H.J. van Gorkom

Spectroscopic properties of the reaction center and of the 47 kDa chlorophyll protein of Photosystem II

The D1-D2-cytochrome b-559 reaction center complex and the 47 kDa antenna chlorophyll protein isolated from spinach Photosystem II were characterized by means of low temperature absorption and fluorescence spectroscopy. The low temperature absorption spectrum of the D1-D2-cytochrome b-559 complex showed two bands in the Qy region located at 670 and 680 nm. On the basis of its absorption maximum and orientation the latter component may be attributed at least in part to P-680, the primary electron donor of Photosystem II. The 47 kDa antenna complex showed absorption bands at 660, 668 and 677 nm and a minor component at 690 nm. The latter transition appeared to be associated with the characteristic low temperature 695 nm fluorescence band of Photosystem II. The 695 nm emission band was absent in the D1-D2 complex, which indicates that it does not originate from the reaction center pheophytin, as earlier proposed. The transition dipole responsible for the main fluorescence at 684 nm from this complex had a parallel orientation with respect to the membrane plane in the native structure. The reaction center preparation contains two spectrally distinct carotenoids with different orientations.

Electron transfer in photosystem II

The picture presently emerging from studies on the mechanism of photosystem II electron transport is discussed. The reactions involved in excitation trapping, charge separation and stabilization of the charge pair in the reaction center, followed by the reactions with the substrates, plastoquinone reduction and water oxidation, are described successively. Finally, a brief discussion on photosystem II heterogeneity is presented.

Spectroscopic properties of chloroplast grana membranes and of the core of photosystem ii

An oxygen-evolving Photosystem II core complex essentially free of the light-harvesting chlorophyll ab protein complex, containing 45 chlorophylls per reaction center was isolated from spinach chloroplasts. Its structural integrity was established by studying its photochemistry and spectral properties. The absorption spectrum measured at 4 K revealed the presence of at least five spectrally distinct chlorophyll a species. The same bands, but in different proportions, were observed in a Photosystem II grana preparation used as starting material for the preparation of the core complex. The relative contributions of these components to the overall absorption were calculated by deconvoluting this spectrum into Gaussian bands. The core complex was enriched in a long-wave band located at 683 nm, which presumably reflects the presence of 8–10 pigment molecules that are closely associated with the reaction center. Low temperature fluorescence emission spectra showed the characteristic Photosystem II emission bands located at 685 nm (F685) and at 695 nm (F685). The two states giving rise to these emissions are in thermal equilibrium down to 70 K. It is suggested that F685 arises from a chlorophyll a species absorbing at 676 nm and that F695 is the result of fluorescence from the photoactive pheophytin a absorbing around 683 nm.

Primary reactions, plastoquinone and fluorescence yield in subchloroplast fragments prepared with deoxycholate.

Abstract 1. In subchloroplast fragments prepared with the detergent deoxycholate the primary reactions of Photosystem II could be studied at room temperature, because the secondary reactions were largely or completely inhibited. 2. The main quencher of chlorophyll fluorescence in these particles was the photosynthetically active pool of plastoquinone in its oxidized form. Its photoreduction in the presence of artificial electron donors was accompanied by a shift of a chlorophyll a absorption band. Its reoxidation in the dark was very slow, even in the presence of ferricyanide. 3. Of all the artificial electron donors tested MnCl 2 was by far the most efficient. 4. Measurements at room temperature of the C550 absorbance change confirmed its correlation with the primary electron acceptor. Its difference spectrum was broader and its extinction coefficient correspondingly lower than at liquid-N 2 temperature. In chloroplasts the C550 concentration was about 1:360 chlorophylls. 5. In the dark C550 was largely in the reduced state and its oxidation by plastoquinone took place in the presence of an artificial electron donor only, suggesting that the redox potential of C550 was increased by accumulated positive charges at the donor side of the reaction center. 6. The free radical 1,1′-diphenyl-2-picrylhydrazyl oxidized C550 directly in a 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-insensitive reaction. A DCMU-insensitive oxidation of C550 was observed at high ferricyanide concentrations as well, but probably in this case an endogenous electron donor was oxidized, which in turn oxidized C550 via the back reaction of the photochemical reaction. 7. The oxidized form of the primary electron donor, P680 + , accumulated in the light in the presence of deoxycholate and a low ferricyanide concentration. In chloroplasts the P680 concentration was about 1:360 chlorophylls. 8. The P 680 absorption difference spectrum and electron spin resonance could be explained by the oxidation of a chlorophyll a dimer. Repeated deoxycholate treatments progressively changed the spectra to those of a monomer. The monomer was still photochemically active. 9. A new interpretation of the difference spectrum of P700 is proposed: it may be the same as that of the difference spectrum of P680 if the bleaching at 700 nm is attributed to a band shift.

Quantum efficiency and antenna size of Photosystems IIα, IIβ and I in tobacco chloroplasts

Abstract Reaction center concentrations were determined in chloroplasts of tobacco, cv John Williams Broadleaf, and its mutants Su/su and Su/su var. Aurea. Quantum yields of the primary reactions of Photosystems I, IIα and IIβ (Melis, A. and Homann, P.H. (1975) Photochem. Photobiol. 21, 431–437) were obtained by measurement of their rate constants and the absorbed energy, under conditions where all three photosystems operated simultaneously and produced almost irreversibly a single charge separation. The concentrations and reaction rates of the photosystems were different in chloroplasts from the wild type and the mutants, but in chloroplasts of each type of plant used essentially all quanta absorbed by chlorophyll caused a charge separation in PS I, PS IIα or PS IIβ. Since the quantum efficiency of each photosystem was close to one, kinetic differences between the photosystems and between different kinds of chloroplasts were only due to differences in antenna size. From the rate constants the number of chlorophyll molecules in the antenna of each photosystem could be calculated. It is argued that PS IIα and PS IIβ must be different, independent structures.

Inhibition of the reoxidation of the secondary electron acceptor of Photosystem II by bicarbonate depletion

In bicarbonate-depleted chloroplasts, the chlorophyll a fluorescence decayed with a halftime of about 150 ms after the third flash, and appreciably faster after the first and second flash of a series of flashes given after a dark period. After the fourth to twentieth flashes, the decay was also slow. After addition of bicarbonate, the decay was fast after all the flashes of the sequence. This indicates that the bicarbonate depletion inhibits the reoxidation of the secondary acceptor R2- by the plastoquinone pool; R is the secondary electron acceptor of pigment system II, as it accepts electrons from the reduced form of the primary electron acceptor (Q-). This conclusion is consistent with the measurements of the DCMU (3-(3,4-dichlorophenyl)-),)-dimethylurea)- induced chlorophyll a fluorescence after a series of flashes in the presence and the absence of bicarbonate, if it is assumed that DCMU not only causes reduction of Q if added in the state QR-, but also if added in the state QT2-.

Energy transfer, charge separation and pigment arrangement in the reaction center of Photosystem II

Abstract Energy transfer and charge separation in the isolated Photosystem II reaction center at room temperature were studied with transient absorption difference spectroscopy upon selective excitation of the reaction center pigments. The measurements were performed with two dye lasers, which had a spectral bandwidth of less than 1 nm, and with an instrument response function of 5 or 18 ps depending on the type of experiment. Small changes with time constants of 0.6 ns and 120 ps are attributed to damaged reaction centers. Selective excitation of the long-wavelength pigments, presumably P680 and the pheophytins, led to charge separation in 3 ps. Selective excitation of the short-wavelength pigments, presumably accessory chlorophylls, led to charge separation in 30 ps with the same quantum efficiency. This excludes equilibration of the excited state between accessory chlorophyll and P680 in less than 30 ps. The overlap of the fluorescence spectrum of accessory chlorophyll with the absorption of P680 is very good and the slow energy transfer is attributed to an about 30 A center-to-center distance, which makes the histidines 118 in helix II of the D1 and D2 proteins likely binding sites of the chlorophylls nearest to the long-wavelength pigments, P680 and pheophytin. Reevaluation of the literature in the light of these data suggests that P680 is a dimer with nearly (anti) parallel Q Y -transition moments of the constituent monomers, making an angle with their connecting axis close to the magic angle, and that the geometry of P680 and the pheophytins is not C 2 -symmetrical around an axis perpendicular to the membrane.

Kok's oxygen clock: What makes it tick? The structure of P680 and consequences of its oxidizing power.

New insights in the structure of P680, the primary electron donor in Photosystem II, are summarized and the implications of its oxidizing power for energy transfer and singlet oxygen production are discussed.

The PSII calcium site revisited.

Oxidation of H2O by photosystem II is a unique redox reaction in that it requires Ca2+ as well as Cl− as obligatory activators/cofactors of the reaction, which is catalyzed by Mn atoms. The properties of the binding site for Ca2+ in this reaction resemble those of other Ca2+ binding proteins, and recent X-ray structural data confirm that the metal is in fact ligated at least in part by amino acid side chain oxo anions. Removal of Ca2+ blocks water oxidation chemistry at an early stage in the cycle of redox reactions that result in O-O bond formation, and the intimate involvement of Ca2+ in this reaction that is implied by this result is confirmed by an ever-improving set of crystal structures of the cyanobacterial enzyme. Here, we revisit the photosystem II Ca2+ site, in part to discuss the additional information that has appeared since our earlier review of this subject (van Gorkom HJ, Yocum CF In: Wydrzynski TJ, Satoh K (eds) Photosystem II: the light-driven water:plastoquinone oxidoreductase), and also to reexamine earlier data, which lead us to conclude that all S-state transitions require Ca2+.

External electric field effects on prompt and delayed fluorescence in chloroplasts

Abstract An electric field pulse was applied to a suspension of osmotically swollen spinach chloroplasts after illumination with a saturating flash in the presence of DCMU. In addition to the stimulation of delayed fluorescence by the electric field, discovered by Arnold and Azzi (Arnold, W.A. and Azzi, R. (1971) Photochem. Photobiol. 14, 233–240) a sudden drop in fluorescence yield was observed. The kinetics of this fluorescence change were identical to those of the integrated delayed fluorescence emission induced by the pulse. The S-state dependence of the stimulated emission was very similar to that of the normal luminescence. We assume that the membrane potential generated by the pulse changes the activation energy for the back reaction in Photosystem II. On this basis, and making use of data we obtained earlier from electrochromic absorbance changes induced by the pulse, the kinetics of the field-induced prompt and delayed fluorescence changes, and also the amplitude of the fluorescence decrease, which was about 12% for a nearly saturating pulse, are explained. Our results indicate that in those reaction centers where a decrease of the activation energy occurs the effect of a pulse can be quite spectacular: the back reaction, which normally takes seconds, is completed in a few hundred microseconds when a sufficiently strong pulse is applied. Measurements of the polarization of the stimulated luminescence supported the interpretation given above. Only 2.8% of the back reaction was found to proceed via transition of reexcited chlorophyll to the ground state, both during the field pulse and in the absence of the field.