Seikichi Izawa

Electron transport and photophosphorylation in chloroplasts as a function of the electron acceptor. II. Acceptor-specific inhibition by KCN

1. 1. Dark pretreatment of chloroplasts with high concentrations of KCN in the presence of a trace of ferricyanide completely blocks subsequent transfer of electrons from water to hydrophilic acceptors such as ferricyanide and methylviologen. In the KCN-treated chloroplasts there is still a largely uninhibited electron flow from water to lipophilic oxidants such as p-benzoquinonediimide (oxidized p-phenylenediamine), duroquinonediimide (oxidized diaminodurene) and 2,5-dimethyl-p-benzoquinone. The cyanide-insensitive electron transport supports phosphorylation with a P/e2 ratio of 0.3 to 0.4. It is concluded (a) that these lipophilic oxidants can accept electrons from an unknown intermediate carrier, X, which precedes the KCN-inhibition site and is inaccessible to hydrophilic oxidants, and (b) that a phosphorylation site is associated with the electron pathway H2O→X. 2. 2. Electron transport from diaminodurene to methylviologen and the associated phosphorylation are both inhibited by KCN. The same is true of cyclic photophosphorylation reactions catalyzed by diaminodurene, reduced dichlorophenolindophenol, pyocyanine, and low concentrations of N-methylphenazonium methosulfate. However, cyclic photophosphorylation can be largely restored by high concentrations of PMS. 3. 3. Isolated plastocyanin reacts readily with high concentrations of KCN (> 10 mM) but only at the relatively high pH values (> 7.5) required for effective KCN treatment of chloroplasts. It is suggested that this copper protein is the site of KCN inhibition in chloroplasts. 4. 4. Photoreduction of p-benzoquinonediimide is extremely sensitive to Photosystem II inhibitors, such as 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and the inhibition is completely independent of light intensity. In contrast, although the reduction of ferricyanide is almost equally sensitive at very low light intensities, the sensitivity decreases as the light intensity increases. It is suggested that the lipophilic reduction site, X, may be located close to Photosystem II.

The stoichiometry of photophosphorylation

Abstract Using chloroplasts from three different plant species in the presence of four different buffer systems we found that the P 2e ratio was consistently above 1.0 if the pH was between 8.4 and 9.4. At pH 8.9 the ratio was usually above 1.25 and indeed no single determination of the ratio fell below 1.1 at this pH unless Tris-HCl was present. Even if the endogenous phosphorylation were subtracted (a procedure which is probably not valid) no single determination would yield a value of P 2e as low as 1.0. Consequently it seems very probable that the theoretical maximum efficiency of photophosphorylation is higher than has been thought.

The number of sites sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea,3-(4-chlorophenyl)-1,1-dimethylurea and 2-chloro-4-(2-propylamino)-6-ethylamino-s-triazine in isolated chloroplasts.

Abstract The absorption of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, 3-(4-chlorophenyl)-1,1-dimethylurea and Atrazine by isolated spinach chloroplasts involves at least three simultaneous processes: (a) An irreversible binding or destruction accounting for about one molecule of inhibitor for every 1000 chlorophyll molecules. This process is not associated with inhibition. (b) A partitioning between the biological and aqueous solvent phases which is independent of inhibitor concentration over the inhibitory range. (c) An absorption which corresponds closely to the degree of inhibition. This process probably represents the formation of the enzyme-inhibitor complex. Estimates of the number of inhibitor-sensitive sites based on the analysis of the partitioning phenomena and estimates based on the Straus-Goldstein analysis of the inhibition kinetics agree. Regardless of which of the three inhibitors is used the number of sites of inhibition seems to be one for every 2500 chlorophyll molecules. The nature of the “photosynthetic unit” is discussed and the question of the minimum size of chloroplast fragments capable of oxygen production is reconsidered in the light of these observations.

Electron transport and photophosphorylation in chloroplasts as a function of the electron acceptor. III. A dibromothymoquinone-insensitive phosphorylation reaction associated with Photosystem II

Abstract Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) is reputed to be a plastoquinone antagonist which prevents the photoreduction of hydrophilic oxidants such as ferredoxin-NADP+. However, we have found that dibromothymoquinone inhibits only a small part of the photoreduction of lipophilic oxidants such as oxidized p-phenylenediamine. Dibromothymoquinone-resistant photoreduction reactions are coupled to phosphorylation, about 0.4 molecules of ATP consistently being formed for every pair of electrons transported. Dibromothymoquinone itself is a lipophilic oxidant which can be photoreduced by chloroplasts, then reoxidized by ferricyanide or oxygen. The electron transport thus catalysed also supports phosphorylation and the P e 2 ratio is again 0.4. It is concluded that there is a site of phosphorylation before the dibromothymoquinone block and another site of phosphorylation after the block. The former site must be associated with electron transfer reactions near Photosystem II, while the latter site is presumably associated with the transfer of electrons from plastoquinone to cytochrome f.

Studies on the energy coupling sites of photophosphorylation. I. Separation of Site I and Site II by partial reactions of the chloroplast electron transport chain

Abstract 1. The transport of electrons from H2O to lipophilic oxidants such as oxidized p-phenylenediamines and 2,5-dimethylquinone, when observed in the presence of the plastoquinone antagonist dibromothymoquinone, has a pH optimum of approximately 7.5 and is independent of the presence or absence of ADP and phosphate. Nevertheless the electron transport supports phosphorylation with an efficiency ( P e 2 ) of 0.3–0.4 and this efficiency is practically pH independent. A reversible proton uptake is associated with the electron transport. The energy coupling site responsible for the phosphorylation, which must be before plastoquinone, we have designated Site II, while the well-known rate-determining coupling site after plastoquinone and before cytochrome f is referred to as Site I. 2. The transport of electrons from reduced 2,6-dichlorophenolindophenol (DCIP) to methylviologen, when observed in the presence of the Photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, is remarkably similar in most respects to the overall Hill reaction (e.g. H2O→methylviologen). The rate of electron flow is markedly stimulated by ADP and phosphate. Electron transport and phosphorylation have the same pH optimum of about 8.5. The P e 2 ratio is also strongly pH dependent, showing a similar pH optimum of 8.0–8.5. However, the absolute value of the P e 2 ratio observed for the partial reaction reduced DCIP→methylviologen is lower than the P e 2 ratio observed for the overall reaction H2O→methylviologen at all pH values. The maximum P e 2 value observed for the reduced DCIP→methylviologen raction is 0.5–0.6 at pH 8.0–8.5 while the maximum value for the H2O→methylviologen reaction under the same conditions is about 1.1. 3. When the P e 2 ratios for the two partial reactions (H2O→dimethylquinone and reduced DCIP→methylviologen) are added together at all pH values from 6 to 9, the resulting curve is very close to the P e 2 -pH profile experimentally obtained for the overall Hill reaction H2O→methylviologen. It seems probable, therefore, that the transport of electrons from reduced DCIP to methylviologen utilizes only the rate-determining coupling Site I while the overall transport of electrons from H2O to methylviologen utilizes both Site I and Site II.

Uncoupling and Energy Transfer Inhibition in Photophosphorylation

Publisher Summary This chapter discusses uncoupling and energy transfer inhibition in photophosphorylation. The uncouplers promote non-phosphorylating electron transport and this lowers the efficiency of phosphorylation. During light-induced electron transport at low pH, chloroplasts shrink, make the medium more alkaline, and acquire the capacity to form adenosine triphosphate (ATP) in a subsequent dark period. Uncouplers might modify the conformational changes, cause rapid hydrogen ion equilibration across the chloroplast membranes, and speed the decay of the ATP-synthesizing ability. Electron transport—which takes place under the conditions that are optimal for phosphorylation—results in conformational change. The same is true of electron transport uncoupled by ethylenediaminetetraacetic acid (EDTA) treatment. The carbonyl cyanide phenylhydrazone uncouplers abolish all conformational changes. Amine-uncoupled electron transport results in chloroplast swelling while atebrin- and chlorpromazine-uncoupled electron transport causes chloroplasts to shrink. Under the conditions of phosphorylation there is little ATPase activity in chloroplasts. Uncouplers rarely increase and often decrease ATPase activity. However, ATPase activity is enhanced by electron transport and sulfhydryl compounds

Photooxidation of ferrocyanide and iodide ions and associated phosphorylation in NH2OH-treated chloroplasts

NH2OH-treated, non-water oxidizing chloroplasts are shown to be capable of oxidizing ferrocyanide and I− via Photosystem II at appreciable rates (⩾ 200 μequiv/h per mg chlorophyll). Using methylviologen as electron acceptor, ferrocyanide oxidation can be measured as O2 uptake, as ferricyanide formation, or as H+ consumption (2 Fe2+ + 2H+ + O2 → 2 Fe3+ + H2O2). I− oxidation can be measured as methylviologen-mediated O2 uptake, or spectrophotometrically, using ferricyanide as electron acceptor. The oxidation product I2 is re-reduced, as it is formed, by unknown reducing substances in the reaction system. The rate-saturating concentrations of these donors are very high: 30 mM with ferricyanide and 15 mM with I−. Relatively lipophilic Photosystem II donors such as catechol, benzidine and p-aminophenol saturate the photooxidation rate at much lower concentrations (< 0.5 mM). It thus seems that the oxidation of hydrophilic reductants such as ferricyanide and I− is limited by permeability barriers. Very likely the site of Photosystem II oxidation is embedded in the thylakoid membrane or is situated on the inner surface of the membrane. The efficiency of phosphorylation (P/e2) is 0.5 to 0.6 with ferrocyanide and about 0.5 with I−. In contrast the P/e2 ratio is 1.0 to 1.2 when water, catechol, p-aminophenol or benzidine serves as electron donor. These differences imply that only one of two phosphorylation sites operate when ferrocyanide and I− are oxidized. Ferrocyanide and I− are also chemically distinct from other Photosystem II donors in that their oxidation does not involve proton release. It is suggested that the mechanism of energy conservation associated with Photosystem II may be only operative when the removal of electrons from the donor results in release of protons (i.e. with water, hydroquinones, phenylamines, etc.).

Studies on the energy coupling sites of photophosphorylation IV. The relation of proton fluxes to the electron transport and ATP formation associated with Photosystem II

1. By using dibromothymoquinone as the electron acceptor, it is possible to isolate functionally that segment of the chloroplast electron transport chain which includes only Photosystem II and only one of the two energy conservation sites coupled to the complete chain (Coupling Site II, observed Pe2 = 0.3–0.4). A light-dependent, reversible proton translocation reaction is associated with the electron transport pathway: H2O → Photosystem II → dibromothymoquinone. We have studied the characteristics of this proton uptake reaction and its relationship to the electron transport and ATP formation associated with Coupling Site II. 2. The initial phase of H+ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H+ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination. 3. Dibromothymoquinone reduction, observed as O2 evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O2 evolution agreeing well with the steady-state rate observed under continuous illumination. 4. Such measurements of the initial rates of O2 evolution and H+ uptake yield an H+e− ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H+e− ratio of 1.7 at pH 7.6.) 5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H+ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, Pi and hexokinase, when added individually, have no effect on proton uptake in this system. 6. The involvement of a proton uptake reaction with an H+e− ratio of 0.5 in the Photosystem II partial reaction H2O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e2) and proton uptake (H+e−) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling.

The swelling and shrinking of chloroplasts during electron transport in the presence of phosphorylation uncouplers

Abstract Illuminated chloroplasts undergo light-scattering changes which are associated with volume changes. These changes can be greatly increased in the presence of electron transport and the increase is quantitatively related to the Hill reaction rate. Chloroplasts treated with the uncoupler atebrin (quinacrine) are capable of very high Hill reaction rates and shrink a great deal during electron transport. The shrinking is reversed rapidly if the light is turned off and even more rapidly if the electron acceptor is exhausted. Chloroplasts treated with the uncoupler methylamine are capable of even higher Hill reaction rates but such chloroplasts swell in the light and shrink again in the dark or after the electron acceptor is exhausted. Both the atebrin-stimulated shrinking and the methylamine-stimulated swelling are partially inhibited by the presence of ATP. Carbonylcyanide phenylhydrazones, like atebrin and methylamine, permit high electron-transport rates by uncoupling the Hill reaction from phosphorylation but they completely abolish the conformational changes of chloroplasts.

The relation of post-illumination ATP formation capacity (Xe) to H+ accumulation in chloroplasts

When methylviologen, pyocyanine or low concentrations of phenazine methosulfate serve as electron carriers, the ratio of post-illumination ATP formation (Xe) to H+ uptake at pH 5–6 is 15−14. However, under the conditions of the experiments only 50–60% of the Xe is trapped as ATP. Therefore the true value of the XeΔH+ ratio probably approaches 12. With these electron carriers there is no Xe formation at pH 8. In the presence of high concentrations of phenazine methosulfate (≧0.1 mM) chloroplasts can develop extremely high levels of Xe even at pH 8 although there is little or no H+ uptake. The highest level of Xe obtained at pH 8 is equivalent to 1 mole ATP trapped per 4 moles of chlorophyll. The value reaches 1 ATP per 3 chlorophyll at pH 6.5 (phenazine methosulfate, 0.3 mM). These phenomena associated with high phenazine methosulfate concentration depend on the presence of 0.05 M NaCl or certain other salts. In the absence of NaCl there is no Xe formation at pH 8 and the familiar pH rise is converted into a light-dependent, reversible pH drop.