Deregulation of electron flow within photosystem II in the absence of the PsbJ protein.

The photosystem II (PSII) complex of photosynthetic oxygen evolving membranes comprises a number of small proteins whose functions remain unknown. Here we report that the low molecular weight protein encoded by the psbJ gene is an intrinsic component of the PSII complex. Fluorescence kinetics, oxygen flash yield, and thermoluminescence measurements indicate that inactivation of the psbJ gene in Synechocystis 6803 cells and tobacco chloroplasts lowers PSII-mediated oxygen evolution activity and increases the lifetime of the reduced primary acceptor Q(A)(-) (more than a 100-fold in the tobacco DeltapsbJ mutant). The decay of the oxidized S(2,3) states of the oxygen-evolving complex is considerably accelerated, and the oscillations of the Q(B)(-)/S(2,3) recombination with the number of exciting flashes are damped. Thus, PSII can be assembled in the absence of PsbJ. However, the forward electron flow from Q(A)(-) to plastoquinone and back electron flow to the oxidized Mn cluster of the donor side are deregulated in the absence of PsbJ, thereby affecting the efficiency of PSII electron flow following the charge separation process.

Regulation of Photosystem II core protein phosphorylation at the substrate level: Light induces exposure of the CP43 chlorophyll a protein complex to thylakoid protein kinase(s).

Light induces conformational changes in the CP43 chl-a-protein antenna complex in isolated PS II core-complexes exposing phosphorylation site(s) to PS II core-associated protein kinase(s), to added solubilized thylakoid protein kinase(s), as well as to tryptic cleavage. The substrate-activation effect is demonstrated by exposure of the PS II cores to light during the kinase assay as well as by preillumination of the PS II cores in which the endogenous kinase(s) has been inactivated by treatment with N-ethylmaleimid. In the latter case, phosphorylation was performed in darkness following addition of the solubilized protein kinase(s). The solubilized protein kinase(s) does not require light activation. The apparent molecular masses of the main protein kinase(s) associated with the PS II cores (about 31-35 kDa and 45 kDa) differ from that of the major protein kinase present in solubilized preparations obtained from spinach thylakoids (64 kDa). The light-induced exposure of CP43 increases with the light intensity in the range of 20-100 mumol photons m(-2) s(-1) as demonstrated by preillumination of N-ethylmaleimid treated cores followed by addition of the solubilized protein kinase(s) and performing the phosphorylation assay in darkness.

Regulation of thylakoid protein phosphorylation at the substrate level: reversible light-induced conformational changes expose the phosphorylation site of the light-harvesting complex II.

Light-dependent activation of thylakoid protein phosphorylation regulates the energy distribution between photosystems I and II of oxygen-evolving photosynthetic eukaryotes as well as the turnover of photosystem II proteins. So far the only known effect of light on the phosphorylation process is the redox-dependent regulation of the membrane-bound protein kinase(s) activity via plastoquinol bound to the cytochrome bf complex and the redox state of thylakoid dithiols. By using a partially purified thylakoid protein kinase and isolated native chlorophyll (chl) a/b light-harvesting complex II (LHCII), as well as recombinant LHCII, we find that illumination of the chl-protein substrate exposes the phosphorylation site to the kinase. Light does not activate the phosphorylation of the LHCII apoprotein nor the recombinant pigment-reconstituted complex lacking the N-terminal domain that contains the phosphothreonine site. The suggested light-induced conformational change exposing the N-terminal domain of LHCII to the kinase is evidenced also by an increase in its accessibility to tryptic cleavage after light exposure. Light activates preferentially the trimeric form of LHCII, and the process is paralleled by chl fluorescence quenching. Both phenomena are slowly reversible in darkness. Light-induced exposure of the LHCII N-terminal domain to the endogenous protein kinase(s) and tryptic cleavage occurs also in thylakoid membranes. These results demonstrate that light may regulate thylakoid protein phosphorylation not only via the signal transduction chain connecting redox reactions to the protein kinase activation, but also by affecting the conformation of the chl-protein substrate.

Photoinactivation of photosystem II induces changes in the photochemical reaction center II abolishing the regulatory role of the QB site in the D1 protein degradation.

The effect of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (diuron) binding at the secondary quinone (QB) binding site of reaction center II (RCII), on the high-light-induced degradation of the RCII proteins D1 and D2, and the core proteins CP43 and CP47 was investigated in vivo in Chlamydomonas reinhardtii. The degradation of the RCII-D2 and the CP43 proteins shows a short lag relative to that of the RCII-D1 protein. Diuron retards but does not prevent the degradation of RCII-D1, D2 and CP43 proteins. The degradation of the CP47 protein is not retarded by diuron. The RCII-D1 protein present in cells photoinactivated in the presence of diuron is subsequently degraded in cells transferred to low light or to darkness. The protein can be replaced (turnover) at least partially under both conditions. The RCII-D1 protein is not degraded during photoinactivation of a cytochrome-bf-defective mutant. Degradation occurs however when the cells are returned to low light permitting slow reoxidation of plastoquinol [Zer, H., Prasil, O. & Ohad, I. (1994) J. Biol. Chem. 269, 17,670-17,676]. Addition of diuron does not prevent the degradation of the protein at this stage. Tryptic digestion of the RCII-D1 protein is partially inhibited by diuron in isolated thylakoids [Trebst, A., Depka, B., Kraft, B. & Johanningmeier, U. (1988) Photosynth. Res. 18, 163-177] but not in thylakoids obtained from photoinactivated cells. We conclude that photoinactivation induces a series of sequential changes in RCII exposing the cleavage site of the RCII-D1 protein to degradation and abolishing the regulatory role of the QB site occupancy by plastoquinone or analog ligands on the cleavage process. The degradation of the RCII-D2 and CP43 proteins may be a secondary process following modification and/or loss of the RCII-D1 protein.

Role of plastoquinol oxidoreduction in regulation of photochemical reaction center IID1 protein turnover in vivo.

The light-induced turnover of the D1 protein subunit of reaction center II (RCII) was investigated in Chlamydomonas reinhardtii y-1 (control) and D6, AC208, and B4 mutants lacking cytochrome b6/f, plastocyanin or photosystem I activity, respectively, and, thus, impaired in light-dependent plastoquinol (PQH2) oxidation. Charge recombination assayed by thermoluminescence measurements indicated similar RCII properties in control and mutant cells. The D1 protein is not degraded in the mutants during photoinactivation; however, RCII-D1 is irreversibly altered, and the protein is degraded when the cells are incubated in low light permitting slow reoxidation of the PQH2 pool. Photoinactivation precedes D1 degradation also in the control cells. Thus, in vivo under physiological conditions photoinactivation and "tagging" of RCII-D1 are resolved from the degradation process. RCII activity in photoinactivated cells may be recovered only following D1 degradation and replacement. Recovery may occur either in the light or dark in the absence of de novo chlorophyll synthesis. The degradation of the photoinactivated RCII-D1 protein is a prerequisite for the synthesis and stable integration of new D1 indicating that tagged D1 is still assembled in the inactive reaction centers. The physiological implication of these results is that oxidation of the PQH2 pool in photoinactivated cells affects RCII-D1 protein degradation and replacement, and, thus, D1 turnover in vivo is regulated by the turnover of PQ at the binding site of the secondary stable electron acceptor quinone of RCII.

Inverse correlation between resistance towards copper and towards the redox-cycling compound paraquat: a study in copper-tolerant hepatocytes in tissue culture.

The essential mediatory role of copper or iron in the manifestation of paraquat toxicity has been demonstrated (Kohen and Chevion (1985) Free Rad. Res. Commun. 1, 79-88; Korbashi, P. et al. (1986) J. Biol. Chem. 261, 12472-12476). Several liver cell lines, characterized by their resistance to copper, were challenged with paraquat and their cross-resistance to paraquat and copper was studied. Cell growth and survival data showed that copper-resistant cells, containing elevated copper, are more sensitive towards paraquat than wild type cells. Copper-deprived resistant cells did not have this sensitivity. Paraquat was also shown to cause a marked degradation of cellular glutathione in all cell lines. Albeit the fact that the basal glutathione levels are higher in copper-resistant than in wild type cells, there is more paraquat-induced degradation of cellular glutathione (GSH + GSSG) in resistant cells. It is suggested that in copper-resistant cells which contain elevated levels of copper, paraquat-induced cellular injury is potentiated even where glutathione levels are elevated. Additionally, in vitro experiments are presented that support the in vivo findings demonstrating a role for copper in glutathione degradation.