Copper(II) inhibition of electron transfer through photosystem II studied by EPR spectroscopy.

EPR spectroscopy was applied to investigate the inhibition of electron transport in photosystem II by Cu2+ ions. Our results show that Cu2+ has inhibitory effects on both the donor and the acceptor side of photosystem II. In the presence of Cu2+, neither EPR signal IIvery fast nor signal IIfast, which both reflect oxidation of tyrosinez, could be induced by illumination. This shows that Cu2+ inhibits electron transfer from tyrosinez to the oxidized primary donor P680+. Instead of tyrosinez oxidation, illumination results in the formation of a new radical with g = 2.0028 +/- 0.0002 and a spectral width of 9.5 +/- 0.3 G. At room temperature, this radical amounts to one spin per PS II reaction center. Incubation of photosystem II membranes with cupric ions also results in release of the 16 kDa extrinsic subunit and conversion of cytochrome b559 to the low-potential form. On the acceptor side, QA can still be reduced by illumination or chemical reduction with dithionite. However, incubation with Cu2+ results in loss of the normal EPR signal from QA- which is coupled to the non-heme Fe2+ on the acceptor side (the QA(-)-Fe2+ EPR signal). Instead, reduction of QA results in the formation of a free radical spectrum which is 9.5 G wide and centered at g = 2.0044. This signal is attributed to QA- which is magnetically decoupled from the non-heme iron. This suggests that Cu2+ displaces the Fe2+ or severely alters its binding properties. The inhibition of tyrosinez is reversible upon removal of the copper ions with EDTA while the modification of QA was found to be irreversible.

Activation/deactivation cycle of redox-controlled thylakoid protein phosphorylation. Role of plastoquinol bound to the reduced cytochrome bf complex.

Signal transduction via light-dependent redox control of reversible thylakoid protein phosphorylation has evolved in plants as a unique mechanism for controlling events related to light energy utilization. Here we report for the first time that protein phosphorylation can be activated without light or the addition of reducing agents by a transient exposure of isolated thylakoid membranes to low pH in darkness. The activation of the kinase after incubation of dark-adapted thylakoids at pH 4.3 coincides with an increase in the plastoquinol: plastoquinone ratio up to 0.25. However, rapid plastoquinol reoxidation ( < 1 min) at pH 7.4 contrasts with the slow kinase deactivation (t 1/2 = 4 min), which indicates that the redox control is not directly dependent on the plastoquinone pool. Use of inhibitors and a cytochrome bf-deficient mutant of Lemna demonstrate the involvement of the cytochrome bf complex in the low-pH induced protein phosphorylation. EPR spectroscopy shows that subsequent to the transient low pH treatment and transfer of the thylakoids to pH 7.4, the Rieske Fe-S center, and plastocyanin become reduced and are not reoxidized while the kinase is slowly deactivated. However, the deactivation correlates with a decrease of the EPR gz signal of the reduced Rieske Fe-S center, which is also affected by quinone analogues that inhibit the kinase. Our data point to an activation mechanism of thylakoid protein phosphorylation that involves the binding of plastoquinol to the cytochrome bf complex in the vicinity of the reduced Rieske Fe-S center.

Temperature-dependent triplet and fluorescence quantum yields of the photosystem II reaction center described in a thermodynamic model.

A key step in the photosynthetic reactions in photosystem II of green plants is the transfer of an electron from the singlet-excited chlorophyll molecule called P680 to a nearby pheophytin molecule. The free energy difference of this primary charge separation reaction is determined in isolated photosystem II reaction center complexes as a function of temperature by measuring the absolute quantum yield of P680 triplet formation and the time-integrated fluorescence emission yield. The total triplet yield is found to be 0.83 +/- 0.05 at 4 K, and it decreases upon raising the temperature to 0.30 at 200 K. It is suggested that the observed triplet states predominantly arise from P680 but to a minor extent also from antenna chlorophyll present in the photosystem II reaction center. No carotenoid triplet states could be detected, demonstrating that the contamination of the preparation with CP47 complexes is less than 1/100 reaction centers. The fluorescence yield is 0.07 +/- 0.02 at 10 K, and it decreases upon raising the temperature to reach a value of 0.05-0.06 at 60-70 K, increases upon raising the temperature to 0.07 at approximately 165 K and decreases again upon further raising the temperature. The complex dependence of fluorescence quantum yield on temperature is explained by assuming the presence of one or more pigments in the photosystem II reaction center that are energetically degenerate with the primary electron donor P680 and below 60-70 K trap part of the excitation energy, and by temperature-dependent excited state decay above 165 K. A four-compartment model is presented that describes the observed triplet and fluorescence quantum yields at all temperatures and includes pigments that are degenerate with P680, temperature-dependent excited state decay and activated upward energy transfer rates. The eigenvalues of the model are in accordance with the lifetimes observed in fluorescence and absorption difference measurements by several workers. The model suggests that the free energy difference between singlet-excited P680 and the radical pair state P680+l- is temperature independent, and that a distribution of free energy differences represented by at least three values of about 20, 40, and 80 meV, is needed to get an appropriate fit of the data.

Characterization of a D1-D2-cyt b-559 complex containing 4 chlorophyll a/2 pheophytin a isolated with the use of MgSO4.

A D1-D2-cyt b-559 complex containing 4 chlorophyll alpha, 1 beta-carotene and 1 cytochrome b-559 per 2 pheophytin a has been isolated from spinach with 30% yield using a Q-Sepharose Fast-Flow anion-exchange column equilibrated with 0.1% Triton X-100, 10 mM MgSO4 and 50 mM Tris-HCl (pH 7.2). The preparation was then stabilized with 0.1% dodecyl-beta-D-maltoside. This method gave a yield 10 times higher than that using a Fractogel TSK-DEAE 650(S) column equilibrated with 0.1% Triton X-100, 30 mM NaCl and 50 mM Tris-HCl (pH 7.2). The PS II RC complex was characterized using absorption and fluorescence spectroscopy at 277 and 77 K. A selective reversible bleaching under reducing conditions with maximum at 682 nm, associated with pheophytin a reduction, and light-induced absorption differences with a lifetime of 1.0 ms, ascribed to the triplet state of P680 were measured and indicated that the isolated D1-D2-cyt b-559 complex is active in charge separation. The results are compared with the data obtained for a PS II RC preparation containing 6 chlorophyll alpha, 2 beta-carotene and 1 cyt b-559 per 2 pheophytin a.

Influence of the H-subunit and Fe2+ on electron transport from I- to QA in Fe(2+)-free and/or H-free reaction centers from Rhodobacter sphaeroides R-26.

From reaction centres (RC) of Rhodobacter sphaeroides R-26 two LM preparations with 0.90 Fe2+/RC (LM) and 0.10 Fe2+/RC (LM/dFe) were prepared. Reconstitution of LM/dFe with the H-subunit and subsequently with Zn2+ yielded LMH/dFe and LMH/dFe and LMH/dFe + Zn preparations, respectively. In these four samples the decay of the primary radical pair P+I- was studied by means of transient absorption spectroscopy and compared with that in native RC. In LMH/dFe the reduction of QA by Bpheo a occurred in 5 ns, with concomitant increase in the yield of PT, the triplet state of the primary donor. In the LM/dFe, LM and LMH/dFe + Zn preparations the decay of I- had the same rate (200 ps)-1 as in native RC. Thus, neither the H-subunit in the RC nor a divalent metal as Fe2+ or Zn2+ are necessary per se for fast reduction of QA. Only demetallation in the presence of the H-subunit slows down the reduction of QA.

Primary photosynthetic processes in Heliobacterium chlorum at 15K.

Absorbance changes induced by 25-ps laser flashes were measured in membranes of Heliobacterium chlorum at 15 K. Absorbance difference spectra, measured at various times after the flash showed negative bands in the Qy region at 812, 793 and 665 nm. The first of these bands was attributed to the formation of excited singlet states of a long-wavelength form of antenna bacteriochlorophyll g (BChl g 808). Absorbance changes of shorter wavelength absorbing antenna BChls g were at least an order of magnitude smaller, indicating rapid excitation energy transfer (i.e. within the time resolution of the apparatus) from these BChls to BChl g 808. Excited BChl g 808 showed a bi-exponential decay with time constants of 50 and 200 ps. The bands at 793 and 665 nm may be attributed to the primary charge separation and reflect the photooxidation of the primary electron donor P-798 and photoreduction of a primary electron acceptor absorbing near 670 nm, presumably a BChl c or Chl a-like pigment. The bleaching of this pigment reversed with a time constant of 300 ps at 15 K and of 800 ps at 300 K. This indicates that electron transfer from the primary to the secondary electron acceptor is approximately 2.5 times faster at 15 K than at room temperature.