Energetics of photosynthesis in Zea mays. I. Studies of the flash-induced electrochromic shift and fluorescence induction in bundle sheath cells

Bundle sheath strands free of mesophyll contamination were isolated from 3–4-week-old leaves of maize (Zea mays L.). Patterns of electron flow in the preparations were studied in the presence of physiological substrates. Relative electron flow rates were estimated from the flash-induced electrochromic band shift changes (P-518) and cytochrome f turnover. Induction of chlorophyll fluorescence was also measured. Little Photosystem II activity was found to be present, the principal pathway of electron flow being Photosystem I-driven cyclic electron transfer. The latter was activated through reductive poising by NADPH, generated via malate decarboxylation or (less efficiently) from dihydroxyacetone phosphate. The actions of these electron donors and of oxygen, nitrite and methyl viologen as electron acceptors in redox poising the Photosystem I-driven cycle were investigated and are discussed in relation to the regulation of photosynthesis in the bundle sheath.     

Two pathways of electron transfer in quinol-mediated cyclic phosphorylation in spinach chloroplasts

Low potential quinones are mediators of cyclic phosphorylation in washed spinach thylakoid membranes if they are prereduced to provide the proper redox poise. Cyclic phosphorylation catalyzed by different quinols varies in its sensitivity to the electron transfer inhibitor 2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether (DNPINT), which is thought to inhibit electron flux from the bound plastoquinone (B) to the plastoquinone pool (Trebst, A., Wietoska, H., Draber, W. and Knops, H.J. (1978) Z. Naturforsch. 33c, 919–927). Cyclic phosphorylation catalyzed by uncharged quinols is extremely sensitive to DNPINT, whereas cyclic phosphorylation catalyzed by negatively charged quinols is approximately two orders of magnitude less sensitive. Many quinols have pK₁ values in the physiological range (pH 7–9). Increasing the concentration of the deprotonated quinol either by raising the assay pH, increasing the mediator concentration, or increasing the fractional reduction of the quinone results in a decrease in the sensitivity of cyclic phosphorylation to DNPINT. At very high DNPINT concentrations, cyclic phosphorylation catalyzed by all quinols (and ferredoxin) is inhibited, but not phenazine methosulfate catalyzed cyclic phosphorylation.These data suggest that the deprotonated form of the quinol can donate electrons directly to the plastoquinone pool, whereas the uncharged quinol most obligately transfer electrons through the bound plastoquinone ‘B’. A second site of DNPINT action after the plastoquinone pool is also observed, which requires much higher DNPINT concentrations for inhibition of phosphorylation.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.     

Regulation of Cyclic Photophosphorylation during Ferredoxin-Mediated Electron Transport : Effect of DCMU and the NADPH/NADP Ratio

Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O₂ (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP⁺ ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.     

Cyclic photophosphorylation reactions catalyzed by ferredoxin,methyl viologen and anthraquinone sulfonate. Use of photochemical reactions to optimize redox poising

The flavin analogue 5-deazariboflavin is a convenient catalyst for the photoreduction of low-potential redox compounds. In an anaerobic medium with Tricine buffer as the electron donor, 5-deazariboflavin is capable of photoreducing both ferredoxin and methyl viologen. We have used this method to conduct a comparative study of the Photosystem I photophosphorylation activities supported by the reduced forms of ferredoxin, methyl viologen and anthraquinone sulfonate. All of these catalysts are capable of generating high rates (200–500 μmol ATP/h per mg chlorophyll) of cyclic photophosphorylation, but only the activity dependent on ferredoxin exhibits sensitivity to antimycin A. This finding suggests that the size of the catalyst and its ability to approach the thylakoid membrane, rather than low-redox potential, governs antimycin A sensitivity. Ferredoxin-catalyzed activity is, however, less sensitive to inhibition by dibromothymoquinone than are the activities supported by methyl viologen and anthraquinone sulfonate. This discrepancy is due to binding of the inhibitor by ferredoxin.     

The relationship of cyclic and non-cyclic electron flow patterns with reduced indophenols to photophosphorylation

Optimal cyclic photophosphorylation with reduced indophenols under anaerobic conditions was shown to require a critical redox balance. Over-reduction inhibited this phosphorylation; addition of oxidizing agents like ferricyanide, air, ferredoxin or ferredoxin plus triphosphopyridine nucleotide relieved the inhibition.

When ascorbate and indophenol served as the electron donor couple for TPN⁺ reduction, only the amount of TPNH formed was dependent on the concentration of TPN⁺. The phosphorylation observed in this system was dependent only on the concentration of indophenol, and on the ability of reduced indophenol to mediate cyclic photophosphorylation. The cyclic electron flow with reduced indophenol was shown to operate simultaneously with the non-cyclic electron flow to TPN⁺. It was concluded that there was no phosphorylation site in the non-cyclic electron flow between ascorbate-indophenol and TPN⁺ and that the phosphorylation observed in this case was due only to cyclic photophosphorylation with the reduced indophenols.

In the light of these results, a working hypothesis with two different sites for cyclic and non-cyclic photophosphorylation is suggested.     

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark.

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg2+. The reaction is inhibited by (I) an antibody to ferredoxin-NADP+ reductases (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q. It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Discrimination of flash-induced fast and slow electric potential components

This work aimed at the resolution of the multi-component electric potential changes induced by single-turnover flash illumination of Photosystem-I-enriched subchloroplast vesicles. If supplemented with ferredoxin and under carefully adjusted redox poising, these vesicles show a pronounced slow-rising and -decaying electric potential component, as monitored by endogenous and exogenous field-sensitive probes, carotenoids and oxonol VI, respectively. The fast and slow potential components can be easily discriminated without the need for computer-assisted deconvolution after selective presaturation of the slow component by preillumination or a transmembrane ΔpH, after selective suppression of the slow component by low valinomycin or uncoupler concentrations or in the absence of ferredoxin. The slow electric potential component, as compared to the fast one, is relatively sensitive to low concentrations of ionophores and uncouplers, detergent, ageing and lower temperatures (4–12°C), is associated with electrogenic proton displacements and is interpreted to respond to a field that is more located on the membrane-bulk interface. Temperature effects show transition temperatures around 20°C for both the rise and decay of the slow potential component. The results provide further evidence that the carotenoids and oxonol VI sense the same (slow) electric field, but may be differently located in the thylakoid membrane.     

The orientation of the magnetic axes of membrane-bound iron-sulfur clusters and a cytochrome b-559 in the green halophilic alga Dunaliella parva

Photosynthetic membrane fragments separated from whole cells of the green alga Dunaliella parva, were oriented by incorporation into multilayers on thin Mylar films. These partially dehydrated films were then examined by EPR spectroscopy for evidence of orientation of paramagnetic components. Five previously identified paramagnetic components, the reduced states of iron-sulfur clusters A and B, the intermediate acceptor X^?, the reduced Rieske iron-sulfur cluster, and oxidized cytochrome b-559, displayed EPR signals showing orientation. In addition, several previously unknown paramagnetic components were also observed to be oriented. Four components, previously characterized in spinach chloroplast preparations, the iron-sulfur clusters A and B, the intermediate acceptor X^?, and cytochrome b-559, were shown to be similar in the green alga, D. parva. The orientations of iron-sulfur clusters A and B, however, were determined unambiguously in this preparation; this was not possible in previous work with spinach. The heme plane orientation of cytochrome b-559 was found to be perpendicular to the membrane plane in agreement with the results in spinach preparations. A new photoinduced EPR signal with g values of 1.88, 1.97 and 2.12 was seen only in the oriented preparations and was indicative of a reduced iron-sulfur cluster with an orientation different from that of iron-sulfur cluster A or B. This suggests the existence of a previously unidentified acceptor in Photosystem I of green plants. These studies clearly show that the orientation of these components in bioenergetic membranes are conserved over a large span of evolutionary development and are, therefore, an important aspect of the mechanism of electron transfer.     

Electron transport pathways in spinach chloroplasts. Reduction of the primary acceptor of Photosystem II by reduced nicotinamide adenine dinucleotide phosphate in the dark

Addition of NADPH to osmotically lysed spinach chloroplasts results in a reduction of the primary acceptor (Q) of Photosystem II. This reduction of Q reaches a maximum of 50% in chloroplasts maintained under weak illumination and requires added ferredoxin and Mg²⁺. The reaction is inhibited by (i) an antibody to ferredoxin-NADP⁺ reductase (EC 1.6.7.1), (ii) treatment of chloroplasts with N-ethylmaleimide in the presence of NADPH, (iii) disulfodisalicylidenepropanediamine, (iv) antimycin, and (v) acceptors of non-cyclic electron transport. Uncouplers of phosphorylation do not affect NADPH-driven reduction of Q.It is proposed that electron flow from NADPH to Q may occur in the dark by a pathway utilising portions of the normal cyclic and non-cyclic electron carrier sequences. The possible in vivo role for such a pathway in redox poising of cyclic electron transport and hence in controlling the ATP/NADPH supply ratio is discussed.     

Studies on the pathway of cyclic electron flow in mesophyll chloroplasts of a C4 plant

1. Cyclic photophosphorylation driven by white light, as followed by ¹⁴CO₂ fixation by mesophyll chloroplast preparations of the C₄ plant Digitaria sanguinalis, was specifically inhibited by disalicylidenepropanediamine (DSPD), antimycin A, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIb), 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDAC), and KCN suggesting that ferredoxin, cytochrome b₅₆₃, plastoquinone, cytochrome f, and plastocyanin are obligatory intermediates of cyclic electron flow. It was found that 0.2 μM DCMU and 40 μM o-phenanthroline blocked noncyclic electron flow, stimulated cyclic photophosphorylation, and caused a partial reversal (40–100%) of the inhibition by DBMIB and antimycin A, but not DSPD.

2. Cyclic photophosphorylation could also be activated using only far-red illumination. Under this condition, however, cyclic photophosphorylation was much less sensitive to the inhibitors DBMIB, EDAC and antimycin A, but remained completely sensitive to DSPD and KCN. Inhibition in far-red light was not increased by preincubating the chloroplasts with the various inhibitors for several minutes in white light.

3. The striking correspondence between the effects of photosystem II inhibitors, DCMU and o-phenanthroline, on cyclic photophosphorylation under white light and cyclic photophosphorylation under far-red light (in the absence of photosystem II inhibitors) suggests that electrons flowing from photosystem II may regulate the pathway of cyclic electron flow.     

Different roles of plastoquinone in the photoreduction of ferredoxin and of membrane-bound iron-sulfur centers of chloroplasts

Photosynthetic electron transport in chloroplasts was inhibited by the plastoquinone antagonist, dibromothymoquinone (DBMIB) in two steps. Lower concentrations of DBMIB inhibited the photoreduction of the bound iron-sulfur centers of photosystem I without inhibiting the photoreduction of ferredoxin. Higher concentrations of DBMIB did inhibit the oxygenic photoreduction (i.e., by water) of ferredoxin and NADP⁺, but their photoreduction was restored, wholly or partly, by each of four chemically diverse uncouplers, similar only in facilitating proton movement across membranes. By contrast, none of the uncouplers alleviated the DBMIB inhibition of the photoreduction of the bound Fe-S centers. These divergent responses to uncouplers are incompatible with the Z scheme but are consistent with the new concept of oxygenic and anoxygenic photosystems in plant photosynthesis (Proc. Natl. Acad. Sci. USA $\text{78}$, 2942–2946, 1981).     

Ferredoxin-NADP+ reductase and ferredoxin of the protozoan parasite Toxoplasma gondii interact productively in Vitro and in Vivo

Toxoplasma gondii possesses an apicoplast-localized, plant-type ferredoxin-NADP(+) reductase. We have cloned a [2Fe-2S] ferredoxin from the same parasite to investigate the interplay of the two redox proteins. A detailed characterization of the two purified recombinant proteins, particularly as to their interaction, has been performed. The two-protein complex was able to catalyze electron transfer from NADPH to cytochrome c with high catalytic efficiency. The redox potential of the flavin cofactor (FAD/FADH(-)) of the reductase was shown to be more positive than that of the NADP(+)/NADPH couple, thus favoring electron transfer from NADPH to yield reduced ferredoxin. The complex formation between the reductase and ferredoxins from various sources was studied both in vitro by several approaches (enzymatic activity, cross-linking, protein fluorescence quenching, affinity chromatography) and in vivo by the yeast two-hybrid system. Our data show that the two proteins yield an active complex with high affinity, strongly suggesting that the two proteins of T. gondii form a physiological redox couple that transfers electrons from NADPH to ferredoxin, which in turn is used by some reductive biosynthetic pathway(s) of the apicoplast. These data provide the basis for the exploration of this redox couple as a drug target in apicomplexan parasites.     

Heparin inhibition of ferredoxin-NADP reductase in chloroplast thylakoid membranes

Heparin, an anionic polysaccharide, inhibited the ferredoxin-catalyzed reduction of NADP in spinach chloroplast thylakoid membranes. Under the same conditions of assay, heparin did not interfere markedly with photoreduction of methyl viologen, anthraquinone sulfonate, or ferredoxin. A kinetic analysis of the heparin-induced interference with NADP photoreduction showed partial competitive inhibition. Heparin also interfered with NADPH oxidation by membrane-bound ferredoxin-NADP reductase (with dichlorophenol-indophenol as the acceptor) by a mechanism that involves partial competitive inhibition. This reaction was sensitive to the presence of salts; increasing ionic strength increases the heparin Ki for inhibition of NADPH oxidation. These results show that heparin binds to ferredoxin-NADP reductase, and in doing so interferes with binding to the reductase by both ferredoxin and NADP(H). Since heparin is redox inactive and does not interfere with the photophosphorylation reaction, it is a useful inhibitor of thylakoid membrane reactions which require the catalytic activity of ferredoxin-NADP reductase.     

Plastoquinones are effectively reduced by ferredoxin:NADP+ oxidoreductase in the presence of sodium cholate micelles. Significance for cyclic electron transport and chlororespiration

The effect of sodium cholate and other detergents (Triton X-100, sodium dodecyl sulphate, octyl glucoside, myristyltrimethylammonium bromide) on the reduction of plastoquinones (PQ) with a different length of the side-chain by spinach ferredoxin:NADP(+) oxidoreductase (FNR) in the presence of NADPH has been studied. Both NADPH oxidation and oxygen uptake due to plastosemiquinone autoxidation were highly stimulated only in the presence of sodium cholate among the used detergents. Sodium cholate at the concentration of 20 mM was found to be the most effective on both PQ-4 and PQ-9-mediated oxygen uptake. The FNR-dependent reduction of plastoquinones incorporated into sodium cholate micelles was stimulated by spinach ferredoxin but inhibited by Mg(2+) ions. It was concluded that the structure of sodium cholate micelles facilitates contact of plastoquinone molecules with the enzyme and creates favourable conditions for the reaction similar to those found in thylakoid membranes for PQ-9 reduction. The obtained results were discussed in terms of the function of FNR as a ferredoxin:plastoquinone reductase both in cyclic electron transport and chlororespiration.     

The accessibility of the chloroplast cytochromes ƒ and b-559 to ferricyanide

(1) (a) A concentration range of ferricyanide ( 0.125–0.5 mM) can be found which in the dark causes oxidation of cytochrome ƒ with two distinct kinetic components of comparable amplitude. The slow oxidation has a half time of 1–2 min. (b) The oxidation of cytochrome ƒ by ferricyanide is rapid and monophasic after the chloroplasts are frozen and thawed. (c) The oxidation of cytochrome b-559 by ferricyanide in the dark is mostly monophasic with a time course similar to that of the fast component in the cytochrome ƒ oxidation. (d) Ascorbate reduction of cytochromes ƒ and b-559 appears monophasic. Reduction of cytochrome b-559 by ascorbate is somewhat faster, and that by hydroquinone somewhat slower, than the corresponding reduction of cytochrome ƒ.

(2) (a) The kinetics of dark ferricyanide oxidation of cytochrome ƒ after actinic preillumination in the presence of an electron acceptor are approximately monophasic with a half time of about 30 s and do not show the presence of the slowly oxidized component observed after prolonged dark incubation. (b) The effect of actinic preillumination in altering the time course of ferricyanide oxidation appears to persist for several minutes in the dark. (c) Preillumination causes an increase in the extent of cytochrome b-559 oxidation by low concentrations of ferricyanide. The increase is inhibited if 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea is present during the preillumination. (d) The presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea during preillumination does not inhibit the amplitude or rate of ferricyanide oxidation of cytochrome ƒ, although the presence of the inhibitor KCN does cause such inhibition.

(3) It is proposed that a significant fraction of the cytochrome ƒ population resides at a position in the membrane relatively inaccessible to the aqueous interface compared to high potential cytochrome b-559. Actinic illumination would cause a structural or conformational change in the cytochrome ƒ and/or the membrane resulting in an increase in accessibility to this fraction of the cytochrome ƒ population.     

Ferredoxin-Dependent Photosynthetic Electron Transport and Coupled Processes of Energy Storage in Chloroplasts of Wheat Plants Grown under Different Levels of Nitrogen Supply

The rates of electron transfer in the presence of natural cofactors, ferredoxin and NADP, which were added in the amounts catalyzing noncyclic or cyclic electron transfer, were studied in thylakoids isolated from 17-day-old wheat seedlings. Upon excitation of both photosystems (PS) of photosynthesis, the potential rate of NADP reduction in thylakoids isolated from plants grown on nitrogen-free nutrient solution did not differ from that in thylakoids from the control plants. However, the P/2e ratio was significantly lower in thylakoids isolated from nitrogen-deficient plants. On the contrary, in the presence of DCMU, the rate of PSI-driven electron transfer from an artificial donor to NADP was considerably higher in these than in the control thylakoids. In the presence of ferredoxin under anaerobic conditions, the rate of phosphorylation coupled to cyclic electron transport was also significantly higher in thylakoids isolated from nitrogen-deficient plants, than in thylakoids isolated from control plants. Our data show that PSI-driven electron transport and cyclic photophosphorylation are activated in nitrogen-starved wheat plants, at least at the initial stages of starvation.     

On an Inhibitor of Ferredoxin Catalyzed Cyclic Photophosphorylation in Thylakoid Membranes*

Dibromo- and diiodo-naphthoquinones are shown to be inhibitors of the cytochrome b₆/f complex in isolated thylakoid membranes from spinach chloroplasts. Dibromo-naphthoquinone inhibits ferredoxin catalyzed cyclic photophosphorylation at 0.1 μM concentrations, but non cyclic e-flow only at 10 μM. It does not inhibit cyclic systems with artifical cofactors, nor non-cyclic electron flow from duroquinol through photosystem I via the cytochrome b₆/f complex. Dibromo-naphthoquinone does however, lower the stoichiometry for ATP formation in the duroquinol donor system. This inhibitory pattern is quite different from that of DBMIB, but very similar to that of antimycin. This antimycin-like behaviour of these inhibitors is interpreted to indicate a) the existence of a Q_c site in the cytochrome b₆/f complex and its obligate function in ferredoxin catalyzed cyclic electron flow and b) a non-essential role of the Q_c site in non-cyclic electron flow, but which — when operative — pumps an extra proton across the thylakoid membrane increasing the ATP yield.     

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe-4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin-NADP(+) reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin-NADP(+) reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.