time of O 2 release in Chlorella, thylakoids and Photosystem II membranes has generally been found to be 0.9 to 3.0 ms, but recently Plijter et al. (Biochim. Biophys. Acta 935 (1988) 299–311) reported it to be between 30 to 130 ms. This work presents a new method, using a Clark electrode, for measuring O 2 release times in leaf discs, thylakoids and Photosystem II membranes. The sample is illuminated with saturating pulses of light of 3 μs to 200 ms duration given at 5 Hz. Presuming the Kok model of four charge transfers per O 2 evolved, the O 2 release time could be calculated from the time needed to observe the evolution of one O 2 less 4-times the turnover time of the charge transfer reaction. Upper bounds for the half times of O 2 release were found to be 6, 11, and 5 ms in thylakoids, leaf discs, and Photosystem II membrane fragments, respectively. In thylakoids, using a bare platinum electrode operated under weak bias conditions, the half-time of O 2 signal rise is 2.7 ms. The rate of rise of this O 2 signal could be slowed by treating the thylakoids with nitrate or formate. The turnover time of the oxygen evolution system, the minimum time between evolution of oxygen molecules, of Photosystem II that is necessary to support light saturated photosynthesis was measured to be 16, 28 and 22 ms in thylakoids, leaf discs and Photosystem II membrane fragments, respectively. The rate limitation for photosynthesis is not the O 2 release time but is the rate of charge transfer through or beyond the plastoquinone pool.

Charge equilibrium between the water-oxidizing complex and the electron donor tyrosine-D in Photosystem II

The decay kinetics for the S2 and S3 states of the water-oxidizing complex have been measured with an unmodulated Joliot-type oxygen electrode in isolated spinach thylakoids. The S2 and S3 states decay biphasically (Vermaas, W.F.J., Renger, G. and Dohnt, G. (1985) Biochim. Biophys. Acta 764, 194–202) with half-decay times of 1–1.5 s and 30–35 s at room temperature. The proportion of the fast phase is negligible in preilluminated thylakoids but increases during dark adaptation to 22–24% for both S2 and S3. This process, t12 ≈ 10 min, is accompanied with the conversion of the S0 state to S1 in about 25% of the centers. Chemical reduction of tyrosine-D+, which gives rise to the EPR Signals IIslow, by dichlorphenolindophenol/ascorbate increases the proportion of the fast decaying phase of S2 and S3 to about 70–80%. The decay of S2 is accompanied by the accumulation of S1 and the decay of S3 results in a transient increase of S2. These data led us to conclude that the fast phase in the S2 and S3 decay is correlated with one-electron donation from tyrosine-D to the water-oxidizing complex located within the same center. This process results in the S3D → S2D+ (→ S1D+) and S2D → S1D+ univalent sequences of deactivating reactions. The electron transfer from tyrosine-D to the S2 and S3 states is strongly temperature-dependent and shows 0.46 and 0.49 eV activation energy, respectively, over the +8 to +37°C temperature range. The deactivation process which is reflected by the slower phase of S2 and S3 decay has an activation energy of 0.65 and 0.76 eV, respectively. An extension of the Kok model of oxygen evolution is also presented taking into account the effect of fast electron donation from tyrosine-D to the water-oxidizing complex.

DIFFERENCE SPECTRA OF THE OXIDIZED INTERMEDIATES IN THE PHOTOSYNTHETIC OXYGEN‐EVOLVING SYSTEM: EVIDENCE FOR A SMALL S0→S1 ABSORPTION CHANGE

Abstract—A consensus has emerged in the recent literature on the fact that the UV difference spectrum of the first oxidation step (S0→ S1) of the photosynthetic oxygen‐evolving complex is significantly different and generally smaller than the spectra of the higher oxidation steps (S0→ S1and S2→ S3). Discrepancies still persist, however, notably in the 300 nm region where the S0→ S1 change was either reported to be markedly smaller than the other changes, or, at variance, to have a similar amplitude. A novel approach is proposed here for estimating the ratio of these changes, requiring no estimate of the Kok model parameters, such as the initial S0/S1ratio, or damping coefficients. This was achieved by comparing the absorption difference between two fully deactivated states, differing only in their S0/S1, distribution, with the flash‐induced changes measured from these states. The results show that, at two wavelengths around 300 nm, the S0→ S1 change is at least 4 times, and probably 5–6 times smaller than the S0→ S1change.

Effect of abscisic acid on the photosynthetic oxygen evolution in barley chloroplasts

In vivo effect of abscisic acid (ABA) on photosynthetic oxygen evolution was investigated in barley chloroplasts. The most important kinetic parameters of O2-producing reactions were changed. The results show inhibition of the O2-flash yields at ABA concentrations of 10 μmol/l and 100 μmol/l and an increase in the degree of damping of the oscillations. ABA has a marked effect on the distribution of the oxygenevolving centers in S0 and S1 states and on sum of the centers (S0+S1) estimated according to the Kok model. In addition, the amplitude and the shape of the initial oxygen burst under continuous illumination are also significantly altered. At a concentration of 100 μmol/l, ABA strongly inhibits Hill reaction activity measured by DCPIP reduction. The results cannot be explained by the hypothesis of socalled "stomata effect". On the other hand, no effects were observed on the investigated parameters in experiments involving ABA applied in vitro to isolated chloroplasts. It is hypothesized that ABA disrupts the granal chloroplasts structure and raises the degree of participation of the cooperative mechanism of O2-evolution connected with the functioning of PS IIβ centers in the stroma situated thylakoids.

Photosystem II activity and triazine resistance in weeds

The oxygen-evolving properties of broken chloroplasts isolated from biotypes of Chemopodium album and Amaranthus retroflexus that were either sensitive or resistant to s-triazine herbicides were compared. The pattern of oxygen flash yields produced by herbicide-sensitive, dark-adapted chloroplasts of either species was reminiscent of that found with spinach chloroplasts. In contrast, dark-adapted chloroplasts isolated from the herbicide-resistant biotypes exhibited a highly damped oxygen flash pattern in which there was significant oxygen released after the first light flash. Analysis of these results with the Kok model of photosystem II (Kok, B., Forbush, B., and McGloin, M. 1970. Photochem. Photobiol. 11: 457–475) suggested that the unusual properties of the resistant organelles were due to the survival of significant amounts of S3 and S2 states during dark adaptation and to a higher proportion of inactive photosystem II reaction centres during each light flash. Deactivation experiments verified the suggestion that the S3 and S2 states more readily survive a 10-min dark period in resistant organelles. Information about electron transport on the oxidizing side of photosystem II was obtained with a modulated oxygen electrode and suggested that there was no difference between the two biotypes in the value of the rate constant of the reaction that limits the rate of electron transport between the water-splitting step and photosystem II.

Fundamental differences between period-4 oscillations of the oxygen and fluorescence yield induced by flash excitation in inside-out thylakoids

The period-4 oscillation pattern of the chlorophyll a fluorescence induced by flash excitation of dark-adapted inside-out thylakoids was compared to the oxygen-yield pattern obtained under the same conditions of flash energy and adaptation to dark. For a quantitative comparison, a least-square best fitting method was used. In the Kok model, besides the miss and the double hit parameters α and β, it has been necessary to introduce a new variable, z, which describes a decrease of the apparent number of active centers by a factor z at each flash of a series. We demonstrate that under similar flash conditions, the oscillation pattern of fluorescence yield (measured 80 ms after each of a series of flashes) fundamentally differs from that of the O 2 yield. After a 3 h dark-adaptation period, the oscillation pattern of the oxygen yield presents a change of phase as a function of flash number, associated with the damping coming from misses ( α ⩾ 0.12). In contrast, under the same conditions, the fluorescence oscillation showed no phase change. The same oscillation pattern was repeated every four flashes with a decreasing amplitude. The numerical fitting of the oscillation pattern of fluorescence yields no misses, no double hits, but a progressive vanishing of fluorescent centers in a proportion 1 − z F. The higher the flask energy the faster the decrease of the oscillating part of fluorescence. The product of flash energy, I, by the flash number necessary to decrease the fluorescence oscillation by half, N 1 2 , was found to be independent of the exciting flash energy. The initial amplitude of the fluorescence oscillation was not light saturated in our experimental conditions, though the flash energy used was at least 5-times higher than necessary to saturate the transitions S i of O 2 evolution (except S 2 → S 3). All these results show that the fluorescence oscillation with period 4 is not related to the S i states of most O 2-evolving centers. The existence of a 4-step reaction series (the S′ i states), functioning in parallel to the S i states, is postulated. The S′ i states seen by fluorescence are controlled by the limited amount of a component stored in the dark, which is exhausted after each flash of a series in a quantity proportional to flash energy.

Flash-induced oxygen yield sequences during the life cycle of Chlorella

(i) The pattern of O2 flash yields in the first 4 hours of the life cycle cannot be described by the simple Kok model without additional assumptions. (ii) The miss coefficient α in the mature cells in significantly higher than that in the autospores, its change occurring at the expense of the single-hit coefficient β. Computer simulation yielded α values of 0.29 and 0.23 and β values of 0.66 and 0.72 in the light and dark, respectively. (iii) The onset of light at the beginning of the cycle drastically changes the equilibrium distribution of the S states in the dark; the ratio S0/S1 increases from 30/70 to 50/50 in 1 h, and is restored not earlier than in the 6th hour. (iv) In the presence of 1 mmol/l p-benzoquinone, the alga shows pronounced and long-lasting oscillations in the O2 yield sequences, independently of the time of the life cycle. This means that the O2-evolving system itself is always present and equally efficient throughout the life cycle. Limits imposed on its activity (mainly in the first 4 hours) are clearly of an external nature. The redox potential of the inner thylakoid space is presumably involved.

Studies on the nature of the water-oxidizing enzyme: III. Spectral characterization of the intermediary redox states in the water-oxidizing enzyme system Y

Spectral changes in the ultraviolet region (250–380 mn) that are caused by redox transitions within the water-oxidizing enzyme system Y were analyzed by measuring time-resolved (1–2 μs) absorption changes in dark-adapted PS II membrane fragments. In order to eliminate effects due to the interference with binary oscillation at the acceptor side the experiments were performed in trypsinized (at pH = 6.0) samples with 0.4 mM K 3[Fe(CN) 6] as exogenous oxidant. In the presence of 10 mM CaCl 2 PS II-membrane fragments trypsinized at pH = 6.0 are fully competent in oxygen evolution compared with the control (measured as average oxygen yield per flash). It was found: (1) Excitation of dark-adapted samples with a train of saturating laser flashes (7 ns full width at half maximum, FWHM) induces absorption changes with resolved kinetics in the micro- and millisecond range. Except for the kinetically unresolved initial amplitude the other transient compoents are strongly dependent (in extent and half-life time) on the flash number. (2) After elimination of the oxygen-evolving capacity by 1 mM hydroxylamine (NH 2OH) binary oscillation are completely lacking of absorption changes at 325 nm. The remarkably smaller absorption change in the first flash can be explained by partial oxidation of acceptor ‘C400’ due to K 3[Fe(CN) 6]. (3) A significantly different pattern of the absorption changes is observed if phenyl- p-benzoquinone is used instead of K 3[Fe(CN) 6]. Based on the oscillations of the initial amplitude of the absorption changes, exogenous quinones are assumed to give rise to a more complicated reaction pattern. (4) The spectral analysis performed on the basis of Kok's model (Kok, B., Forbush, B. and McGloin, P.M. (1970) Photochem. Photobiol. 11, 457–475) by the use of α, β and the apparent [ S 0] [ S 1] ratio experimentally determined via oxygen-yield measurement leads to the separation of the difference spectra Δε( S i → S i + 1 ) for the redox transitions in system Y. The obtained difference spectra are almost identical for the S 0 → S 1 and S 2 → S 3 transitions, while for S 1 → S 2 a markedly different spectrum has been observed. The deviations are especially pronounced in the range around 325 nm. (5) The relaxation kinetics of absorption changes at 325 nm that are characterized by a 1 ms half-life time oscillate in their amplitude synchronously with the oxygen yield in a flash train. The amplitude of these kinetics as a function of wavelength corrected for the Z ox Z difference spectrum closely resembles the calculated spectrum for the S 3 → S 0 transition. Based on the experimental data a model is proposed for the molecular mechanism of photosynthetic water oxidation that is an extension of our previous hypothesis (Renger, G. (1977) FEBS Lett. 81, 223–228). Accordingly, the catalytic site for water oxidation is assumed to be a binuclear manganese center which undergoes redox reactions at the manganese centers only during S 1 → S 2 and S 3 → S 0 transitions. The implications of this model are discussed.

Binding and release kinetics of inhibitors of Q −A oxidation in thylakoid membranes

Oxygen flash yield patterns of dark adapted thylakoid membranes as measured with a Joliot-type O 2-electrode indicate that inhibitors that block the oxidation of the reduced primary quinone Q − A of Photosystem II vary greatly in the rate of binding to and release from the inhibitor / Q B binding environment. The ‘classical’ Photosystem-II herbicides like diuron and atrazine exhibit slow binding and release kinetics, whereas, for example, phenolic inhibitors, o-phenanthroline and synthetic quinones are exchanging quite rapidly with Q B (about once per second or faster at inhibitor concentrations causing about 50% inhibition of O 2 evolution). No general relationship between the efficiency of the inhibitor and the exchange rate is observed; it depends mainly on the type of inhibitor. Based on the classical Kok model, equations are derived in order to calculate oxygen yields evolved by thylakoids in single-turnover flashes as a function of the rate constants of inhibitor binding to and release from the inhibitor / Q B binding environment in the presence of an oxidized or semireduced Q A · Q B or Q A · inhibitor complex. Fitting of theoretical and experimental values yields that o-phenanthroline binds much faster to an oxidized than to a semireduced Q A · Q B complex. This fits very well with the hypothesis that the Q − B affinity to the site is much higher than that of Q B. In the case of i-dinoseb, however, inhibitor / quinone exchange seems to occur mainly in the semiquinone state. Possibilities to explain this result are discussed.

The influence of anions on oxygen evolution by isolated spinach chloroplasts

A study has been made of the onset of chloride deprivation on the oxygen-evolving characteristics of isolated spinach chloroplasts. Using a modulated oxygen electrode it is found that the type of inhibition depends on the anion replacing chloride in the bathing medium. With nitrate a large increase in phase lag accompanies a relatively small inhibition which can be shown to be consistent with a decrease in the rate constant of the reaction which limits the rate of electron transport between water and Photosystem II. With sulphate there is a very small phase change but a larger inhibition which suggests that replacing chloride with sulphate in an electron-transport chain shuts off that chain. With acetate there is a moderate increase in phase lag and the largest inhibitory effect. The phase-lag increase suggests that acetate is affecting the same chloride-sensitive site as nitrate. However, the inhibition cannot be explained by this effect alone and points to the existence of a second chloride-sensitive site. Of the four forward reactions associated with the Kok model of oxygen evolution (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) only S∗ 3 → S 0 is slowed down when chloride is replaced by nitrate. This reaction is not slowed down by replacing chloride with sulphate.

Computation of S-state binding energy and wave functions in a Saxon—Wood potential

Forty-three years ago, Kok and coworkers introduced a phenomenological model describing period-four oscillations in O2 flash yields during photosynthetic water oxidation (WOC), which had been first reported by Joliot and coworkers. The original two-parameter Kok model was subsequently extended in its level of complexity to better simulate diverse data sets, including intact cells and isolated PSII-WOCs, but at the expense of introducing physically unrealistic assumptions necessary to enable numerical solutions. To date, analytical solutions have been found only for symmetric Kok models (inefficiencies are equally probable for all intermediates, called “S-states”). However, it is widely accepted that S-state reaction steps are not identical and some are not reversible (by thermodynamic restraints) thereby causing asymmetric cycles. We have developed a mathematically more rigorous foundation that eliminates unphysical assumptions known to be in conflict with experiments and adopts a new experimental constraint on solutions. This new algorithm termed STEAMM for S-state Transition Eigenvalues of Asymmetric Markov Models enables solutions to models having fewer adjustable parameters and uses automated fitting to experimental data sets, yielding higher accuracy and precision than the classic Kok or extended Kok models. This new tool provides a general mathematical framework for analyzing damped oscillations arising from any cycle period using any appropriate Markov model, regardless of symmetry. We illustrate applications of STEAMM that better describe the intrinsic inefficiencies for photon-to-charge conversion within PSII-WOCs that are responsible for damped period-four and period-two oscillations of flash O2 yields across diverse species, while using simpler Markov models free from unrealistic assumptions.

A Study on Oxygen Evolution and on the S-State Distribution in Thylakoid Preparations of the Filamentous Blue-Green Alga Oscillatoria chalybea

When thylakoid preparations of the filamentous blue-green alga Oscillatoria chalybea are exposed to short (2 or 8 μs) saturating light flashes, the oxygen evolution pattern can be distinguished in several respects from the one usually observed in Chlorella. Thus, it appears that a substantial electrochemical signal is already seen under the first flash with maximal flash yield always occurring under the fourth flash. This refers to dark adapted preparations (up to 60 min). Fitting of such an experimental sequence in the 4-state Kok model yields an S-state population consisting of 36-41% S0, 40-49% S1, 1-10% S2 and up to 13% S3. No abnormality under the first flash is seen in such preparations. Characteristic for sequences with Oscillatoria prepara­tions is a high level of misses which are in the region of 25 per cent, whereas double hits do not seem to play a substantial role in the damping of such sequences. The existence of metastable S3, anyway inconsistent with the coherent Kok model, is not confirmed by mass spectrometry. No 18O2 seems to be evolved under the first flash from Oscillatoria thylakoids suspended in 50% H2 18O. although, when judged from the absolute amperometric signal amplitude, mass spectrometric detection of O2 should have been possible. With the same method we are fully able to detect 18O2 under the second flash in Chlorella vulgaris. In Chlorella this is true for experimental conditions in which the amperometric signal amplitude under the second flash is even smaller than those under the first or second flash in Oscillatoria. The attempt to correlate the amperometrie signal observed under the first flash with a photoinhibition of respiration in our pro­karyotic organism was not successful. However, the attempt to incorporate the phenomenon in the coherent Kok model shows that the Oscillatoria sequence fully resembles those with Chlorella, if the first flash signal and 40-50% of the signal observed under the second flash is simply removed from the sequence. The remaining sequence exhibits the usual properties known from Chlorella or higher plant chloroplasts. If one assumes contribution of the reduced state S-1 to the dark population of S-states, a fit in the five rank Kok model yields correct adjustments with a S-state distribution of 6-20% S-1, 31-40% S0, 49-54% S1, 0% S2 and 0% S3 which would be fully consistent with the Kok model and corresponds to the distribution observed with Chlorella or higher plant chloroplasts. The question what the first electrochemical signal is due to remains unanswered.

The interaction of nitrite with photosynthetic electron transport under anaerobic conditions

Chlorella cells were examined in a modulated oxygen polarograph under aerobic and anaerobic conditions. At light intensities below about 600 ergs · cm −2 · s −1 of 650 nm light, the oxygen yield and phase lag are lower under anaerobic conditions. Addition of 25 mM sodium nitrite increases both these parameters to values close to those found in the presence of oxygen. It is proposed that nitrite is reduced by Photosystem I thus diverting electrons from the cyclic electron transport pathway. The intersystem electron transport chain becomes more oxidized and this suppresses a backflow of electrons to the oxidizing side of Photosystem II, hence increasing the oxygen yield and the phase lag. The removal of oxygen from the bathing medium also alters the response of dark adapted Chlorella to a series of saturating light flashes. In terms of the Kok model of Photosystem II (Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475) there is a large increase in the parameter α. Addition of nitrite reverses this change and virtually restores the response seen in the presence of oxygen. The deactivation of the S 2 state is greatly speeded up in the absence of oxygen but the addition of nitrite again reverses this.