Hann-Jörg Eckert

Two sites of photoinhibition of the electron transfer in oxygen evolving and tris-treated PS II membrane fragments from spinach

Photoinhibition was analyzed in O2-evolving and in Tris-treated PS II membrane fragments by measuring flash-induced absorption changes at 830 nm reflecting the transient P680+ formation and oxygen evolution. Irradiation by visible light affects the PS II electron transfer at two different sites: a) photoinhibition of site I eliminates the capability to perform a ‘stable’ charge separation between P680+ and QA- within the reaction center (RC) and b) photoinhibition of site II blocks the electron transfer from YZ to P680+. The quantum yield of site I photoinhibition (2–3×10-7 inhibited RC/quantum) is independent of the functional integrity of the water oxidizing system. In contrast, the quantum yield of photoinhibition at site II depends strongly on the oxygen evolution capacity. In O2-evolving samples, the quantum yield of site II photoinhibition is about 10-7 inhibited RC/quantum. After selective elimination of the O2-evolving capacity by Tris-treatment, the quantum yield of photoinhibition at site II depends on the light intensity. At low intensity (<3 W/m2), the quantum yield is 10-4 inhibited RC/quantum (about 1000 times higher than in oxygen evolving samples). Based on these results it is inferred that the dominating deleterious effect of photoinhibition cannot be ascribed to an unique target site or a single mechanism because it depends on different experimental conditions (e.g., light intensity) and the functional status of the PS II complex.

Analysis of the electron transfer from Pheo− to QA in PS II membrane fragments from spinach by time resolved 325 nm absorption changes in the picosecond domain

Absorption changes at 325 nm (ΔA 325) induced by 15 ps laser flashes (λ = 650 nm) in PS II membrane fragments were measured with picosecond time‐resolution. In samples with the reaction centers (RCs) kept in the open state (P I QA) the signals are characterized by a very fast rise (not resolvable by our equipment) followed by only small changes within our time window of 1.6 ns. In the closed state (P I Q− A) of the reaction center the signal decays with an average half‐life time of about 250 ps. It is shown that under our excitation conditions (E = 2 × 1014 photons/cm2 per pulse) subtraction of the absorption changes in closed RCs (ΔA closed 325) from those in open RCs (ΔA open 325) leads to a difference signal which is dominated by the reduction kinetics of QA. From the rise kinetics of this signal and by comparison with data in the literature it is inferred that QA becomes reduced by direct electron transfer from Pheo− with a time constant of about 350 ± 100 ps.

Effects of hydrogen/deuterium exchange on photosynthetic water cleavage in PS II core complexes from spinach

H/D isotope exchange effects on P680+· reduction by Yz and electron abstraction from the water oxidizing complex (WOC) in redox state S3 by YZ OX were analyzed in PS II core complexes from spinach by measurements of laser flash induced absorption changes at 820 nm and 355 nm. The results obtained reveal: (1) the rate of YZ oxidation by P680+· is almost independent of the substitution of exchangeable protons by deuterons; and (2) the reaction between YZ OX and the WOC in S3 exhibits a kinetic H/D isotope exchange effect of similar magnitude as that recently observed in PS II membrane fragments [Renger, G., Bittner, T. and Messinger, J. (1994) Biochem. Soc. Trans. 22, 318–322]. Based on these results it is inferred that photosynthetic dioxygen formation comprises the cleavage of at least one hydrogen bond.

KINETIC STUDIES ON THE STABILIZATION OF THE PRIMARY RADICAL PAIR P680+ Pheo‐ IN DIFFERENT PHOTOSYSTEM II PREPARATIONS FROM HIGHER PLANTS*

Abstract— The stabilization of the primary radical pair P680+ pheophytin (Pheo)‐ through rapid electron transfer from Pheo to the special plastoquinone of photosystem II (PS II), QA, was analyzed on the basis of time‐resolved (40 ps) UV‐absorption changes detected in different PS II preparations from higher plants. Lifetime measurements of1Chi* fluorescence by single photon counting and a numerical analysis of the redox reactions revealed (1) at exciton densities required for light saturation of the stable charge separation, annihilation processes dominate the excited state decay leading to very similar lifetimes of 1Chi* in systems with open and closed reaction centers and (2) the difference of absorption changes induced by actinic flashes of comparatively high photon density in samples with open and photochemically closed reaction centers, respectively, provides a suitable measure of the rate constant of QA formation. Conclusion 2 was confirmed in PS II membrane fragments by measurements at three wavelengths (280 nm, 292 nm and 325 nm) where the difference spectrum of Q‐A formation exhibits characteristic features. The numerical evaluation of the experimental data led to the following results: (1) the rate constant of Q‐A formation was found to be (300 ± 100 ps)‐1 in PS II membrane fragments and PS II core complexes deprived of the distal and proximal antenna and (2) an iron depletion treatment of membrane fragments does not affect these kinetics. The implications of these results are briefly discussed in terms of the PS II reaction pattern.

Studies on the recombination kinetics of the radical pair P680+Pheo− in isolated PS II core complexes from spinach

Yield and decay kinetics of the laser flash-induced primary radical pair P680+Pheo− were analyzed by measuring flash-induced absorption changes at 820 nm (ΔA820) and fluorescence decay kinetics in PS II core complexes under different redox conditions for the primary plastoquinone acceptor, QA. If QA is chemically reduced in the dark by Na2S2O4, the yield of long-lived (τ > 1 ns) P680+Pheo− states is 30% with lifetimes of 4 ns (about 23 of the total decay) and 30 ns (about 13 of the total decay). The fluorescence data suggest that the 30 ns radical pair and probably the 4 ns radical pair as well decay predominately by recombination to P680∗Pheo. Irradiation of PS II core complexes in the presence of Na2S2O4 for only 10 s with intense visible light increases the yield of the 30 ns primary radical pair by a factor of 3. This result can be explained by a double reduction of QA and subsequent protonation leading to QAH2. At short irradiation times this effect is reversible because subsequent addition of K3[Fe(CN)6] fully restores the capability for a stable charge separation giving rise to P680+QA formation. However, longer irradiation of Na2S2O4-treated PS II core complexes leads to an irreversible impairment of the primary charge separation.

Excitation energy transfer in intact cells and in the phycobiliprotein antennae of the chlorophyll d containing cyanobacterium Acaryochloris marina.

The cyanobacterium Acaryochloris marina is unique because it mainly contains Chlorophyll d (Chl d) in the core complexes of PS I and PS II instead of the usually dominant Chl a. Furthermore, its light harvesting system has a structure also different from other cyanobacteria. It has both, a membrane-internal chlorophyll containing antenna and a membrane-external phycobiliprotein (PBP) complex. The first one binds Chl d and is structurally analogous to CP43. The latter one has a rod-like structure consisting of three phycocyanin (PC) homohexamers and one heterohexamer containing PC and allophycocyanin (APC). In this paper, we give an overview on the investigations of excitation energy transfer (EET) in this PBP-light-harvesting system and of charge separation in the photosystem II (PS II) reaction center of A. marina performed at the Technische Universität Berlin. Due to the unique structure of the PBP antenna in A. marina, this EET occurs on a much shorter overall time scale than in other cyanobacteria. We also briefly discuss the question of the pigment composition in the reaction center (RC) of PS II and the nature of the primary donor of the PS II RC.

Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy.

The fluorescence decay spectra and the excitation energy transfer from the phycobiliproteins (PBP) to the chlorophyll-antennae of intact cells of the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina were investigated at 298 and 77 K by time- and wavelength-correlated single photon counting fluorescence spectroscopy. At 298 K it was found that (i) the fluorescence dynamics in A. marina is characterized by two emission peaks located at about 650 and 725 nm, (ii) the intensity of the 650 nm fluorescence depends strongly on the excitation wavelength, being high upon excitation of phycobiliprotein (PBP) at 632 nm but virtually absent upon excitation of chlorophyll at 430 nm, (iii) the 650 nm fluorescence band decayed predominantly with a lifetime of 70 +/- 20 ps, (iv) the 725 nm fluorescence, which was observed independent of the excitation wavelength, can be described by a three-exponential decay kinetics with lifetimes depending on the open or the closed state (F(0) or F(m)) of the reaction centre of Photosystem II (PS II). Based on the results of this study, it is inferred that the excitation energy transfer from phycobiliproteins to Chl d of PS II in A. marina occurs with a time constant of about 70 ps, which is about three times faster than the energy transfer from the phycobilisomes to PS II in the Chl a-containing cyanobacterium Synechococcus 6301. A similar fast PBP to Chl d excitation energy transfer was also observed at 77 K. At 77 K a small long-lived fluorescence decay component with a lifetime of 14 ns was observed in the 640-700 nm spectral range. However, it has a rather featureless spectrum, not typical for Chl a, and was only observed upon excitation at 400 nm but not upon excitation at 632 and 654 nm. Thus, this long-lived fluorescence component cannot be used as an indicator that the primary PS II donor of Acaryochloris marina contains Chl a.

Effects of trypsin and bivalent cations on P-680+-reduction, fluorescence induction and oxygen evolution in Photosystem II membrane fragments from spinach

Abstract Laser-flash-induced absorption changes at 830 nm, fluorescence-induction curves and the average oxygen yield per flash have been measured in spinach Photosystem II membrane fragments as a function of trypsin treatment and its modification by CaCl2. The following was found. (i) The relative contribution of the nanosecond relaxation to the overall decay kinetics of 830 nm absorption changes reflecting the P-680+-reduction decreases as a function of incubation time with trypsin. Simultaneously, mild treatment at pH = 6.0 markedly increases the extent of 200 μs kinetics that highly revert back to nanosecond kinetics by CaCl2 addition. After harsher trypsin treatment (pH = 7.5) pH-dependent 2–20 μs kinetics appear that cannot be reverted to nanosecond kinetics by CaCl2. (ii) The CaCl2-induced restoration of nanosecond kinetics is mainly due to a Ca2+-induced effect rather than to a functional role of Cl−. Sr2+ can substantially substitute for Ca2+, whereas Mg2+, Mn2+ and monovalent ions are almost inefficient. (iii) A quantitative correlation between the extent of the nanosecond kinetics and the average oxygen yield per flash was not observed. (iv) If CaCl2 is present in the assay medium for trypsin treatment the samples are markedly protected to proteolytic degradation. This effect mainly refers to the reaction pattern of the acceptor side. Other bivalent cations can substitute Ca2+ for its protective function. (v) The CaCl2-induced protection to proteolytic attack is extremely sensitive to a very short trypsin pretreatment that does hardly affect the shape of the fluorescence induction curve. The results are discussed in relation to the functional and structural organization of Photosystem II.

Characterization of the water oxidizing complex of photosystem II of the Chl d-containing cyanobacterium Acaryochloris marina via its reactivity towards endogenous electron donors and acceptors

Acaroychloris (A.) marina is a unique oxygen evolving organism that contains a large amount of chlorophyll d (Chl d) and only very few Chl a molecules. This feature raises questions on the nature of the photoactive pigment, which supports light-induced oxidative water splitting in Photosystem II (PS II). In this study, flash-induced oxygen evolution patterns (FIOPs) were measured to address the question whether the S(i) state transition probabilities and/or the redox-potentials of the water oxidizing complex (WOC) in its different S(i) states are altered in A. marina cells compared to that of spinach thylakoids. The analysis of the obtained data within the framework of different versions of the Kok model reveals that in light activated A. marina cells the miss probability is similar compared to spinach thylakoids. This finding indicates that the redox-potentials and kinetics within the WOC, of the reaction center (P680) and of Y(Z) are virtually the same for both organisms. This conclusion is strongly supported by lifetime measurements of the S(2) and S(3) states. Virtually identical time constants were obtained for the slow phase of deactivation. Kinetic differences in the fast phase of S(2) and S(3) decay between A. marina cells and spinach thylakoids reflect a shift of the E(m) of Y(D)/Y(D)(ox) to lower values in the former compared to the latter organisms, as revealed by the observation of an opposite change in the kinetics of S(0) oxidation to S(1) by Y(D)(ox). A slightly increased double hit probability in A. marina cells is indicative of a faster Q(A)(-) to Q(B) electron transfer in these cells compared to spinach thylakoids.

Effect of monochromatic UV-B radiation on electron transfer reactions of Photosystem II.

The adverse effect of low intensity, small band UV-B irradiation (λ = 305 ± 5 nm, I = 300 mW m−2) on PS II has been studied by comparative measurements of laser flash-induced changes of the absorption at 325 nm, ΔA325(t), as an indicator of redox changes in QA, and of the relative fluorescence quantum yield, F(t)/Fo, in PS II membrane fragments. The properties of untreated control were compared with those of samples where the oxygen evolution rate under illumination with continuous saturating light was inhibited by up to 95%. The following results were obtained: a) the detectable initial amplitude (at a time resolution of 30 μs) of the 325 nm absorption changes, ΔA325, remained virtually invariant whereas the relaxation kinetics exhibit significant changes, b) the 300 μs kinetics of ΔA325 dominating the relaxation in UV-B treated samples was largely replaced by a 1.3 ms kinetics after addition of MnCl2, c) the extent of the flash induced rise of the relative fluorescence quantum yield was severely diminished in UV-B treated PS II membrane fragments but the relaxation kinetics remain virtually unaffected. Based on these results the water oxidizing complex (WOC) is inferred to be the primary target of UV-B impairment of PS II while the formation of the ‘stable’ radical pair P680+·QA−● is almost invariant to this UV-B treatment.