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.     

Period-four oscillations of the flash-induced oxygen formation in photosynthesis*

In this minireview, the earlier researches that led to the discovery of the period-four oscillations of the flash-induced oxygen formation are presented. It also includes the background of the classical model proposed by Bessel Kok, in which the formation of oxygen requires the sequential accumulation of four positive charges on the donor side of the same reaction center. This revised version was published online in August 2006 with corrections to the Cover Date.     

Oxygen-evolving system and secondary quinonic acceptors are highly reduced in dark adapted Euglena cells: A thermoluminescence study

Characteristics of thermoluminescence glow curves were compared in three types of Euglena cells: (i) strictly autotrophic, Cramer and Myers cells; (ii) photoheterotrophic cells sampled from an exponentially growing culture containing lactate as substrate repressing the photosynthetic activity; (iii) semiautotrophic cells, sampled when the lactate being totally exhausted, the photosynthesis was enhanced.In autotrophic and semiautotrophic cells, composite curves were observed after series of two or more actinic flashes fired at –10°C, which can be deconvoluted into a large band peaking in the range 12–22°C and a smaller one near 40°C, This second band presents the characteristics of a typical B band (due to S_2/3Q_B ^- recombination), whereas the first one resembled the band, shifted by -15–20°C, which is observed in herbicide resistant plants. The amplitude of this major band, which was in all cases very low after one flash, exhibited oscillations of period four but rapidly damping, with maxima after two and six flashes. In contrast, photoheterotrophic Euglena displayed single, non-oscillating curves with maxima in the range 5–10°C.In autotrophic and semiautotrophic cells, oxidizing pretreatments by either a preillumination with one or more (up to twenty-five) flashes, or a far-red preillumination in the presence of methylviologen, followed by a short dark period, induced thermoluminescence bands almost single and shifted by +3–5°C, or +12°C, respectively. In autotrophic cells, far-red light plus methyl viologen treatment induced a band peaking at 31°C, as in isolated thylakoids from Euglena or higher plants, while it had barely any effect in photoheterotrophic cells.Due to metabolic activities in dark-adapted cells, a reduction of redox groups at the donor and acceptor sides of PS II dark-adapted cells is supposed to occur. Two different explanations can be proposed to explain such a shift in the position of the main band in dark-adapted autotrophic control. The first explanation would be that in these reducing conditions a decreasing value of the equilibrium constant for the reaction: S_nQ_A ^-Q_BS_nQ_AQ_B ^-, would determine the shift of the main TL band towards low temperatures, as observed in herbicide resistant material. The second explanation would be that the main band would correspond to peak III already observed in vivo and assigned to S_2/3Q_B ^2- recombinations.Abbreviations CM Cramer and Myers - D₁ a 32 kDa protein component of the PS II reaction center, psbA.gene product - D₂ a 34 kDa protein component of the PS II reaction center, psbD gene product - FR lar-red illumination - Lexpo and Lstat cells from lactate culture samples at exponential and stationary phase of growth - MV methylviologen - pBQ parabenzoquinone - PQ plastoquinone - PS II photosystem II - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - TL thermoluminescence     

Transient peaks in the delayed luminescence from Scenedesmus obtusiusculus induced by phosphorus starvation and carbon dioxide deficiency

Cells of the unicellular green alga Scenedesmus obtusiusculus (Chod.) were starved of phosphorus for 24, 48, 72 and 96 h, and the decay kinetics of the delayed luminescence from the differently starved cells was monitored for several minutes. Cells starved for 24 h showed similar delayed luminescence decay kinetics and accumulated output of photons as control cells after excitation with white light. Two transient peaks (with several components) in the decay kinetics of delayed luminescence were observed after 48 h of phosphorus starvation but not after 72 or 96 h. The amplitude of the transient peaks varied depending on the length of the excitation period with white light and on the length of the dark period preceding light excitation. High CO₂ availability induced no transient peak, whereas low CO₂ availability induced a high transient peak. Transient peaks could not be induced by excitation with light of 660 or 680 nm and only a single transient peak developed using 700 nm light. The kinetics of the delayed luminescence was changed, and the accumulated output of photons was decreased when the pH of the medium was changed from 7.2 to 9.5, both in cells starved for phosphorus for 96 h and in controls. The data indicate that a complicated metabolic pattern is involved in the mechanisms giving rise to the observed transient peaks in the delayed luminescence. The main factors may be a reduction in the translocation of trioses from chloroplasts, a concomitant reduction in Calvin cycle activities and changes in the amount of ATP and reducing agents available.     

Structured near-infrared Magnetic Circular Dichroism spectra of the Mn4CaO5 cluster of PSII in T. vulcanus are dominated by Mn(IV) d-d ‘spin-flip’ transitions

Photosystem II passes through four metastable S-states in catalysing light-driven water oxidation. Variable temperature variable field (VTVH) Magnetic Circular Dichroism (MCD) spectra in PSII of Thermosynochococcus (T.) vulcanus for each S-state are reported. These spectra, along with assignments, provide a new window into the electronic and magnetic structure of Mn₄CaO₅. VTVH MCD spectra taken in the S₂ state provide a clear g = 2, S = 1/2 paramagnetic characteristic, which is entirely consistent with that known by EPR. The three features, seen as positive (+) at 749 nm, negative (?) at 773 nm and (+) at 808 nm are assigned as ⁴A → ²E spin-flips within the d³ configuration of the Mn(IV) centres present. This assignment is supported by comparison(s) to spin-flips seen in a range of Mn(IV) materials. S₃ exhibits a more intense (?) MCD peak at 764 nm and has a stronger MCD saturation characteristic. This S₃ MCD saturation behaviour can be accurately modelled using parameters taken directly from analyses of EPR spectra. We see no evidence for Mn(III) d-d absorption in the near-IR of any S-state. We suggest that Mn(IV)-based absorption may be responsible for the well-known near-IR induced changes induced in S₂ EPR spectra of T. vulcanus and not Mn(III)-based, as has been commonly assumed. Through an analysis of the nephelauxetic effect, the excitation energy of S-state dependent spin-flips seen may help identify coordination characteristics and changes at each Mn(IV). A prospectus as to what more detailed S-state dependent MCD studies promise to achieve is outlined.     

A portable multi-flash kinetic fluorimeter for measurement of donor and acceptor reactions of Photosystem 2 in leaves of intact plants under field conditions

A newly-developed field-portable multi-flash kinetic fluorimeter for measuring the kinetics of the microsecond to millisecond reactions of the oxidizing and reducing sides of photosystem 2 in leaves of intact plants is described and demonstrated. The instrumental technique is a refinement of that employed in the double-flash kinetic fluorimeter (Joliot 1974 Biochim Biophys Acta 357: 439–448) where a low-intensity short-duration light pulse is used to measure the fluorescence yield changes following saturating single-turnover light pulses. The present instrument uses a rapid series of short-duration (2 s) pulses to resolve a complete microsecond to millisecond time-scale kinetic trace of fluorescence yield changes after each actinic flash. Differential optics, using a matrix of optical fibers, allow very high sensitivity (noise levels about 0.05% F_max) thus eliminating the need for signal averaging, and greatly reducing the intensity of light required to make a measurement. Consequently, the measuring pulses have much less actinic effect and an entire multi-point trace (seven points) excites less than 1% of the reaction centers in a leaf. In addition, bu combining the actinic and measuring pulse light in the optical fiber network, the tail of the actinic flash can be compensated for, allowing measurements of events as rapidly as 20 s after the actinic flash. This resolution makes practical the routine measurement of the microsecond turnover kinetics of the oxygen evolving complex in leaves of intact plants in the field. The instrument is demonstrated by observing flash number dependency and inhibitor sensitivity of the induction and decay kinetics of flash-induced fluorescence transients in leaves of intact plants. From these traces the period-two oscillations associated with the turnover of the two-electron gate and the period-four oscillations associated with the turnover of the oxygen evolving complex can be observed. Applications of the instrument to extending our knowledge of chloroplast function to the whole plant, the effects on plants of environmental stress, herbicides, etc, and possible applications to screening of mutants are discussed.Abbreviations DCMU 3-(3,4-Dichlorophenol)-1,1-dimethylurea - PS 2 photosystem 2 - PS 1 photosystem 1 - P₆₈₀ primary electron donor of the PS 2 reaction center - Q_A primary acceptor quinone of PS 2 - Q_B secondary acceptor quinone of PS 2 - CCCP carbonyl cyanide-m-chlorophenylhydrazone - Y_z donor to P₆₈₀ ⁺ - F₀ level of fluorescence with all PS 2 centers open - F_max maximum level of fluorescence with all PS 2 centers closed - P₆₈₀Q_A Open reaction centers with P₆₈₀ reduced and Q_A oxidized (low fluorescence) - P₆₈₀Q_A ^- Closed reaction centers, in which P₆₈₀ is reduced (high fluorescence) - P₆₈₀ ⁺Q_A ^- Closed reaction centers, in which P₆₈₀ is oxidized (low fluorescence)     

Control of misses in oxygen evolution by the oxido-reduction state of plastoquinone in Dunaliella tertiolecta

The way misses happen in oxygen evolution is subject to debate (Govindjee et al. 1985). We recently observed a linear lowering of the miss probability with the flash number (Meunier and Popovic 1989). Therefore, we investigated in Dunaliella tertiolecta the link between the average miss probability and the redox state of plastoquinone after n flashes. The effect of flashes was to oxidize the plastoquinone pool; we found that the oxidation of plastoquinone highly correlated (linear regression: R ²=0.996) with the lowering of the miss probability. The flash frequency was found to affect both the miss probability and the redox state of plastoquinone. When pre-flashes were given using a high flash frequency (10 Hz), the plastoquinone pool was oxidized and misses were low; however, if long dark intervals between flashes were used, the oxidizing effect of flashes was lost and the misses were high. We could not explain our results by assuming equal misses over all S-states; but unequal misses, caused by deactivations, were coherent with our results. We deduced that chlororespiration was responsible for the reduction of plastoquinone in the dark interval between flashes. We compared oxygen evolution with and without benzoquinone, using a low flash frequency (0.5 Hz) for maximum misses. Benzoquinone lowered the misses from 34% to 3%, and raised the amplitude of oxygen evolution by more than a factor of two (2). From this we deduced that the charge carrier C postulated to explain misses (Lavorel and Maison-Peteri 1983) did not account for more than 3% of miss probability in Dunaliella tertiolecta. These results indicate that the misses in oxygen evolution are controlled by the redox state of plastoquinone, through deactivations.     

Evidence for a linear variation of the miss and single hit S-state probabilities with the flash number,measured by oxygen evolution in Dunaliella tertiolecta

Oxygen evolution in Dunaliella tertiolecta under flashing light was measured with a bare electrode, at a 10 Hz frquency. The sigma coefficients of the oxygen evolution recurrence law (Thibault (1978) J Theor Biol 73, 271) were determined using groups of nine consecutive points. The S-state transition probabilities were computed from the sigma coefficients and plotted as a function of the flash number of the first of the points used. Low standard deviations over the sigma coefficients resulted from the use of our system (Meunier & Popovic (1988) Rev Sci Instr 59, 486). We observed a linear lowering of the miss probability with time, and a linear rise of the single-hit probability with the same absolute value of the slope. The hypothesis that the slopes were zero was statistically tested and was rejected with a 99.9% confidence interval. Our work demonstrates that, to be accurate, an oxygen evolution model has to take the variations in the properties of S-states into account.International Journal of Fracture 76 (1995) R37     

P680+ reduction in oxygen-evolving Photosystem II core complexes

The kinetics of P680⁺ reduction in oxygen-evolving spinach Photosystem II (PS II) core particles were studied using both repetitive and single-flash 830 nm transient absorption. From measurements on samples in which PS II turnover is blocked, we estimate radical-pair lifetimes of 2 ns and 19 ns. Nanosecond single-flash measurements indicate decay times of 7 ns, 40 ns and 95 ns. Both the longer 40 ns and 95 ns components relate to the normal S-state controlled Y_z P680⁺ electron transfer dynamics. Our analysis indicates the existence of a 7 ns component which provides evidence for an additional process associated with modified interactions involving the water-splitting catalytic site. Corresponding microsecond measurements show decay times of 4 s and 90 s with the possibility of a small component with a decay time of 20–40 s. The precise origin of the 4 s component remains uncertain but appears to be associated with the water-splitting center or its binding site while the 90 s component is assigned to P680⁺-Q_A ^– recombination. An amplitude and kinetic analysis of the flash dependence data gives results that are consistent with the current model of the oxygen-evolving complex.Abbreviations PS II- Photosystem II- - P680- primary donor (Chl-a_II dimer) of PS II - Y_z- Tyr 161 donor to P680 - Q_A- quinone secondary acceptor to P680 - LHC- light-harvesting chlorophyll protein of PS II - BBY- Berthold, Babcock and Yocum PS II membrane fragment preparation - PPBQ- phenyl-p-benzoquinone     

S-state dependence of the miss probability in Photosystem II

The oxygen production of dark-adapted Photosystem II upon illumination by a series of single-turnover flashes shows a damped period four oscillation with flash number. The damping is attributed to `misses' resulting from a statistical probability that a reaction center fails to produce a stable charge separation after a saturating flash. The origin of misses is of interest because its probable dependence on flash number, in principle, affects the quantitative interpretation of all measurements on phenomena associated with the period four oscillation. We show that the kinetics of chlorophyll fluorescence yield transients induced by a flash series can be used to estimate the relative amplitudes of the miss probability on each flash. It is concluded that a major part of the misses must be caused by failure of the reduction of the oxidized primary electron donor chlorophyll P680⁺ by the secondary donor tyrosine Y_Z before the charge separation is lost by recombination. The probability of this failure is found to increase with the oxidation state of the oxygen-evolving complex: more than half of it occurs upon charge separation in the S₃ state, which is attributed to the presence of Y_Z ^ox S₂ in Boltzmann equilibrium with Y_ZS₃. This revised version was published online in June 2006 with corrections to the Cover Date.