Influence of Ca(2+) on the thylakoid lumen violaxanthin de-epoxidase activity through Ca(2+) gating of H(+) flux at the CF(o) H(+) channel.

Part of the chloroplast photoprotection response to excess light absorption involves formation of zeaxanthin (and antheraxanthin) via the violaxanthin deepoxidase enzyme, the activity of which requires lumen acidity near or below pH 6.0. Clearly, the violaxanthin de-epoxidase activity is strongly regulated because at equivalent energization levels (including the parameters of H(+) accumulation and ATP formation rates), there can be either low or high violaxanthin de-epoxidase enzyme activity. This work shows that the factor or factors responsible for regulating the violaxanthin deepoxidase correlate directly with those which regulate the expression of membrane-localized or delocalized proton gradient (Delta[Formula: see text] (H+)) energy coupling. The most clearly identified factor regulating switching between localized and delocalized energy coupling modes is Ca(2+) binding to the lumen side of the thylakoid membrane; in particular, Ca(2+) binding to the 8 kDA subunit III of the CF(o) H(+) channel. The activity of violaxanthin deepoxidase in pea (Pisum sativa) and spinach (Spinacea oleracea) thylakoids is shown here to be strongly correlated with conditions known from previous work to displace Ca(2+) from the CF(o) H(+) channel and thus to modulate the extent of lumenal acidification while maintaining a fairly constant rate of ATP formation.

Influence of osmolality of the medium on photosynthetic electron transport, proton fluxes and photophosphorylation in isolated thylakoids.

The high osmotic potential inhibition of photosynthetic electron transport was determined to be related to membrane compaction rather than to an effect of primary thylakoid volume changes. Osmotic inhibition of proton fluxes and phosphorylation were entirely due to osmotic inhibition of electron transport. The ATPase activity, the nature of coupling and the rate constant of proton efflux were not influenced by osmotic pressure, while the rate constant and the extent of proton influx were inhibited by osmotic pressure.

Destruction of vitamin K1 of cultured carrot cells by ultraviolet radiation and its effect on plasma membrane electron transport reactions.

The effect of ultraviolet radiation on plasma membrane electron transport reactions was studied in cultured carrot cells. It was found that a 90 min treatment inhibited transmembrane hexacyanoferrate reduction greater than 50%. Extraction of lipophilic quinones from irradiated cells showed that vitamin K1 and coenzyme Q were totally destroyed, while control unirradiated cells showed the presence of 0.4 mumole vitamin K1 g dry wt.-1. The addition of exogenous vitamin K1 in concentrations of 1-10 microM partially restored plasma membrane electron transport with impermeable hexacyanoferrate as the electron acceptor. Total restoration of activity was given by growing irradiated cells in vitamin K1 supplemented growth media for 6 days. This shows that vitamin K1 may function as a member of the transplasma membrane electron transport chain in cultured carrot cells.

Inhibition of violaxanthin deepoxidation by ultraviolet-B radiation in isolated chloroplasts and intact leaves.

The effect of pretreatment with ultraviolet-B (UV-B) light (280-320 nanometers) on the enzymatic conversion of the diepoxyxanthophyll violaxanthin to the epoxy-free zeaxanthin occurring in thylakoid membranes was investigated. When isolated chloroplasts of pea (Pisum sativum) were exposed to UV-B, a biologically effective fluence of 7000 joules per square meter caused about 50% inhibition of the activity of the violaxanthin deepoxidase, measured as the first order rate constant of the absorbance change at 505 nanometers. The dose requirement for the inhibition of the deepoxidase in intact leaves, however, was about 2 orders of magnitude higher. The inhibition of the rate constant was observed for both the dark deepoxidation at pH 5, and for the light-driven deepoxidation induced by the lumen acidification due to electron transport from H(2)O to methylviologen or due to a photosystem I partial reaction with duroquinol as the electron donor. The availability of violaxanthin was not directly affected by UV-B radiation, as shown for UV-B-treated chloroplasts by the final extent of the 505 nanometer change measured in the dark at pH 5 or by the partial photosystem I reaction. A significant decrease in the violaxanthin availability was observed when lumen acidification was caused by electron transport from H(2)O to methylviologen. That effect was probably caused by the wellknown UV-B inhibition of photosystem II with a subsequent decreased ability to reduce the plastoquinone pool, the redox state of which is believed to regulate the final amount of converted violaxanthin.

Evidence that localized energy coupling in thylakoids can continue beyond the energetic threshold onset into steady illumination.

Energy transduction from proton gradients into ATP formation in chloroplast thylakoids has been hypothesized to be driven equally efficiently by localized domain delta mu H+ or by a delocalized delta mu H+ (Beard, W. A. and Dilley, R. A. (1988) J. Bioenerg. Biomembr. 20, 129-154). An important question is whether the apparent localized protonmotive force energy coupling mode can be observed only in the dark-to-light transient in the flash excitation protocol commonly used, or whether the localized energy coupling gradient can be maintained under conditions of continuous illumination ATP formation. The assay in the previous work was to use permeable amines, added to thylakoids in the dark, and observe the effect of the amine on the length of the energization lag (number of single-turnover flashes) required to initiate ATP formation in the dark-to-light transition. Amine buffers delayed the ATP onset in high-salt-stored membranes but did not delay the onset with low salt-stored membranes. This work tested whether permeable amines show the different effects in low- or high-salt-stored thylakoids which had attained a steady-state ATP formation rate (in continuous light) for 20-40 s prior to adding the amine. Hydroxyethylmorpholine was the preferred amine for such experiments, a suitable choice inasmuch as it behaves similarly to pyridine in the flash-induced ATP formation onset experiments, but it permeates more rapidly than pyridine and it has a higher pKa, which enhances its buffering effects. With high-salt-stored thylakoids, 0.5 or 1.0 mM hydroxyethylmorpholine added after 40 s of continuous illumination caused a marked, but transient, slowing of the ATP formation rate, but little or no slowing of the rate was observed with low-salt-stored thylakoids (at similar phosphorylation rates for the two thylakoid samples). Those data indicate that in continuous illumination conditions the proton gradient driving ATP formation in thylakoids from the low-salt-stored treatment did not equilibrate with the lumen, but in thylakoids stored in high-salt the delta mu H+ freely equilibrated with the lumen. That suggestion was supported by measurement of the luminal pH under coupling conditions by the [14C]methylamine distribution method using low- or high-salt-stored thylakoids. Further supportive evidence was obtained from measuring the effect of permeable amine buffers on H+ uptake under coupled and basal conditions with both types of thylakoid.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional size of photosynthetic electron transport chain determined by radiation inactivation.

Radiation inactivation technique was employed to determine the functional size of photosynthetic electron transport chain of spinach chloroplasts. The functional size for photosystem I+II (H(2)O to methylviologen) was 623 +/- 37 kilodaltons; for photosystem II (H(2)O to dimethylquinone/ferricyanide), 174 +/- 11 kilodaltons; and for photosystem I (reduced diaminodurene to methylviologen), 190 +/- 11 kilodaltons. The difference between 364 +/- 22 (the sum of 174 +/- 11 and 190 +/- 11) kilodaltons and 623 +/- 37 kilodaltons is partially explained to be due to the presence of two molecules of cytochrome b(6)/f complex of 280 kilodaltons. The molecular mass for other partial reactions of photosynthetic electron flow, also measured by radiation inactivation, is reported. The molecular mass obtained by this technique is compared with that determined by other conventional biochemical methods. A working hypothesis for the composition, stoichiometry, and organization of polypeptides for photosynthetic electron transport chain is proposed.