Richard A. Dilley

The pH Dependence of Violaxanthin Deepoxidation in Isolated Pea Chloroplasts.

The absorbance change at 505 nm was used to monitor the kinetics of violaxanthin deepoxidation in isolated pea (Pisum sativum) chloroplasts under dark conditions at various pH values. In long-term measurements (65 min) a fast and a slow exponential component of the 505-nm absorbance change could be resolved. The fast rate constant was up to 10 times higher than the slow rate constant. The asymptote value of the fast kinetic component was twice that of the slow component. The pH dependency of the parameters of the fast kinetic component was analyzed from pH 5.2 to pH 7.0. It was found that the asymptote value dropped slightly with increasing pH. The rate constant was zero at pH values greater than 6.3 and showed maximum values at pH values less than 5.8. Hill plot analysis revealed a strong positive cooperativity for the pH dependency of the fast rate constant (Hill coefficient nH = 5.3). The results are discussed with respect to published activity curves of violaxanthin deepoxidation.

Photophosphorylation as a function of illumination time. II. Effects of permeant buffers

(1) The amounts of orthophosphate, bicarbonate and tris (hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid. (2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KC1 are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl) aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KC1, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrepancies occur with all of the other buffers mentioned. (3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KC1, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase. (4) We suggest that the protons produced by electron transport may be used directly for phosphorylation without even entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.

Photoinactivation of chloroplasts already inhibited on the oxidizing side of photosystem II

Abstract We have investigated the irreversible inhibition produced in the photosynthetic electron-transport chain by illuminating Cl − -free or Tris-washed chloroplasts. Our data indicate that the site of this inhibition is on the oxidizing side of Photosystem II, either at the reaction center, P-680, or its immediate electron donor, Z; the possibility that the primary photoinduced lesion is at the second stable Photosystem II electron acceptor, Q B , has been excluded. Comparison of our data with those in the literature lead us to postulate that the photoinactivation under study here is mechanistically the same as photoinhibition in living plants (Powles, S.B. (1984) Annu. Rev. Plant Physiol. 35, 15–44), the former occurring on an accelerated time scale.

Photophosphorylation as a function of illumination time. I. Effects of permeant cations and permeant anions

(1) Very brief periods of illumination do not initiate photophosphorylation in isolated chloroplast lamellae. The time of illumination required before any phosphorylation can be detected is inversely proportional to the light intensity. At very high intensities, phosphorylation is initiated after illumination for about 4 ms. (2) There is no similar delay in the initiation of electron transport. The rate of electron transport is very high at first but declines at about the time the capacity for ATP synthesis develops. When the chloroplasts are uncoupled with gramicidin the high initial rate persists. (3) Various ions which permeate the thylakoid membrane (K+ or Rb+ in the presence of valinomycin, SCN-, I-, or C1O4-) markedly increase the time of illumination required to initiate phosphorylation. Potassium ions in the presence of valinomycin increase the delay to a maximum of about 50 ms whereas thiocyanate ions increase the delay to a maximum of about 25 ms. The effects of K+ with valinomycin and the effect of SCN- are not additive. Permeant ions and combinations of permeant ions have little or no effect on phosphorylation during continuous illumination. (4) The reason for the threshold in the light requirement and the reason for the effect of permeant ions thereon are both obscure. However, it could be argued that the energy for phosphorylation initially resides in an electric potential gradient which is abolished by migration of ions in the field, leaving a more slowly developing proton concentration gradient as the main driving force for phosphorylation during continuous illumination. If so, the threshold in the presence of permeant ions should depend on internal hydrogen ion buffering.

A partial reaction in Photosystem II: Reduction of silicomolybdate prior to the site of dichlorophenyldimethylurea inhibition

Silicomolybdate functions as an electron acceptor in a Photosystem II water oxidation (measured as O2 evolution) partial reaction that is 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) insensitive, that is, reduction os silicomolybdate occurs at or before the level of Q, the primary electron acceptor for Photosystem II. This report characterizes the partial reaction with the principal findings being as follows: 1. Electron transport to silicomolybdate significantly decreased room temperature Photosystem I side of the DCMU had no effect on the fluorescence level, consistent with silicomolybdate accepting electrons at or before Q. In the absence of DCMU, silicomolybdate is also reduced at a site on the Photosystem I side of the DCMU block, prior to or at plastoquinone, since the plastoquinone antagonist dibromothymoquinone (DBMIB) did not affect the electron transport rate. 3. Electron transport from water to silicomolybdate (+ DCMU) is not coupled to ATP formation, nor is there a measurable accumulation of protons within the membrane (measured by amine uptake). Silicomolybdate is not inhibitory to phosphorylation per se since neither cyclic nor post-illumination (XE) phosphorylation were inhibited. 4. Uncouplers stimulated electron transport from water to silicomolybdate in the pH range of 6 to 7, but inhibited at pH values near 8. These data are consistent with the view that when electron flow is through the abbreviated sequence of water to Photosystem II to silicomolybdate (+ DCMU), conditions are not established for the water protons to be deposited within the membrane. Experiments reported elsewhere (Fiaquinta, R.T., Dilley, R.A. and Horton, P.(19741 J. Bioenerg. 6, 167-177) and these data, are consistent with the hypothesis that electron transport between Q and plastoquinone energizes a membrane conformational change that is required to interact with the water oxication system so as to result in the deposition of water protons either within the membrane itself or within the inner oxmotic space.

Intrathylakoid pH in Isolated Pea Chloroplasts as Probed by Violaxanthin Deepoxidation

Light-driven violaxanthin deepoxidation was measured in isolated pea (Pisum sativum) chloroplasts without ATP synthesis (basal conditions) and with ATP synthesis (coupled conditions). Thylakoids stored in high salt (HS) or low salt (LS) storage medium were tested. In previous experiments, HS thylakoids and LS thylakoids were related to delocalized and localized proton coupling, respectively.Light-driven deepoxidase activity was compared to the pH dependence of deepoxidase activity established in dark reactions. At an external pH of 8, light-driven deepoxidation indicated effective pH values close to pH 6 for all reaction conditions. Parallel to deepoxidation, the thylakoid lumen pH was estimated by the fluorescent dye pyranine.In LS thylakoids under coupled conditions the lumen pH did not drop below pH 6.7. At pH 6.7, no deepoxidase activity is expected based on the pH dependence of enzyme activity. The results suggest that deepoxidation activity is controlled by the pH in sequestered membrane domains, which, under localized proton coupling, can be maintained at pH 6.0 when the lumen pH is far above pH 6.0. The extent of violaxanthin conversion (availability), however, appeared to be regulated by lumenal pH. Dithiothreitol-sensitive nonphotochemical quenching of chlorophyll fluorescence was dependent on zeaxanthin and not related to lumenal pH. Thus, zeaxanthin-dependent quenching[mdash]known to be pH dependent[mdash]appeared to be triggered by the pH of localized membrane domains.

Kinase-induced changes in electron transport rates of spinach chloroplasts

Abstract Phosphorylation of spinach chloroplast membranes by the endogenous protein kinase caused oppositely directed changes in the rates of photosystems I and II partial electron transport reactions. When assayed at a limiting actinic light intensity, the rate of the photosystem II reaction decreased, and that of photosystem I increased, by approximately 15–20%. These effects could only be accurately measured, however, after minimizing the photoinhibition of electron transport that resulted from the illumination used to activate the chloroplast protein kinase. The data support the hypothesis that phosphorylation of the light-harvesting chlorophyll protein results in a change in the distribution of absorbed light energy between the two photosystems.

Purification and partial sequence of the Mr 10,000 phosphoprotein from spinach thylakoids

The Mr 10,000 phosphoprotein was purified from photosystem II particles by solubilization of the particles in 5% (w/v) dodecyl dimethylamine oxide, centrifugation in 10% (w/v) sucrose, and three chromatography steps. The purified phosphoprotein showed a unique NH2 terminus indicating a highly purified polypeptide. The amino acid sequence for the first nine residues is NH2-Ala-Thr-Gln-Thr-Val-Glu-Ser-Ser-Ser . . . COOH. The amino acid composition was determined and could also be used to help distinguish the polypeptide from other known thylakoid proteins. The sequence and composition data indicated that the Mr 10,000 phosphoprotein is neither the hydrophobic 8-kDa subunit of the energy coupling complex nor cytochrome b-559, but rather a unique, as yet unidentified, polypeptide associated with photosystem II.

Selective inhibition of the spinach thylakoid LHC II protein kinase

Abstract Treatment of spinach thylakoids with the adenosine affinity inhibitor 5′- p -fluorosulfonylbenzoyl adenosine (FSBA) resulted in at least 95% inhibition of phosphorylation of the light-harvesting protein complex of Photosystem II (LHC II), while the M r 10 000 polypeptide showed a 35% decrease in phosphorylation. This residual kinase activity after FSBA treatment appears to have the same properties as the control, since phosphorylation of the M r 10 000 polypeptide subsequent to FSBA treatment could be achieved with either light or reducing conditions in the dark. [ 14 C]FSBA labelled several polypeptides, but only the M r 50 000 band was protected against the label by prior addition of ADP or adenosine, making it a possible candidate for the LHC II kinase. FSBA had no effect on electron transport, and [ 14 C]FSBA did not label LHC II or the M r 10 000 polypeptide, indicating that the FSBA was not interfering with activation of the kinase or modifying the substrates, but rather acting at the level of the LHC II protein kinase. Inhibition of LHC II phosphorylation by FSBA resulted in the elimination of the slow ATP-induced decrease in variable fluorescence, a parameter believed to be associated with phosphorylation of the LHC II. The half-times and time-course for inhibition of LHC II phosphorylation and inhibition of the ATP-induced decrease of fluorescence yield were identical, consistent with the concept that LHC II phosphorylation plays a major role in this fluorescence change.

Photophosphorylation not coupled to DCMU-insensitive photosystem II oxygen evolution

Summary An abbreviated Photosystem II electron transport sequence from water to silicomolybdate plus ferricyanide functions in the presence of DCMU. This electron transfer was not coupled to photophosphorylation, and therefore places phosphorylation associated with Photosystem II (Site II) after the site of DCMU inhibition. Silicomolybdate per se was not inhibitory to phosphorylation in either the cyclic or noncyclic (water to methylviologen) electron transport mode.