Redox state chloroplast

Horton, P., Allen, J.F., Black, M.T. and Bennett, J. 1981. Regulation of phosphorylation of chloroplast membrane polypeptides by the redox state of the plastoquinones. FEBS Lett., 125.193-196. 

It is also noted that rusticyanin has three additional histidines (five in all). An increase in reduction potential for Rieske s [2Fe-2S] protein (350 mV) as compared to that for chloroplast [2Fe-2S] ferrodoxins (-400 mV) has been explained by the coordination of two histidines instead of two cysteines (76). In the case of the high-potential [4Fe-4S] protein, the reduction potential of 350 mV, compared to that for [4Fe-4S] centers in bacterial ferrodoxins (-400 mV), is accounted for by a different redox state change. This is made possible by H bonding and/or the more buried nature of the [4Fe-4S] cluster (77, 78). On present evidence, neither of these possibilities would seem to explain the high E° of rusticyanin. Another so far unexplained difference in the case of rusticyanin is its stability at pH 2, which is the working pH in its in vivo reaction with an acid-resistant cytochrome and aqua Fe (47, 48). An X-ray crystal structure of rusticyanin is required to help understand these different properties. 

To explain the Emerson enhancement effect, Govindjee and Rahinowitch ° obtained evidence from the action spectra of the enhancement effect the presence of two distinct forms of Chl-a in vivo presumably present in two different pigment systems. Soon, new experimental evidence in support of the two-photo-system concept was reported independently by Govindjee etal and by Kautsky eta/. from evidence obtained from fluorescence and by Kok and Hoch Duysens " and Witt based on measurements of absorbance changes. We present here two results based on absorbance changes associated with changes in the redox state of electron carriers in the chloroplast. 

In the meantime, Witt and colleagues observed similar effects on the redox state of cytochrome / when red and far-red light were alternately used for illumination. They also observed similar two-light effects with respect to a species that was later identified as plastoquinone, as well as another species, P700 discovered earlier by Kok. Fig. 17 (A) shows the spectroscopic results of the two-light effect on P700 in broken spinach chloroplasts. Illumination of chloroplasts with 720-nm light of 0.5- ec duration... 

Fig. 7. (A) Time course of phosphorylation of LHC II during the state 1-to-state 2 (left) and state 2-to-state 1 (right) transitions in pea chloroplasts. (B) Redox titration of P incorporation into LHC II and ATP-induced fluorescence decrease. (A) adapted from Telfer, Allen, Barber and Bennett (1983) Thylakoidprotein phosphorylation during state 1-state 2 transitions in osmotically shocked pea chloroplasts. Biochim Biophys Acta. 722 176-181 (B) from Horton, Allen, Black and Bennett (1981) Regulation of phosphorylation of chloroplast membrane polypeptides by the redox state of plastoquinone. FEBS Lett 125 194.

As described earlier, the Chl-a fluorescence yield ofchloroplasts is influenced by the redox state ofthe electron acceptor Q, and thus the amplitude of fluorescence yield may be used to monitor the midpoint potential ofQ by means of a redox titration. Such a redox titration was first carried out by Cramer and Butler in 1969 with spinach chloroplasts at pH 7. Two transitions, presumably representative of two quenchers of fluorescence or electron acceptors, were observed in the titration process, with one of value between -20 and -35 mV, and another with a much more negative value, between -270 and -320 mV. The quencher Q, which had been held for the previously described, light-induced fluorescence yield changes, was ascribed to tbe more positive quenching transition. 

As discussed in Chapter 16, the fluorescence yield changes in chloroplasts reflect the redox state of the stable primary electron acceptor of photosystem II. In 1972, Warren Butler proposed that the oxidized... 

Fig. 10. Absorbance-change transients at 820 nm in PS-1 particles containing PMSHj(PMS plus dithionite) induced by a series of four saturating dye-laser flashes. The redox states of the PS-1 reaction center and the reactions accompanying the series of laser flash-induced absorbance changes are illustrated at right. See text for further details. Figure source. Sauer, Mathis, Acker and van Best (1978) Electron acceptors associated with P-700 in triton solubilized photosystem I particles from spinach chloroplasts. Biochim Biophys Acta 503 127.

Because the redox state of the complex ZQB controls fluorescence yield, Z must be inactivated in order to probe QB interactions. A pretreatment of chloroplasts with high concentrations of hydroxylamine will completely destroy the oxidizing side of PS II (Z) and produce some reduction of B (13). The experimental protocol was the following first, a chloroplast suspension was treated in the dark with 10 mM hydroxylamine and was measured immediately. After a 5 min dark incubation period, the chloroplasts received a strong actinic illumination that gave rise to the QB form. After complete relaxation (15 min), DCMU-type inhibitors were added producing Q B and difference between the two values was evaluated in relation to the inhibitor concentrations and a half-effect determined. 

There is a significant difference, however, between the effect of pH that we report here (the fast phase has a pH-independent Xy its proportion increases at high pH) and the effects observed earlier with chloroplasts or PS-II particles (the fast phase has a pH-dependent half-time). In fact, the pH effect is probably related to the protonation of chemical groups close to Z. but its precise origin is difficult to analyze. The different behaviour observed here may be due to the absence of several polypeptides which normally participate in the PS-II reaction center core. If our interpretation is correct, it also follows that Z is active in only a fraction of the reaction centers. This could be due to its redox state (it might stay partly oxidized in our experiments), or to a partial denaturation of some of the centers. 

Using the protocol of titrations of qE against the ionophoric uncoupler nigericin in p>ea and spinach chloroplasts, and using 9-aminoacridine fluorescence as a monitor for the proton gradient, we have investigated some relationships between ApH, "redox state", and zeaxanthin content, in their effects up>on the magnitude of qE. 

Assay of thylakoid phosphorylation in vivo showed that the LHCII polypeptides 13 and 17 are not phosphorylated in the dark nor in light incubated cells, while LHCn polypeptides 11 and 12 as well as the 32-35 kDa polypeptides are weakly phosphorylated Fig, 3). These results are in agreement with previously published data using a different cyt.bredox controlled phosphorylation of thylakoid polypeptides in Chlamydomonas cells may be influence by the chlororespiration pathway affecting the redox state of the chloroplast membranes (7), it was of interest to test the kinase(s) activity in isolated thylakoids in vitro. Results of such an experiment, shown in Fig.4, demonstrate that the LHCII polypeptides 13 and 17 are not phosphorylated even if reductants known to Fig. 2. Absence of cyt. be/f polypeptides in thylakoids activate the ledox controlled kinase were present, marker "O difference was found i" the... 

Gasexchange of attached leaves of Phaseolus vulgavis and 820 nm absorbance changes, monitoring the redox state of P700 were measured simultaneously as in [4]. To supress photorespiration O2 was reduced to 2% when CO2 was low and net e"-transport from H2O to PGA) was calculated from CO2-uptake. Intact chloroplasts were isolated from Spinacia oleracea. For determination of the activation state of enzymes their activity was measured immediately after osmotic rupture of chloroplasts as in [5]. 

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