Light-induced oxidation-reduction reactions in a cell-free preparation from the blue-green alga Nostoc muscorum: The role of cytochrome f, cytochrome b558, C550, and P700 in noncyclic electron transport

1. Cytochrome f (λ_max = 554 nm, E_m = +0.35 V) and cytochrome b₅₅₈ (λ_max = 558 nm, E_m = +0.35 V) were photooxidized by Photosystem I and photoreduced by Photosystem II in a cell-free preparation from the blue-green alga Nostoc muscorum. The steady-state oxidation levels of both cytochromes were affected by noncyclic electron acceptors and by inhibitors of noncyclic electron transport. These results are consistent with the hypothesis that the mechanism of NADP reduction by water involves a Photosystem II and a Photosystem I light reaction operating in series and linked by a chain of electron carriers that includes cytochrome f and cytochrome b₅₅₈.2. Phosphorylation cofactors shifted the steady-state of cytochrome f to a more reduced level under conditions of noncyclic electron transport but had no effect on cytochrome b₅₅₈. These observations suggest that the noncyclic phosphorylation site lies before cytochrome f (on the Photosystem II side) and that cytochrome f is closer to this site than is cytochrome b₅₅₈.3. A Photosystem II photoreduction of C550 at 77 °K was observed, suggesting that in blue-green algae, as in other plants, C550 is closely associated with the primary electron acceptor for Photosystem II. A Photosystem I photooxidation of P700 at 77 °K was observed, consistent with P700 serving as the primary electron donor of Photosystem I.     

NADH and NADPH as electron donors to respiratory and photosynthethic electron transport in the blue-green alga,Aphanocapsa

In the blue-green alga, Aphanocapsa, light inhibits respiration. This can be observed with spheroplasts when O₂ uptake is measured with NADH or NADPH as electron donor. However, NAD(P)H oxidation is unaffected by illumination. Furthermore, it was possible to demonstrate electron transfer from NAD(P)H to Photosystem I. Thus, the inhibition of respiratory oxygen uptake by light is explained by a competition of cytochrome oxidase and Photosystem I for reduction equivalents. Based on studies with inhibitors, electron transfer from NAD(P)H to Photosystem I involves the chloroplast cytochrome b₆-f complex.     

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.).     

The role of reagents accelerating the deactivation reactions of water-splitting enzyme system Y (ADRY reagents) in destabilizing high-potential oxidizing equivalents generated in chloroplast Photosystem II

A class of compounds, usually referred to as ADRY reagents, destabilize intermediates in the photosynthetic water-oxidizing process. The effects of these species on the reduction kinetics of Z^?, the oxidized donor to P-680, have been monitored in Tris-washed chloroplasts by following the decay of EPR Signal IIf. In the presence of ADRY reagents (e.g., sodium picrate, carbonyl cyanide m-chlorophenylhydrazone) this process follows an exponential time course, the decay half-time of which decreases as the ADRY reagent concentration increases. From this pseudo-first-order behavior, the second-order rate constants for four commonly used ADRY reagents have been extracted. The ADRY-induced acceleration in Z^? reduction proceeds independently of conditions imposed on the acceptor side of Photosystem II and shows no synergism with exogenous electron donors. These observations are most easily rationalized in terms of a model which proposes direct reduction of Z^? by the ADRY reagent followed by regeneration of the reduced ADRY reagent in a nonspecific reaction with membrane components such as carotenoids, chlorophyll or protein. A comparison of the second-order rate constants we obtain for ADRY reagents in their reaction with Z^? in Tris-washed chloroplasts with those obtained from the literature for the ADRY- reagent induced deactivation of states S₂ and S₃ in oxygen-evolving chloroplasts reveals a close similarity between the two processes. From this observation, a general model for the action of ADRY reagents in destabilizing the high-potential oxidizing equivalents generated in Photosystem II is proposed.     

Contrasting effects of plastocyanin on the photoreduction and photooxidation of cytochrome f in chloroplasts

Removal of plastocyanin from Photosystem I subchloroplast particles had no effect on the Photosystem I photooxidation of cytochrome f. Chloroplasts depleted of plastocyanin by sonication lost the ability to reduce cytochrome f in Photosystem II light. Addition of plastocyanin restored the photoreduction of cytochrome f. These results are consistent with a plastocyanin site on the reducing side of cytochrome f.     

Inhibition of energy-transducing functions of chloroplast membranes by lipophilic iron chelators

Lipophilic metal chelators inhibit various energy-transducing functions of chloroplasts. The following observations were made.1. Photophosphorylation coupled to any known mode of electron transfer, i.e. whole-chain noncyclic, the partial noncyclic Photosystem I or Photosystem II reactions, or cyclic, is inhibited by several lipophilic chelators, but not by hydrophilic chelators.2. The light- and dithioerythritol-dependent Mg²⁺-ATPase was also inhibited by the lipophilic chelators.3. Electron transport through either partial reaction, Photosystem I or Photosystem II was not inhibited by lipophilic chelators. Whole-chain coupled electron transport was inhibited by bathophenanthroline, and the inhibition was not reversed by uncouplers. The diketone chelators diphenyl propanedione and nonanedione inhibited the coupled, whole-chain electron transport and the inhibition was reversed by uncouplers, a pattern typical of energy transfer inhibitors.The electron transport inhibition site is localized in the region of plastoquinone → cytochrome f. This inhibition site is consistent with other recent work (Prince et al. (1975) FEBS Lett. 51, 108 and Malkin and Aparicio (1975) Biochem. Biophys. Res. Commun. 63, 1157) showing that a non-heme iron protein is present in chloroplasts having a redox potential near +290 mV. A likely position for such a component to function in electron transport would be between plastoquinone and cytochrome f, just where our data suggests there to be a functional metalloprotein.4. Some of the lipophilic chelators induce H⁺ leakiness in the chloroplast membrane, making interpretation of their phosphorylation inhibition difficult. However, 1–3 mM nonanedione does not induce significant H⁺ leakiness, while inhibiting ATP formation and the Mg²⁺-ATPase. Nonanedione, at those concentrations, causes a two- to four-fold increase in the extent of H⁺ uptake.5. These results are consistent with, but do not prove, the involvement of a non-heme iron or a metalloprotein in chloroplast energy transduction.     

Effect of phenolic herbicides on the oxygen-evolving side of Photosystem II. Formation of the carotenoid cation

Phenolic herbicides were added to suspensions of spinach chloroplasts or to oxygen-evolving Photosystem II membranes. Flash absorption spectroscopy at 21°C around 1000 nm reveals that these chemicals lead to a flash-induced absorption increase attributed to the radical-cation of a carotenoid. The herbicides studied can be arranged in the following order of decreasing efficiency for the reported effect: i-dinoseb, bromonitrothymol, trinitrophenol, ioxynil, dinitroorthocresol, 2,4-dinitrophenol. A similar effect was not observed with atrazine, DCMU or o-phenanthroline. For a given herbicide concentration, the amount of flash-induced carotenoid cation increases sharply when the pH is lowered below 5.5. A similar effect does not take place with other molecules which induce the formation of a carotenoid cation: tetraphenylboron, FCCP, 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT-2p). The previous effects are observed in both oxygen-evolving Photosystem II and in preparations in which oxygen evolution is inhibited with alkaline Tris. In untreated material, the carotenoid cation is formed with a half-time of 10–35 μs. After Tris treatment, this half-time is a little longer at low than at high pH. These results indicate the existence of a specific site where phenolic inhibitors interact in the oxygen-evolving site of Photosystem II     

Lack of site-specificity of spinach chloroplast coupling factor 1

The irreversible inhibition of chloroplast phosphorylation by either sulfate anions, or N-ethylmaleimide, is energy dependent. Chloroplasts must first be illuminated in the presence of the inhibitors and a mediator of electron flow, for the subsequent phosphorylation to show any inhibition. Both inhibitors affect the chloroplast coupling factor 1.Electron transport only through Photosystem I can be used to activate either of these inhibitions. The subsequent inhibition in a second light reaction is the same whether ATP synthesis is supported by Photosystem I, or by Photosystem II electron transport. The reverse experiment, activating inhibition by electron transport only through Photosystem II, is possible in the case of sulfate. Again, the inhibition is expressed whether Photosystem II or Photosystem I electron flow supports ATP synthesis. We conclude that the two electron transport regions probably generate the same high energy state which is able to activate all members of a functionally uniform coupling factor population. These enzyme molecules must catalyze phosphorylation coupled to electron transport through either region of the chain. The results tend to discredit models requiring a separate group of coupling factor molecules unique to each part of the chain.     

Effect of NADP+ on light-induced cytochrome changes in membrane fragments from a blue-green alga

The effect of NADP⁺ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP⁺ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH₂) to NADP⁺. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH₂ as the electron donor, NADP⁺ had no effect on the light-induced redox changes of cytochromes: with or without NADP⁺, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP⁺ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b₅₅₉. Cytochrome b₅₅₉ appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP⁺) for the electron flow from water.     

Photochemical activity and components of membrane preparations from blue-green algae. I. Coexistence of two photosystems in relation to chlorophyll a and removal of phycocyanin

Nostoc muscorum (Strain 7119) cells were disrupted and the accessory pigment phycocyanin was removed from membrane fragments by digitonin treatment. The phycocyanin-depleted membrane fragments retained both Photosystem I and Photosystem II activity, as evidenced by high rates of NADP⁺ photoreduction either by water or by reduced 2,6-dichlorophenolindophenol, indicating that phycocyanin is not an essential component for electron transport activity.No separation of the two photosystems was effected by the digitonin treatment. Even drastic digitonin treatments failed to diminish significantly the remarkably stable electron transport from water to NADP⁺.Action spectra and relative quantum efficiency measurements demonstrated the existence of both Photosystem I and Photosystem II in membrane fragments which contained chlorophyll a as the only significant light-absorbing pigment.     

Effects of magnesium and chloride ions on light-induced electron transport in membrane fragments from a blue-green alga

The effects of magnesium and chloride ions on photosynthetic electron transport were investigated in membrane fragments of a blue-green alga, Nostoc muscorum (Strain 7119), noted for their stability and high rates of electron transport from water or reduced dichlorophenolindophenol to NADP⁺. Magnesium ions were required not only for light-induced electron transport from water to NADP⁺ but also for protection in the dark of the integrity of the water-photooxidizing system (Photosystem II). Membrane fragments suspended in the dark in a medium lacking Mg²⁺ lost the capacity to photoreduce NADP⁺ with water on subsequent illumination. Chloride ions could substitute, but less effectively, for each of these two effects of magnesium ions. By contrast, the photoreduction of NADP⁺ by DCIPH₂ was independent of Mg²⁺ (or Cl^?) for the protection of the electron transport system in the dark or during the light reaction proper. Furthermore, high concentrations of MgCl₂ produced a strong inhibition of NADP⁺ photoreduction with DCIPH₂ without significantly affecting the rate of NADP⁺ photoreduction with water. The implications of these findings for the differential involvement of Photosystem I and Photosystem II in the photoreduction of NADP⁺ with different electron donors are discussed.     

Polylysine-enhanced effectiveness of plastocyanin in photosystem I

1. Chloroplast fragments from either Chlamydomonas reinhardi or spinach, which lack plastocyanin, or from Euglena gracilis depleted of cytochrome c552, require a large excess of exogenously added plastocyanin or cytochrome c552 to restore Photosystem I activity.2. In the presence of a small amount of polylysine, Photosystem I activity of chloroplast fragments is stimulated greatly by plastocyanin or cytochrome c552, and the reaction is saturated at a lower concentration of these proteins. Higher concentrations of polylysine inhibit Photosystem I activity; the inhibition is not reversed by plastocyanin or cytochrome c552.3. Salt protects chloroplast fragments from stimulation by polylysine plus plastocyanin or cytochrome c552, and also reverses this stimulation.4. The data suggest that polylysine, at low concentration, enhances binding of plastocyanin or cytochrome c552 to chloroplast membranes, thereby increasing the effective concentration at their site of function. The total inhibition of Photosystem I activity, independent of the presence of plastocyanin or cytochrome c552, at higher polylysine concentrations is similar probably to that observed previously in chloroplasts which retain their plastocyanin.     

Flash-induced absorption changes of the primary donor of photosystem II at 820 nm in chloroplasts inhibited by low pH or tris-treatment

A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O₂ evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.     

A major site of bicarbonate effect in system II reaction. Evidence from ESR signal IIvf, fast fluorescence yield changes and delayed light emission

In order to determine the major site of bicarbonate action in the electron transport complex of Photosystem II, the following experimental techniques were used: electron spin resonance measurements of Signal II_vf, measurements of chlorophyll a fluorescence yield rise and decay kinetics, and delayed light emission decay. From data obtained using these experimental techniques the following conclusions were made: (1) absence of bicarbonate causes a reversible inactivation of up to 40% of Photosystem II reaction center activity; (2) there is no significant effect of bicarbonate on electron flow from the charge accumulating S state to Z; (3) there is no significant effect of bicarbonate on electron flow from Z to P-680⁺; (4) electron flow from Q^? to the intersystem electron transport pool is inhibited by from 4- to 6-fold under bicarbonate depletion conditions.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

Role of β-carotene in the reaction centres of Photosystems I and II of spinach chloroplasts prepared in non-polar solvents

Spinach chloroplasts have been prepared nonaqueously using non-polar solvents (n-hexane, CCl₄, n-heptane) and the β-carotene content extracted in a controlled manner. This procedure is reproducible and does not result in large structural or spectral changes of the chloroplasts. The organisation of the chlorophyll-proteins is unaltered, as fragmentation with digitonin results in the appearance of the same fractions as found previously for aqueously-prepared chloroplasts, including the pink zone containing cytochromes f and b₆ in the ratio 1:2. The chloroplasts possess both Photosystem I activity (P-700 photo-bleaching, and NADP⁺ photoreduction) and Photosystem II activity (parabenzoquinone reduction with Mn²⁺ as electron donor, and chlorophyll fluorescence induction). Use of moderate intensity red illumination has allowed a study of the role of β-carotene in photochemistry separate from its roles in energy transfer and photoprotection.Removal of the fraction of β-carotene closely associated with the Photo-system I reaction centre caused the rate of NADP⁺ photoreduction to fall to a low, but significantly non-zero level. Thus, in the complete absence of β-carotene, photochemistry can still be observed, however the specific association of β-carotene with the reaction centre is required for maximal rates. We propose that β-carotene bound at the reaction centre decreases the rate of transfer of excitation energy away from the reaction centre, and increases the rate of photochemistry. It is possible that this occurs via formation of an exciplex between ground state β-carotene and chlorophyll in the first excited state.     

The kinetics of light-induced changes of C-550, cytochrome b559 and fluorescence yield in chloroplasts at low temperature

The kinetics of the photoreduction of C-550, the photooxidation of cytochrome b₅₅₉ and the fluorescence yield changes during irradiation of chloroplasts at ?196 °C were measured and compared. The photoreduction of C-550 proceeded more rapidly than the photooxidation of cytochrome b₅₅₉ and the fluorescence yield increase followed the cytochrome b₅₅₉ oxidation. These results suggest that fluorescence yield under these conditions indicates the dark reduction of the primary electron donor to Photosystem II, P680⁺, by cytochrome b₅₅₉ rather than the photoreduction of the primary electron acceptor.The photoreduction of C-550 showed little if any temperature dependence over the range of ?196 to ?100 °C. The amount of cytochrome b₅₅₉ photooxidized was sensitive to temperature decreasing from the maximal change at temperatures between ?196 to ?160 °C to no change at ?100 °C. To the extent that the reaction occurred at temperatures between ?160 and ?100 °C the rate was largely independent of temperature. The rate of the fluorescence increase was dependent on temperature over this range being 3–4 times more rapid at ?100 than at ?160 °C. At ?100 °C the light-induced fluorescence increase and the photoreduction of C-550 show similar kinetics. The temperature dependence of the fluorescence induction curve is attributed to the temperature dependence of the dark reduction of P680⁺.The intensity dependence of the photoreduction of C-550 and of the photooxidation of cytochrome b₅₅₉ are linear at low intensities (below 200 μW/cm²) but fall off at higher intensities. The failure of reciprocity in the photoreduction of C-550 at the higher intensities is not explained by the simple model proposed for the Photosystem II reaction centers.