Joseph Neumann

The mechanism of the oxidation of ascorbate and Mn2+ by chloroplasts: The role of the radical superoxide

1. 1. The mechanism of the photooxidation of ascorbate and of Mn2+ by isolated chloroplasts was reinvestigated. 2. 2. Our results suggest that ascorbate or Mn2+ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn2+ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin. 3. 3. In the absence of ascorbate or Mn2+ the superoxide formed dismutases either spontaneously or enzymatically producing O2 and H2O2. In the presence of ascorbate or Mn2+, however, the superoxide is reduced to H2O2 with no formation of O2. Consequently, in the absence of reducing compounds, in the reaction H2O to low potential acceptor one O2 (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn2+ or other suitable reductants up to three molecules O2 can be taken up per four electrons transported. 4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O2; the addition of ascorbate or Mn2+ to this system results in a two fold stimulation of O2 uptake, with no stimulation of NADPH oxidation. The stimulation of O2 uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O2 uptake in the system H2O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase. 5. 5. In Class II chloroplasts in the system H2O → NADP upon the addition of ascorbate or Mn2+ an apparent inhibition of O2 evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts. 6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP2e in this system (when the rate of electron flow is based on the rate of O2 uptake) should be revised in the upward direction.

Proton conductance of the thylakoid membrane: Modulation by light

where JH is the proton flux and XB the protonmotive force [l] . I, is obtained from the slope of the curve of proton flux versus proton-motive force. We have found that the proton conductance of the thylakoid membrane, is very low at low light intensities, and increases by several orders of magnitude with increase in light intensity. The sharp increase in proton conductance occurs when XH reaches a certain threshold value. This threshold for proton conduction is related to the critical ApH required for ATP synthesis; the latter requirement was documented previously [2]. The results presented indicate that the increase in conductance is associated with the coupling factor of the chloroplast (CF,), while the proton conductance of the membrane proper is negligible.

Antimycin A as an uncoupler and electron transport inhibitor in photoreactions of chloroplasts

Abstract 1. The effect of antimycin A on photoreactions of isolated chloroplasts in the μM range was shown to depend strongly on chloroplast concentration, pH of reaction and duration of preincubation. 2. Antimycin A was shown to be an uncoupler of photophosphorylation and an inhibitor of electron transport. The latter activity sets in at lower concentrations, but both activities overlap to a considerable extent. 3. The inhibition of electron transport by antimycin A is potentiated by high pH and by the presence of uncouplers. 4. The uncoupling activity of antimycin A was manifested by inhibiting ATP synthesis (noncyclic and cyclic), stimulation of electron flow and stimulation of the dark decay of the proton pump. 5. The two effects of antimycin A can be separated by the addition of serum albumin, which presumably removes part of the inhibitor from the reaction mixture.

Interaction between ferredoxin and ferredoxin-NADP reductase from chloroplasts

Abstract The photochemical reduction of pyridine nucleotides in isolated chloroplasts was shown to proceed via ferredoxin and ferredoxin-NADP reductase ( Shin et , al. , 1963 . The latter enzyme is a flavoprotein which can also mediate the transfer of electrons in reverse, from NADPH to ferredoxin (Lazzarini and San Pietro, 1962) , or to other acceptors such as: dyes (Avron and Jagendorf, 1956) , NAD (Keister et , al. , 1960) and chloroplast cytochrome f , (Zanetti and Forti, 1966) . This communication reports observations indicating that ferredoxin inhibits several activities of the flavoprotein and forms a complex with it.

On the mechanism of action of silicomolybdic acid in chloroplasts.

Giaquinta et al. [l] described recently an electron acceptor system in isolated chloroplasts, comprised of silicomolybdic acid plus ferricyanide whose reduction was insensitive to 3-(3,4-dichlorophenyl)1, ldimethylurea (DCMU). According to these authors silicomolybdic acid is reduced by Q and reduces chemically ferricyanide. Girault and Galmiche studied previously [2,3] the interaction of another heteropolyion, silicotungstic acid, with the chloroplast membrane. These authors concluded [3] that in the presence of silicotungstic acid the chloroplast membrane is changed and consequently ferricyanide is photoreduced in a DCMU insensitive reaction. In this study we have shown: (a) that pretreating the chloroplasts with silicomolybdic acid and removing the bulk of it, causes a change in the properties of the electron transport chain; such chloroplasts can photoreduce ferricyanide (and other electron acceptors) in a DCMU insensitive reaction; (b) that the changes in the chloroplast membrane by pretreatment with silicomolybdic acid require specific conditions and (c) the chloroplasts in which Q has been exposed by silicomolybdic acid can photoreduce this compound.

Circular dichroism studies of the complex between ferredoxin and ferredoxin-NADP reductase

Abstract Circular dichroism (CD) spectra are presented of ferredoxin, ferredoxin-NADP reductase and their complex. A change in CD occurs on complex formation which is consistent with a decrease in the Cotton effects due to the ferredoxin. This change is interpreted as due to a decrease in interaction in ferredoxin between the iron-sulphur chromophore group and the protein. No significant changes in the EPR ∗ signal of reduced ferredoxin were detected in the presence of the reductase.

Evidence for system I mediated non-cyclic photophosphorylation in chloroplasts

Electron transport from ascorbate t DPIP** to NADP, which is a system I mediated reaction, supports ATP formation [ I] . However, ascorbate and DPIP can support ATP formation even in the absence of an electron acceptor [2-41 . The extent of coupling in the system DPIPH, to NADP as measured by the ratio ATPIe can vary considerably and under some experimental conditions can be lower than 0.2 [4,5] . On the basis of these and other observations, several workers maintain that ATP formation in the presence of DPIPH, is of the cyclic type, (both in the presence and absence of an electron acceptor), resembling PMS. According to this formulation, the site of ATP formation is located on the ‘cyclic’ part of the electron transport chain which connects ‘X’ with the linear electron transport chain [6] . However, other observations [7-101 indicate that in the system DPIPH, to NADP (or MV) there is a coupling site on the linear electron transport chain, namely between the site of electron donation and P,,,. Since DPIP (in its oxidized form) can be reduced by photosystem I, [ 11,121 one can visualize a ‘pseudocyclic’ system I mediated electron transport from DPIPH, to the photosystem, to DPIP. The latter might be identical to system I mediated non-

Photophosphorylation in stable chloroplast fragments from the alga Chlamydomonas reinhardi

Abstract Stable chloroplast fragments exhibiting high reproducible rates of photophosphorylation and good coupling ratios can be obtained from the alga Chlamydomonas reinhardi using a simple procedure based on brief sonication of young cells in a medium of high osmolarity containing serum albumin. Chloroplast fragments prepared by this method exhibit long-term stability; Hill activity remains constant for at least 26 h while cyclic and non cyclic photophosphorylation activities exhibit a half time stability constant of about 24 h. Chloroplast fragments isolated by this method are insensitive to uncoupling of photophosphorylation by ammonium chloride. It is suggested that in the presence of ammonium chloride membrane potential may act as the driving force for photophosphorylation.

Photosystem 2 mediated electron transport and phosphorylation with ferricyanide and dibromothymoquinone the uncoupling activity of dibromothymoquinone

Abstract 1. (1) In lettuce chloroplasts, ferricyanide can be photoreduced at two sites in the electron transport chain. At pH 7 and below, ferricyanide is preferentially photoreduced by Photosystem 2 (PS2), whereas at higher pH values it is photoreduced primarily by Photosystem 1 (PS1). 2. (2) When ferricyanide is photoreduced by Photosystem 2 only (either at low pH or at any pH in KCN treated chloroplasts), the ATP/e 2 value is reduced from 1.0 to about 0.5. 3. (3) Dibromothymoquinone (DBMIB) while inhibiting electron flow to PS1 acts as an electron acceptor for PS2. At pH 6.0 addition of DBMIB to ferricyanide causes an increase in electron flow and an inhibition of ΔpH. The former is due to the fact that both ferricyanide and DBMIB are photoreduced, whereas the latter is probably due to the fact that ( a ) DBMIB is reduced inside the thylakoid, and reoxidized outside the thylakoid, dissipating the proton gradient formed by its reduction and ( b ) DBMIB reduction competes to some extent with ferricyanide reduction. Addition of DBMIB to ferricyanide causes a decrease in the P/e 2 ratio; consequently true values for P/e 2 at “coupling site II” cannot be obtained in the presence of ferricyanide plus DBMIB, but can be obtained with ferricyanide in KCN-treated chloroplasts or in lettuce chloroplasts with ferricyanide alone, at low pH values.

A differential effect of 3-(3'4' dichlorophenyl)-1,1 dimethyl urea and atrazine on fluorescence kinetics in chloroplasts

It was found that DCMU had a differential effect at two concentration ranges on variable fluorescence kinetics in isolated chloroplasts. The increase in fluorescence rate at low concentrations of DCMU was abolished by preincubation of chloroplasts with ferricyanide or formate, treatments which were shown to convert Fe in the PS II reaction center (i.e., the FeQA complex) into a non-oxidizable form, but it was not affected by Tris treatment. Increase in fluorescence kinetics (at the initial linear rate) at high concentrations of DCMU was found to be abolished by Tris treatment but it was only marginally affected by ferricyanide or formate treatments. The effect of Tris could be abolished by addition of hydroquinone-ascorbate, which restored electron flow to the pool of secondary acceptors.Contrary to the effect of DCMU, no such differential concentration dependence of the variable fluorescence kinetics was found for atrazine.The increase in fluorescence kinetics (at the initial linear rate) at a low concentration rate of DCMU is presumably restricted to units which contain an oxidizable Fe in the FeQA complex. Increase in fluorescence kinetics (at the initial linear rate) at high DCMU concentration is probably related to the effect of DCMU at the QB site.