Modification of the water oxidizing complex in leaves of the dgd1 mutant of Arabidopsis thaliana deficient in the galactolipid digalactosyldiacylglycerol.

The primary biochemical defect in the genetically well characterized dgd1 mutant of Arabidopsis thaliana causes a 90% reduction in the relative amount of the galactolipid digalactosyldiacylglycerol (DGDG). To study the effect of this DGDG deficiency on photosystem II (PS II), time-resolved transients of laser-flash-induced changes of the relative fluorescence quantum yield Fvar,rel(t) were measured in whole leaves from wild-type and the dgd1 mutant. The results obtained reveal (i) in untreated leaves the decay kinetics of Fvar, rel(t) reflecting QA.- reoxidation by endogenous plastoquinone are very similar in wild-type and the dgd1 mutant at room temperature, (ii) the Arrhenius plot of the temperature dependence of electron transfer from QA.- to QB exhibits a break point at about 19 degrees C in wild-type and about 12 degrees C in the dgd1 mutant, (iii) in leaves treated with DCMU the slow reoxidation of QA.- by the PS II donor side is blocked to a much higher extent in the dgd1 mutant (about 50%) compared to wild-type (about 10%), and iv) the normalized amplitude of Fvar,rel(t = 1 micros) reflecting the percentage of fast P680.+ reduction by YZ exhibits a characteristic period four oscillation in wild-type while this feature is strongly damped in the dgd1 mutant. Presumably, the severe DGDG deficiency is causing the thermal down shift of a lipid phase transition that affects the QA.- reoxidation by QB. Most strikingly, the properties of the WOC are modified as a result of reduced DGDG content. Thus, the lipid DGDG appears to be of structural relevance for the WOC.

Kinetics of S2 and S3 reduction by tyrosine Y(D) and other endogenous donors as a function of temperature in spinach PS II membrane fragments with a reconstituted plastoquinone pool.

The characteristic period four oscillation patterns of oxygen evolution induced by a train of single-turnover flashes were measured as a function of temperature in dark-adapted photosystem II (PS II) membrane fragments that were reconstituted with native plastoquinone-9 (PQ-9) by a recently developed procedure [Kurreck, J., Seeliger, A. G., Reifarth, F., Karge, M., & Renger, G. (1995) Biochemistry 34, 15721-15731]. The following results were obtained: (a) within the range 0-35 degrees C, the probabilities of misses (alpha) and double-hits (beta) and the dark population of redox state S1 exhibit similar dependencies on the temperature; (b) below a characteristic temperature theta(c) these parameters remain virtually independent of temperature, above theta(c) (theta(c) = 20 degrees C for alpha and beta; theta(c) = 30 degrees C for S1) the values of alpha and beta increase whereas S1 decreases; and (c) the dark decay of S2 and S3 via fast and slow kinetics owing to reduction of the water oxidase by Y(D) and other endogenous electron donor(s), respectively, exhibits comparatively strong temperature dependencies with the following activation energies: E(A)(S2(fast)) = 60 +/- 10 kJ/mol, EA(S3(fast)) = 55 +/- 10 kJ/mol, E(A)(S2(slow)) = 80 +/- 5 kJ/mol, and E(A)(S3(slow)) = 75 +/- 5 kJ/mol. These values of PQ-9 reconstituted PS II membrane fragments are very similar to those that were previously reported for thylakoids [Messinger, J., Schröder, W. P., & Renger, G. (1993) Biochemistry 32, 7658-7668]. These findings reveal that the reaction coordinates of feeding electrons by endogenous electron donors into the water oxidizing complex (WOC) that attains the redox states S2 and S3 is virtually invariant to Triton X-100 treatment used in the isolation procedure of PS II membrane fragments from thylakoids. Implications of these findings are discussed.

Reconstitution of the endogenous plastoquinone pool in photosystem II (PS II) membrane fragments, inside-out-vesicles, and PS II core complexes from spinach.

The possibility of reconstituting a functionally competent endogenous plastoquinone pool in photosystem II (PS II) membrane fragments, inside-out-vesicles (ISO-vesicles), and PS II core complexes was analyzed by measuring (i) the characteristic period four oscillation of the oxygen yield due to excitation of dark-adapted samples with a train of short flashes and (ii) laser flash-induced transients of the relative quantum yield of chlorophyll fluorescence. The data obtained revealed that (a) an endogenous pool capacity comparable to that of intact thylakoids can be restored in PS II membrane fragments and ISO-vesicles by a sonication treatment using native plastoquinone-9, (b) a more pronounced oxygen oscillation pattern arises in PS II core complexes after application of the same reconstitution procedure, (c) the extent of the endogenous pool restoration at a ratio of 15 quinone molecules per PS II in the reconstitution assay strongly depends on the nature of the quinone molecule [maximum effects can be only achieved with PQ-9, while at the same concentration ubiquinone-45 (UQ-9) is almost inefficient], and (d) a sonication step is required for stable insertion of PQ-9 into PS II preparations. Measurements of the reconstruction degree as a function of the structure of different quinones with selected properties lead to the conclusion that specific binding domains exist in PS II in addition to the QB site. These domains exhibit a surprisingly high specificity for the type of quinone that can be bound. On the basis of a comparison of the results obtained, the structure of the quinone head group seems to be more important than the large hydrophobic side chain and/or the general lipophilicity of the compound.

Involvement of the CP47 protein in stabilization and photoactivation of a functional water-oxidizing complex in the cyanobacterium Synechocystis sp. PCC 6803.

Oscillation patterns of the oxygen yield per flash induced by a train of single-turnover flashes were measured as a function of dark incubation and different pre-illumination conditions in several autotrophic mutant strains of Synechocystis sp. PCC 6803 carrying short deletions within the large, lumen-exposed hydrophilic region (loop E) of the chlorophyll a-binding photosystem II protein CP47. A physiological and biochemical characterization of these mutant strains has been presented previously [Eaton-Rye, J. J., & Vermaas, W. F. J. (1991) Plant Mol. Biol. 17, 1165-1177; Haag, E., Eaton-Rye, J. J., Renger, G., & Vermaas, W. F. J. (1993) Biochemistry 32, 4444-4454], and some functional properties were described recently [Gleiter, H. M., Haag, E., Shen, J.-R., Eaton-Rye, J. J., Inoue, Y., Vermaas, W. F. J., & Renger, G. (1994) Biochemistry 33, 12063-12071]. The present study shows that in several mutants the water-oxidizing complex (WOC) became inactivated during prolonged dark incubation, whereas the WOC of the wild-type strain remained active. The rate and extent of the inactivation in the mutants depend on the domain of loop E, where 3-8 amino acid residues were deleted. The most pronounced effects are observed in mutants delta(A373-D380) and delta(R384-V392). A competent WOC can be restored from the fully inactivated state by illumination with short saturating flashes. The number of flashes required for this process strongly depends on the site at which a deletion has been introduced into loop E. Again, the most prominent effects were found in mutants delta(A373-D380) and delta(R384-V392). Interestingly, the number of flashes required for activation was reduced by more than an order of magnitude in both mutants by the addition of 10 mM CaCl2 to the cell suspension. On the basis of a model for photoactivation proposed by Tamura and Cheniae (1987) [Biochim. Biophys. Acta 890, 179-194], a scheme is presented for the processes of dark inactivation and photoactivation in these mutants. The results presented here corroborate an important role of the large hydrophilic domain (loop E) of CP47 in a functional and stable WOC.