pH dependent changes in the reactivity of the primary electron acceptor of System II in spinach chloroplasts to external oxidant and reductant

Changes in the rates of dark oxidation and reduction of the primary electron acceptor of System II by added oxidant and reductant were investigated by measuring the induction of chlorophyll fluorescence under moderate actinic light in 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea-inhibited chloroplasts at pH values between 3.6 and 9.5. It was found that:

1. (1) The rate of dark oxidation of photoreduced primary acceptor was very slow at all the pH values tested without added electron acceptor.

2. (2) The rate was accelerated by the addition of ferricyanide in the whole pH range. It was dependent approximately on the 0.8th power of the ferricyanide concentration.

3. (3) The rate constant for the oxidation of the primary acceptor by ferricyanide was pH-dependent and became high at low pH. The value at pH 3.6 was more than 100 times that at pH 7.8.

4. (4) The pH-dependent change in the rate constant was almost reversible when the chloroplasts were suspended at the original pH after a large pH change (acid treatment).

5. (5) An addition of carbonylcyanide m-chlorophenylhydrazone or heavy metal chelators had little effect on the rate of dark oxidation of the primary acceptor by ferricyanide.

6. (6) The dark reduction of the primary acceptor by sodium dithionite also became faster at low pH.

From these results it is concluded that at low pH the primary acceptor of System II becomes accessible to the added hydrophilic reagents even in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea.     

Surface potential and reaction of membrane-bound electron transfer components. I. Reaction of P-700 in sonicated chloroplasts with redox reagents

Salt- or pH-induced change of the rate of reduction of the phtooxidized membrane bound electron transfer components, P-700, by ionic and nonionic reductants added in the outer medium was studied in sonicated chloroplasts.

The rate with the negatively charged reductants increased with the increase of salt concentration at a neutral pH or with the decrease of medium pH. Salts of divalent cations were much more effective than those of monovalent cations. A trivalent cation was even more effective. The rate with a nonionic reductant was little affected by salts.

The change of the reduction rate was analyzed using the Gouy-Chapman theory, which explains the change of reduction rate by the changes of activities of ionic reductants at the charged membrane surface where the reaction takes place. This analysis gave more useful parameters and explained more satisfactorily the case with high-valence cation salts than the Brönsted type analysis. The values for the surface charge density and the surface potential of the membrane surface in the vicinity of P-700 estimated from the analysis were lower than those estimated for the surface in the vicinity of Photosystem II primary acceptor, suggesting the heterogeneity of the thylakoid surface.

The salt-induced surface potential change was shown to affect the activation energy of the reaction between P-700 and the ionic reagent.     

Surface potential and reaction of membrane-bound electron transfer components. I. Reaction of P-700 in sonicated chloroplasts with redox reagents.

Salt- or pH-induced change of the rate of reduction of the photoxidized membrane bound electron transfer components, P-700, by ionic and nonionic reductants added in the outer medium was studied in sonicated chloroplasts. The rate with the negatively charged reductants increased with the increase of salt concentration at a neutral pH or with the decrease of medium pH. Salts of divalent cations were much more effective than those of monovalent cations. A trivalent cation was even more effective. The rate with a nonionic reductant was little affected by salts. The change of the reduction rate was analysed using the Guoy-Chapman theory, which explains the change of reduction rate by the changes of activities of ionic reductants at the charged membrane surface where the reaction takes place. This analysis gave more useful parameters and explained more satisfactorily the case with high-valence cation salts than the Br?nsted type analysis. The values for the surface charge density and the surface potential of the membrane surface in the vicinity of P-700 estimated from the analysis were lower than those estimated for the surface in the vicinity of Photosystem II primary acceptor, suggesting the heterogeneity of the thylakoid surface. The salt-induced surface potential change was shown to affect the activation energy of the reaction between P-700 and the ionic reagent.     

Energy metabolism in the cyanobacterium Plectonema boryanum. Participation of the thylakoid photosynthetic electron transfer chain in the dark respiration of NADPH and NADH

The oxidation of NADPH and NADH was studied in the light and in the dark using sonically derived membrane vesicles and osmotically shocked spheroplasts. These two types of cell-free membrane preparations mostly differ in that the cell and thylakoid membranes are scrambled in the former type and that they are more or less separated in the latter type of preparations. In the light, using both kinds of preparations, each of NADPH and NADH donates electrons via the plastoquinone-cytochrome $\text{b}\text{c}$ redox complex (Qbc redox complex) to the thylakoid membrane-bound cytochrome c-553 preoxidized by a light flash and to methylviologen via Photosystem I. NADPH donates electrons to the thylakoid membrane via a weakly rotenone-sensitive dehydrogenase to a site that is situated beyond the 3(3′,4′-dichlorophenyl)-1,1-dimethylurea sensitive site and before plastoquinone. Ferredoxin and easily soluble cytoplasmic proteins are presumably not involved in light-mediated NADPH oxidation. Inhibitors of electron transfer at the Qbc redox complex as the dinitrophenylether of 2-iodo-4-nitrothymol, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2-n-heptyl-4-hydroxy-quinone-N-oxide are effective, but antimycin A and KCN are not. The oxidation of NADH showed comparable sensitivity to these inhibitors. However, the oxidation of NADH is antimycin-A-sensitive regardless of the kind of membrane preparation used, indicating that in this case electrons are donated to a different site on the thylakoid membrane. In the dark, NADPH and NADH donate electrons at sites that behave similar to those of light-mediated oxidation, indicating that the initial steps of electron transfer are situated at the thylakoid membranes. However, NADPH oxidation is in some cases not sensitive to inhibitors active at the Qbc redox complex. It is concluded that O₂ reduction takes place at two different sites, one partly developed in vitro, situated near the rotenone-sensitive NADPH dehydrogenase, and another, highly KCN-sensitive one, situated beyond the Qbc redox complex and used in vivo. The terminal oxygen-reducing step of NADPH and NADH oxidation in the dark showed a preparation-dependent sensitivity for KCN, more than 80% inhibition in sonically derived membrane vesicles and less than 30% inhibition in osmotically shocked spheroplasts. From this result we tentatively conclude that the highly KCN-sensitive oxidase is not necessarily located at the thylakoid membrane and could be located at the cytoplasmic membrane.     

Photosynthetic electron transport and electrochromic effects at sub-zero temperatures

Spinach chloroplasts, suspended in a liquid medium containing ethyleneglycol, showed reversible absorbance changes near 700 and 518 nm due to P-700 and “P-518” in the region from ?35 to ?50 °C upon illumination. The kinetics were the same at both wavelengths, provided absorbance changes due to Photosystem II were suppressed. At both wavelengths, the decay was slowed down considerably, not only by the System I electron acceptor methyl viologen, but also by silicomolybdate. The effect of the latter compound is probably not due to the oxidation of the reduced acceptor of Photosystem I by silicomolybdate, but to the enhanced accessibility of the acceptor to some other oxidant.In the presence of both an electron donor and acceptor for System I, a strong stimulation of the extent of the light-induced absorbance increase at 518 nm was observed. The most effective donor tested was reduced N-methylphenazonium methosulphate (PMS). The light-induced difference spectrum was similar to spectra obtained earlier at room temperature, and indicated electrochromic band shifts of chlorophylls a and b and carotenoid, due to a large potential over the thylakoid membrane, caused by sustained electron transport. It was estimated that steady-state potentials of up to nearly 500 mV were obtained in this way; the potentials reversed only slowly in the dark, indicating a low conductance of the membrane. This decay was accelerated by gramicidin D. The absorbance changes were linearly proportional to the membrane potential.     

The inhibition of photosynthetic electron transport in spinach chloroplasts by low osmolarity.

The reversible inhibition, by low osmolarity, of the rate of electron transport through photosystem 1 has been investigated in spinach chloroplasts. By use of different electron donor systems to photosystem 1, inhibitors of plastocyanin, and by measurement of the extent of photooxidation of the photosystem 1 reaction center P700, the inhibition site has been localized on the electron donor side of this photosystem. From comparison of the influence of impermeant and permeant salts on the electron transport rate, and from the effect of ionic strength on the oxidation of externally added plastocyanin by subchloroplast preparations, it is concluded that low ionic strength within the thylakoids inhibits the photooxidation of endogenous plastocyanin by P700. The results are taken as evidence that plastocyanin is oxidized by P700 at the internal (lumen) side of the osmotic barrier in the thylakoid membrane.     

Chlorophyll fluorescence induction in anaerobic Scenedesmus obliquus

Fluorescence time curves (Kautsky effect) were studied in anaerobic Scenedesmus obliquus, with an apparatus capable of simultaneous recording of O₂ exchange, and far-red actinic illumination. Results, as interpreted in terms of electron transport reactions, suggest: In the course of becoming anaerobic, fluorescence induction undergoes a series of changes, indicating at least three different effects of the absence of O₂ on electron transport. (1) Immediately on removal of O₂, once the pool of intermediates between the two photo-systems is reduced by light reaction II, electron flow stops, resulting in high fluorescence yield and a cessation of O₂ evolution. O₂ appears to regulate linear electron flow and cyclic feedback of electrons to the intermediate pool. (2) An endogenous reductant formed anaerobically reduces the System II acceptors in the dark. The time course of this reduction is at least biphasic, indicative of inhomogeneity of the primary acceptor pool. Prolonged dark anaerobic treatment induces maximal initial fluorescence which decays rapidly in light and with a System I action spectrum. (3) Anaerobic treatment eventually results in deactivation of the oxidizing side of System II, limiting System II even when the acceptors are oxidized by System I pre-illumination.     

Oxaloacetate inhibition of succinate oxidation in tightly coupled liver mitochondria with ferricyanide as an electron acceptor

When ferricyanide is used as an artificial electron acceptor, succinate oxidation by tightly coupled liver mitochondria becomes inhibited after 1–3 min. No inhibition occurs in the presence of rotenone or glutamate establishing that oxaloacetate causes the inhibtion. Oxygen consumption by mitochondria oxidizing succinate does not become inhibited in the absence of rotenone suggesting that oxaloacetate accumulates to a greater extent when ferricyanide is added than when oxygen is the terminal acceptor. Higher levels of oxaloacetate in the ferricyanide reaction are apparently due to an increased rate of synthesis rather than a decreased rate of removal. Thus it appears that when succinate is the substrate and oxygen the terminal acceptor a control mechanism exists which blocks oxidation of malate. When ferricyanide is added as an artificial electron acceptor this control is lost and oxaloacetate accumulates to inhibit succinate oxidation.     

Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface potential.

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membrane surface potential, which becomes more negative with increasing phosphorylation stoichiometry and decreasing ionic strength. This lowers the membrane surface pH compared to the bulk pH, increasing K(eq) and the rate of MII formation (k(1)) while decreasing the back rate constant (k(-)(1)) of the MI-MII relaxation. MII formation has been observed to depend on bulk pH with a fractional stoichiometry of 0.6-0.7 H(+)/MII. We find that the apparent fractional H(+) dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface pH compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior consistent with titration of a single H(+) binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5 degrees C. We show evidence that suggests this same site has an intrinsic pK of 5.0 prior to light activation and its protonation before activation greatly enhances the rate of MII formation.     

Electron paramagnetic study of electron transport in photosynthetic systems. X. Effect of magnesium ions on the structural state of thylakoid membranes and the kinetics of electron transport between the two photosystems in bean chloroplasts

The effect of Mg2+-ions on the physical state of thylakoid membrane and kinetics of electron transport between two photosystems were studied. The rate of electron transport from photosystem 2 to P700+ and the activity of photosystem 2 were obtained from the kinetics of P700 redox transients induced by flashes of white light (t1/2 = 7 musec or 0.75 msec) fired simultaneously with the background continuous far-red light (707 nm). The spin-labeled stearic acids (I1.14 and I12.3) were used as indicators of Mg2+-induced structural changes. Addition of MgCl2 stimulates incorporation of spin-labels into the lipid region of the thylakoid membrane. It was found that Mg2+-ions modify the ESR spectrum of I12.3. The results evidence that the screening of charged groups on the thylakoid membrane surface induces structural changes in the lipid region of the membrane. We have concluded that these structural changes result in reorientation of lipid molecules in the thylakoid membrane. There is a correlation between Mg2+-induced structural changes and electron transport in chloroplasts. Addition of Mg2+-ions stimulates the photochemical activity of photosystem 2 by increasing the amount of active reaction centres and modifies the rate constant of electron transport from photosystem 2 to P700+. It has been demonstrated that ion regulation of electron transport in more effective in the oxidising side than in the reducing side of plastoquinone shuttle.     

Surface potential and reaction of the membrane-bound electron transfer components. II. Integrity of the chloroplast membrane and reaction of P-700

Electrostatic characteristics of the membrane in the vicinity of P-700 were estimated by analyzing the salt and detergent effects on its reaction rate with ionic reagents using the Gouy-Chapman diffuse double layer theory in various preparations of chloroplasts. Upon disruption of thylakoid membranes by sonic treatment or by treatment with digitonin, the reaction rate markedly increased, while the estimated surface charge density became smaller. It was concluded that the membrane surface which determines the reaction rate between P-700 and the ionic reagents changed as the disruption of thylakoid structure. The outer thylakoid surface had more negative charges than the inner one. Changes in the electrical potential profile across the thylakoid membrane during the illumination were also discussed from these results.     

Photosynthetic electron transport and phosphorylation reactions in thylakoid membranes from the blue-green alga Anacystis nidulans

Thylakoid membranes were prepared from the blue-green alga, Anacystis nidulans with lysozyme treatment and a short period of sonic oscillation. The thylakoid membrane preparation was highly active in the electron transport reactions such as the Hill reactions with ferricyanide and with 2,6-dichlorophenolindophenol, the Mehler reaction mediated by methyl viologen and the system 1 reaction with methyl viologen as an electron acceptor and 2,6-dichlorophenolindophenol and ascorbate as an electron donor system. The Hill reaction with ferricyanide and the system 1 reaction was stimulated by the phosphorylating conditions. The cyclic and non-cyclic phosphorylation was also active.These findings suggest that the preparation of thylakoid membranes retained the electron transport system from H₂O to reaction center 1, and that the phosphorylation reaction was coupled to the Hill reaction and the system 1 reaction.     

Surface potential and reaction of the membrane-bound electron transfer components. II. Integrity of the chloroplast membrane and reaction of P-700

Electrostatic characteristics of the membrane surface in the vicinity of P-700 were estimated by analyzing the salt and detergent effects on its reaction rate with ionic reagents using the Gouy-Chapman diffuse double layer theory in various preparations of chloroplasts.

Upon disruption of thylakoid membranes by sonic treatment or by treatment with digitonin, the reaction rate markedly increased, while the estimated surface charge density became smaller.

It was concluded that the membrane surface which determines the reaction rate between P-700 and the ionic reagents changed as the disruption of thylakoid structure. The outer thylakoid surface had more negative charges than the inner one.

Changes in the electrical potential profile across the thylakoid membrane during the illumination were also discussed from these results.     

Use of artificial electron acceptors in oxidation reactions catalyzed by acetic acid bacteria

Summary The ability of several electron acceptors to promote the Gluconobacter oxydans catalyzed oxidation of glycerol was investigated. p-Benzoquinone was the most effective electron acceptor. The reaction rate obtained with p-benzoquinone was higher than the maximal rate with the natural electron acceptor, oxygen, in all the oxidation reactions tested.     

Inside-out membrane vesicles isolated from spinach thylakoids

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The separation was obtained by partition in an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.     

Oxygen can be replaced by artificial electron acceptors in reactions catalyzed by alcohol oxidase

For the first time, spectrometric and electrochemical studies demonstrated the possibility of using artificial electron acceptors in reactions catalyzed by alcohol oxidase. We report kinetic parameters of homogenous catalytic oxidation of formaldehyde by organic redox compounds belonging to different structural classes (toluidine blue, methylene blue, 2,6-dichlorophenolindo-phenol, and p-benzoquinone) and replacing dioxygen in these reactions. p-Benzoquinone, having the highest redox potential, proved to be the most efficient artificial electron acceptor of all compounds studied.     

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.).     

叶绿体结构状态与光化学活性的关系

叶绿体被膜阻碍以Fecy为受体的电子传递,而对以DCIP为受体的电子传递无妨。被膜完整度越高则P/O值也越高。类囊体膜结构的完整度高,则电子传递速率和P/O值也比膜结构受到破损时高。类囊体膜结构的完整度对保持PS Ⅱ活性是必要的,随着完整度的降低,PS Ⅱ在电子传递中所占比重相应减少。