Water-splitting enzyme

H. Kuhl, J. Kruip, A. Seidler, A. Krieger-Liszkay, M. Bunker, D. Bald, J.A. Scheidig, M. Rogner (2000) Towards structural determination of the water-splitting enzyme. Purification, crystallization, and preliminary crystallographic studies of photosystem II from a thermophilic cyanobacterium. J. Biol. Chem., 275 20652-20659... 

Rutherford, A. W., Photosystem II, the water-splitting enzyme. Trends Biochem. Sci. 14 227, 1989. A readable account of current results and speculations on the oxygen-evolving complex. 

In-vitro approach Data are available in abundance concerning metal effects on isolated chloroplasts (for a review, see Clijsters and Van Assche, 1985). All the metals studied were found to be potential inhibitors of photosystem 2 (PS 2) photosystem 1 (PS 1) was reported to be less sensitive. From the in-vitro experiments, at least two potential metal-sensitive sites can be derived in the photosynthetic electron transport chain the water-splitting enzyme at the oxidising side of PS 2, and the NADPH-oxido-reductase (an enzyme with functional SH-groups) at the reducing side of PS 1 (Clijsters and Van Assche, 1985). Moreover, in vitro, non cyclic photophosphorylation was very sensitive to lead (Hampp et al., 1973 b) and mercury (Honeycutt and Korgmann, 1972). Both cyclic and non-cyclic photophosphorylation were proven to be inhibited by excess of copper (Uribe and Stark, 1982) and cadmium (Lucero et al, 1976). 

In-vivo metal application Reports of metal effects on electron transport and photophosphorylation after application of toxic amounts of metals to intact plants are less frequent. Cadmium and zinc inhibited PS 2 activity in Lycopersicon esculen-tum (Bazinsky et al, 1980) and Phaseolus vulgaris, respectively (Van Assche and Clijsters, 1983). In the green alga Euglena gracilis (De Filippis et al, 1981 b), PS 2 was sensitive to cadmium, copper and zinc. In the three species mentioned above, the water-splitting enzyme was the site of action. 

Z is reduced in the native system by electrons coming from the water-splitting enzyme and the kinetics of Z reduction are affected by the charge storage state (the so-called S states) of the enzyme. The differential kinetics of reduction for each S state were first observed by EPR [179] and correspond to the values obtained for the kinetics of S state turnover measured by absorption changes in the UV [180]. 

In the zeolite, owing to the lack of multimolecular degradation, oxidation of water by [Ru(bpy)3] + becomes possible. The reaction is slow, and hydroxyl radicals, hydrogen peroxide and superoxide are created as intermediates. In plant photosynthesis, an Mn-based water-splitting catalyst system in which the reaction with water only occurs after four electrons have been stored in the water-splitting enzyme... 

Fig. 1. Major protein complexes in the green-plant photosynthetic membrane (top) and the photosystem-ll RC complex (bottom). PC=plastocyanin, Fd=ferredoxin, CF, CFo=coupling factors the small numbers are the molecular weights of proteins in kDa. PS-II RC-core model adapted from Rutherford (1989) Photosystem II, the water-splitting enzyme. Trends in Biochem Sci 14 228.

Saygin and HT Witt (1975) Optical characterization of intermediates in the water-splitting enzyme system of photosynthesis-possible states and configuration of manganese and water. Biochim Biophys Acta 893 452-469... 

Fig. 2. (A) Flash-induced absorbance changes at 367 nm in PS-II particles isolated from Synechococcus at pH 7 and in the presence of 2X10 M silicomolybdate, (B) Spectra produced by each of four consecutive flashes in PS-II particles containing silicomolybdate. Figure source Saygin and Witt (1987) Optical characterization of intermediates in the water-splitting enzyme system of photosynthesis - possible slates and configuration of manganese and water. Biochim Biophys Acta 893 456, 457.

Fig. 6. (A) The effect of ANT-2p on the TL bands in dark-adapted chloroplasts in the absence of DCMU after being illuminated with one 5- s flash and then rapidly frozen to 77 K. (B) The effect of ANT-2p on the TL bands in chloroplasts in the presence of DCMU, without (a) and with ANT-2p (b and c). See text for discussion. Figure source Renger and Inoue (1983) Studies on the mechanism of ADRY agents (agents accelerating the deactivation reactions of water-splitting enzyme system Y) on thermoluminescence emission. Biochim Biophys Acta 725 148.

ADRY = Acceleration of the Deactivation Reactions of the water-splitting enzyme system Y. 

Thus, the formation of cryptohydroxyl radicals is energetically favoured in comparison to the water oxidation into free hydroxyl radical. At the present stage of knowledge it is premature to speculate which type of the above mentioned mechanism for crypto-hydroxyl-radical formation could be realized in the photosynthetic water splitting enzyme system Y. 

The immediate electron donor to Chl-ajj" is a tyrosine (6,7). The spectrum of the tyrosine oxidation in the UV (Fig. 5)(7) appears in the ns range simultaneously with that of the re-reduction of Chl-ajj". Tyr extracts an electron from the water splitting enzyme S. 

A PERIOD-FOUR INFRARED SIGNAL FROM ACTIVE WATER-SPLITTING ENZYME... 

Our attention was drawn to the signal which was exclusively observed with active particles and which is stable over several seconds. If its molecular origin were to be in the water-splitting enzyme, a characteristic flash pattern were to be observed upon excitation in a train of flashes. Fig. 3 shows the IR signal observed at 1740 cm" with a train of flashes (3a) and in a sequence of single flashes (3b). 

The reaction of the photosystem to DCMU strongly depends upon the redox-state of the electron-acceptors Without preillumination the primary acceptor Q is not completely reduced. Therefore, the first maximum of the oxygen-outburst still can be observed (left side, Fig.2) and the highest oxidation-state of the water-splitting enzyme can be reached. The peak also can be simulated by the calculation if 80 % reduction of Q is taken into account. The peak vanishes, if Q is completely reduced by preillumination (lower part of Fig.2). Preillumination shows no effect on the induction of the fluorescence. DCMU increases the fluorescence-intensity. There is nearly no dynamic because all acceptors Q are immediately reduced by excited reaction-centers of PS II and hence the propability of prompt fluorescence is increased. 

APPLICATION OF THE ELECTRON-DONATOR HYDROXILAMINE NH OH delivers electrons to P680 . Z nomore can be oxidized, therefore the oxydation of the water-splitting enzyme is suppressed. As can be seen from Fig. 4, the addition of hydroxilamine reduces the oxygen-developement and the first oxygen-peak is nearly absent depending from the applied concentration of hydroxilamine. The fluorescence smoothly reaches a constant value. A steady state of electron-flow from hydroxil-amine to the Calvin-Cycle via PS I is reached. The electron-delivery by... 

In addition, ABDAC apparently has a stabilizing effect on the water splitting enzyme, as it improves not only the functioning of the oxy-... 

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