Johan J. Plijter

Kinetics of manganese redox transitions in the oxygen-evolving apparatus of photosynthesis

The kinetics of the S-state transitions of the oxygen-evolving complex were analyzed in dark-adapted, oxygen-evolving Photosystem-II preparations supplied with the electron acceptor 2,5-dichloro-p-benzoquinone. The kinetics of flash-induced absorbance changes at 350 nm, largely due to the successive S-state transitions S0 → S1, S1 → S2, S2 → S3 and S3 →; S0, confirm the +1, +1, +1, −3 sequence of manganese oxidation reported earlier (Dekker, J.P., Van Gorkom, H.J., Wensink, J. and Ouwehand, L. (1984) Biochim. Biophys. Acta 767, 1–9), and reveal half-times of 30, 110, 350 and 1300 μs, respectively, for these transitions.

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.

The primary reaction of photosystem II in the D1‐D2‐cytochrome b‐559 complex

Flash‐induced absorbance changes in the picosecond and nanosecond time range have been measured in the ‘D1‐D2‐cyt b‐559 complex’ isolated from photosystem II membranes. The results indicate the efficient formation of the primary radical pair P680+ pheophytin−, which had a lifetime of about 36 ns, and the presence of unconnected chlorophyll in this preparation. It is concluded that the complex contains the active photosystem II reaction center, and that this reaction center contains at most 4 chlorophyll a molecules.

Picosecond absorbance difference spectroscopy on the primary reactions and the antenna-excited states in Photosystem I particles

Abstract Absorbance difference spectra at various delay times, and kinetics of absorbance changes induced by a 35 ps excitation pulse at 532 nm, were measured of relatively intact Photosystem I particles from spinach containing about 70 chlorophyll a molecules per photoactive primary electron donor P-700. The excitation pulse produced absorbance changes due to the formation of singlet- and triplet-excited antenna chlorophyll a , and, in the case of active reaction centers, also those due to the oxidation of P-700. The formation of excited chlorophyll a was accompanied by the bleaching of the Q y ground state absorption band and by the appearance of a rather flat absorption increase in the region 550–900 nm. The lifetime of singlet-excited chlorophyll a was found to be 40 ± 5 ps. When the iron-sulfur centers were prereduced (photo)chemically, the formation of a radical pair consisting of P-700 + and a chlorophyllous anion was observed. The absorbance-difference spectrum calculated for the reduction of the acceptor was similar to that measured earlier (Shuvalov, V.A., Klevanik, A.V., Sharkov, A.V., Kryukov, P.G. and Ke, B. (1979) FEBS Lett. 107, 313–316), and indicated that the acceptor is a chlorophyll a species absorbing around 693 nm. The lifetime of the radical pair was at least 25 ns. If, however, the acceptor complex was in the oxidized state before the flash, only the oxidation of P-700 was observed. No direct evidence was obtained for the reduction of the chlorophyllous acceptor, implying that if such an anion is formed, it must be reoxidized within 50 ps.

Primary-charge separation and excitation of chlorophyll a in photosystem II particles from spinach as studied by picosecond absorbance-difference spectroscopy

Abstract Photosystem II particles from spinach, containing about 80 chlorophyll a molecules per reaction center, have been investigated with picosecond absorbance-difference spectroscopy. The 35 ps excitation pulse at 532 nm produced absorbance changes due to the formation of singlet excited antenna chlorophyll a and to the primary-charge separation in the reaction centers. The appearance of excited chlorophyll a was accompanied by the bleaching of the ground state Qy absorption band and by the formation of a rather flat absorption band in the region 550–900 nm. At high flash intensity its average lifetime was found to be several tens of picoseconds. In the reaction center charge separation was observed between the primary electron donor P-680 and pheophytin a. Reduction of pheophytin a was accompanied by an absorbance increase between 640 and 675 nm and a bleaching around 685 nm. Electron transfer to a secondary acceptor occurred with a time constant of 250–300 ps. If this secondary acceptor was reduced chemically, the primary radical pair decayed by charge recombination in about 2 ns.

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.

Kinetics of the oxygen-evolving complex in salt-washed photosystem II preparations

The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro-p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z+ confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl2, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P+Q− recombination and a slow Z+ decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl2. These results are interpreted as a light-induced release of strongly bound Ca2+ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca2+.

The influence of the oxidation state of the oxygen-evolving complex of Photosystem II on the spin-lattice relaxation time of Signal II as determined by electron spin-echo spectroscopy

The spin-lattice relaxation time of Signal II, which arises from two plastoquinol cation radicals, D+ and Z+, has been measured with electron spin-echo spectroscopy in Photosystem II preparations with inactivated and with intact oxygen-evolving complex. In Tris- and subsequently EDTA-washed Photosystem II preparations the spin-lattice relaxation times of D+ and Z+ are equal and remain unchanged if the pH is increased from 6.0 to 8.3. In preparations in which the oxygen-evolving complex is not inactivated by the Tris washing, the spin-lattice relaxation times of D+ and Z+ are affected by the redox state of the oxygen-evolving complex. At pH 6.0 the spin-lattice relaxation time decreases with higher redox state of the manganese cluster. The relaxation behavior at pH 8.3 indicates that at this pH the stability of the S1 state is decreased such that in dark-adapted samples nearly 100% of the systems is in the S0 state. This was confirmed by optical experiments where the period-four oscillation in the absorption at 350 nm was monitored as a function of the flash number.

Oxygen release may limit the rate of photosynthetic electron transport; the use of a weakly polarized oxygen cathode

Abstract The kinetics of photosynthetic oxygen evolution upon flash illumination were measured electrochemically at a much less negative cathode potential than used in conventional oxygen polarography, so that the electrode current was an essentially non-disturbing probe of the oxygen concentration. This method allows a good signal-to-noise ratio at sub-millisecond time resolution with stationary suspensions of photosynthetic material and avoids a strong inhibition of photosynthesis occurring under some conditions of measurement with a bare cathode. The results contradict the generally accepted notion that oxygen release promptly follows the 1.2 ms reduction of the oxygen-evolving complex after its four-step photooxidation. A much slower process has to take place before oxygen is detected and before a next cycle of four photoreactions in Photosystem II can be completed successfully: the next three photooxidations of the complex are unaffected, but the fourth is lost for oxygen evolution. This indicates that oxygen is released only slowly from the site of water oxidation, and that water oxidation is required to stabilize the fourth charge separation. Half-times of 30–130 ms were measured for oxygen release in different batches of Photosystem II membranes, chloroplasts and algae. In some conditions oxygen release may be a significant rate-limiting step in photosynthesis.

Destabilization by high pH of the S1‐state of the oxygen‐evolving complex in photosystem II particles

The S‐state distribution in a dark‐adapted photosystem II preparation, isolated from spinach chloroplasts, can be changed by pH. The period 4 oscillation of the oxygen‐releasing reaction, as measured by UV absorbance changes, is shifted by one flash at pH 8.3. This is not due to the generation of the EPR signal II on the first flash, or to the loss of a positive charge to some other electron donor after the first flashes. It is concluded that at pH 8.3 the S1‐state is reduced to S0. The half‐time of the reduction of S1 to S0 is 45 s, and the reduction is reversed in the dark if the pH is readjusted to pH 6.0. The oxygen release at pH 8.3 is irreversibly slowed down to about 3.2 ms.