On the site of function of the Rieske iron-sulfur center in the chloroplast electron transport chain.

A photosynthetic mutant (strain 1073) of Lemna perpusilla was previously shown to have a block in the electron transport chain between plastoquinone and cytochrome f ((1976) Plant Physiol. 57, 577--579). Electron paramagnetic resonance analysis of chloroplasts from this mutant indicates that the g = 1.89 signal of a reduced iron-sulfur center (the 'Rieske' iron-sulfur center) is absent. The absence of this signal indicates the Rieske center is either absent from or defective in the mutant, and this result is consistent with this iron-sulfur center functioning between plastoquinone and cytochrome f in the electron transport chain of chloroplasts.     

On the interaction of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB) with bound electron carriers in spinach chloroplasts

2,5-Dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), when added to chloroplasts as the sole electron donor, is an effective reducing agent. Low concentrations of 2,5-dibromo-3-methyl-6-isopropylbenzoquinone reduce cytochrome f, plastocyanin, and P700 in the dark but do not reduce the high-potential form of cytochrome b₅₅₉. 2,5-Dibromo-3-methyl-6-isopropylbenzoquinone appears to interact at or near the site of function of the “Rieske” iron-sulfur center, as evidenced by a shift in the g value of the electron paramagnetic resonance signal of the reduced center.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Appearance of Membrane-bound Iron-Sulfur Centers and the Photosystem I Reaction Center during Greening of Barley Leaves

Dark-grown barley (Hordeum vulgare) etioplasts were examined for their content of membrane-bound iron-sulfur centers by electron paramagnetic resonance spectroscopy at 15K. They were found to contain the high potential iron-sulfur center characterized (in the reduced state) by an electron paramagnetic resonance g value of 1.89 (the “Rieske” center) but did not contain any low potential iron-sulfur centers. Per mole of cytochrome f, dark-grown etioplasts and fully developed chloroplasts had the same content of the Rieske center. During greening of etioplasts under continuous light, low potential bound iron-sulfur centers appear. In addition, the photosystem I reaction center, as measured by the photooxidation of P700 at 15K, also became functional; during greening the appearance of a photoreducible low potential iron-sulfur center paralleled the appearance of P700 photoactivity.     

Photoreactions of cytochrome b6 in systems using resolved chloroplast electron-transfer complexes

Photoreactions of cytochrome b₆ have been studied using resolved chloroplast electron-transfer complexes. In the presence of Photosystem (PS) II and the cytochrome b₆-f complex, photoreduction of the cytochrome can be observed. No soluble components are required for this reaction. Cytochrome b₆ photoreduction was found to be inhibited by quinone analogs, which inhibit at the Rieske iron-sulfur center of the cytochrome complex, by the addition of ascorbate and by depletion of the Rieske center and bound plastoquinone from the cytochrome complex. Photoreduction of cytochrome b₆ can also be demonstrated in the presence of the cytochrome complex and PS I. This photoreduction requires plastocyanin and a low-potential electron donor, such as durohydroquinone. Cytochrome b₆ photoreduction in the presence of PS I is inhibited by quinone analogs which interact with the Rieske iron-sulfur center. These results are discussed in terms of a Q-cycle mechanism in which plastosemiquinone serves as the reductant for cytochrome b₆ via an oxidant-induced reductive pathway.     

The relationship of the cyclic and non-cyclic electron transport pathways in chloroplasts

Light-induced redox changes of plastocyanin, the Rieske iron-sulfur center, and P-700 have been studied in situ in spinach chloroplasts. Plastocyanin and the Rieske center behaved in an analogous manner in that their steady states were fully oxidized in the light in the presence or absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea when an electron acceptor is present. After illumination under conditions of non-cyclic electron transfer from water to an electron acceptor, followed by a short dark period, the steady state of both shifted to a more reduced level. A 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive photoreduction of the Rieske center was observed in ferricyanide-washed chloroplast fragments. With reduced ferredoxin as electron donor, it was possible to demonstrate a reduction in the dark of these electron carriers and of P-700; this reduction was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea but was inhibited by antimycin A. These findings are discussed in relation to a function for these electron carriers in the cyclic electron transport pathway in chloroplasts and to their function in the non-cyclic electron transport pathway.     

Electron transport from cytochrome b6-f complexes to Photosystem I reaction center complexes in Synechococcus sp. Is cytochrome c-553 a mobile electron carrier?

Numbers of the Photosystem I reaction center complexes and the cytochrome b₆-f complexes with which a cytochrome c-553 molecule can interact within the limiting time of photosynthetic electron transport were examined by measuring flash-induced absorption changes of P-700, cytochrome c-553 and cytochrome f in the thermophilic cyanobacterium Synechococcus sp. The addition of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) did not affect the common 2 ms half-time of P-700, cytochrome c-553 and cytochrome f reduction, which is ascribed to electron transfer from the plastoquinone pool. The inhibitor decreased, however, amounts of the three electron carriers which underwent the 2 ms reduction in the order of cytochrome f, cytochrome c-553 and P-700. On excitation with weak flashes which oxidized only a small fraction of cytochrome c-553 molecules present in cells, P-700 remained in the oxidized state after the flashes was reduced with electrons from the Rieske center or plastoquinone but not from cytochrome c-553. The ratios of cytochrome c-553 to cytochrome f oxidized at various flash intensities were constant and similar to the ratio of the two cytochromes present in cells. It is concluded that cytochrome c-553 cannot exchange electrons with large numbers of the Photosystem I reaction center complexes and the cytochrome b₆-f complexes in the limiting time, but has a mobility sufficient to mediate electron transfer between the two complexes, which are present at an unbalanced ratio in Synechococcus cells.     

Evidence for a Block between Plastoquinone and Cytochrome f in a Photosynthetic Mutant of Lemna with Abnormal Flowering Behavior

Mutant strain 1073 of Lemna perpusilla is concluded to be blocked between plastoquinone and cytochrome f in the photosynthetic electron transport system. The location of the block is based on the following observations of activities in chloroplasts isolated from the mutant and wild-type plants. (a) Relative to wild type, electron flow rates from water to ferricyanide, 2,6-dichlorophenol indophenol or NADP were very low in the mutant, but rates of photosystem I-dependent electron flow and cyclic phosphorylation were high. (b) Chlorophyll a fluorescence induction curves for mutant and wild type were similar. (c) Silicomolybdate and lipophilic acceptors in the mutant were photoreduced at rates comparable to wild type. (d) Cytochrome f of the mutant chloroplasts was not reduced by red light, but was oxidized by red or far red light. (e) Reduction of the primary electron acceptor of photosystem II (Q) by ATP-driven reverse electron flow was not observed in the mutant.     

Modification of the apparent redox reaction between cytochrome f and the rieske iron-sulfur protein

We have investigated the role of cytochrome f and the Rieske FeS protein in spinach chloroplasts using the quinone analogue 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). UHDBT inhibits electron transport at two different sites in spinach chloroplasts. Fluorescence yield measurements monitoring the redox state of Q, the first stable primary acceptor of Photosystem II, and polarographic measurements of electron transport show that at low concentrations UHDBT inhibits near Q. At higher concentrations UHDBT inhibits at a second site. Electron transfer from durohydroquinone to methyl viologen is inhibited (50% inhibition at 21 μM) but not the reaction dichlorophenolindophenol to methyl viologen. Spectroscopic measurements of the kinetics of cytochrome f show that UHDBT inhibits the dark reduction rate of the cytochrome following a 100 ms flash (50% inhibition at 15 μM). By contrast, the oxidation kinetics of cytochrome f following a single-turnover flash are altered little by UHDBT; the initial rates are indistinguishable, and the half-time increases from 220 μs in the control to 285 μs in the presence of 15 μM UHDBT, largely because the extent of the cytochrome f oxidation is enhanced 1.4-fold in the presence of the inhibitor. In a single-turnover flash in the absence of UHDBT, we observe 38–48% of the total cytochrome f turning over, while in the presence of UHDBT we observe 60–69% of the cytochrome turning over. We interpret these results in terms of a linear rapid donor pool to Photosystem I, FeS → cytochrome f → plastocyanin → P-700, in which UHDBT inhibits by interacting with the Rieske FeS center. We conclude that the enhanced extent of cytochrome f oxidation in the presence of UHDBT is due to the removal of the Rieske FeS center from the rapid donor pool. As a consequence, removal of a single electron from the pool results in a greater cytochrome f oxidation. These results indicate that the Rieske FeS center and cytochrome f equilibrate in a time period comparable to the oxidation time of the cytochrome.     

Reaction kinetics of P-700, cytochrome c-553 and cytochrome f in the cyanobacterium, Synechococcus sp

Absorption changes invoked by short flashes in the Soret band region were measured in the thermophilic cyanobacterium Synechococcus sp. and photoresponses of P-700, cytochrome c-553 and cytochrome f were resolved with the aid of a microcomputer. Cytochrome c-553 was oxidized very rapidly with a half-time of less than 20 μs, while the half oxidation time of cytochrome f was 35–45 μs. The two cytochromes were reduced monophasically with half-time of 2 ms after a lag lasting a few milliseconds. The reduction kinetics of P-700 showed three exponential phases with half-times of 40 μs, 200 μs and 2 ms, which are ascribed to electron donation from cytochrome f, the Rieske iron-sulfur protein and plastoquinone, respectively. The results support the following sequence and rates of linear electron transport at the physiological temperature of the cyanobacterium: P-700

cytochrome c-553

cytochrome f

Rieske protein

plastoquinone.     

Co-operation between different targeting pathways during integration of a membrane protein

Membrane protein assembly is a fundamental process in all cells. The membrane-bound Rieske iron-sulfur protein is an essential component of the cytochrome bc₁ and cytochrome b₆f complexes, and it is exported across the energy-coupling membranes of bacteria and plants in a folded conformation by the twin arginine protein transport pathway (Tat) transport pathway. Although the Rieske protein in most organisms is a monotopic membrane protein, in actinobacteria, it is a polytopic protein with three transmembrane domains. In this work, we show that the Rieske protein of Streptomyces coelicolor requires both the Sec and the Tat pathways for its assembly. Genetic and biochemical approaches revealed that the initial two transmembrane domains were integrated into the membrane in a Sec-dependent manner, whereas integration of the third transmembrane domain, and thus the correct orientation of the iron-sulfur domain, required the activity of the Tat translocase. This work reveals an unprecedented co-operation between the mechanistically distinct Sec and Tat systems in the assembly of a single integral membrane protein.     

Characterization of Nuclear Mutants of Maize Which Lack the Cytochrome f/b-563 Complex

Two high fluorescent, nuclear recessive mutants of maize (Zea mays L.), designated hcf-2 and hcf-6, are described which are missing the chloroplast cytochrome f/b-563 complex. Thylakoids from the mutants show a block in whole chain electron transport activity (H₂O to methyl viologen), while retaining activities associated with photosystem II (H₂O to phenylenediamine) and photosystem I (diaminodurene to methyl viologen). Chemically induced, optical difference spectra indicate a loss of cytochromes f and b-563. Cytochrome b-559 is present in both high and low potential forms. EPR analyses of thylakoid membranes of hcf-6 reveals the lack of a signal (g = 1.90) associated with the Rieske Fe-S center. Additionally, hcf-6 is lacking EPR signals at g = 6 (attributable to the high spin ferric heme of cytochrome b-563) and g = 2.5 (unidentified). The mutant retains signals at g = 2.9 (cytochrome b-559) and at g = 4.3 and 9 (both signals probably arising from a storage form of ferric iron).

Thylakoid polypeptides are examined using polyacrylamide gel electrophoresis. hcf-2 and hcf-6 have identical profiles, showing losses of polypeptides with apparent molecular masses of 33 (cytochrome f), 23 (cytochrome b-563), and 17.5 kilodaltons. The protein associated with the Rieske Fe-S center could not be determined from the gel profiles. Additionally, both mutants show an increase in a band with a molecular mass of 31 kilodaltons.

     

Site-specific Inhibition of Photophosphorylation in Isolated Spinach Chloroplasts by Mercuric Chloride

Photophosphorylation associated with noncyclic electron transport in isolated spinach (Spinacia oleracea) chloroplasts is inhibited to approximately 50% by low concentrations of HgCl₂ (less than 1 μmole Hg²⁺/mg chlorophyll) when the electron transport pathway includes both sites of energy coupling. Reactions involving only a part of the electron transport system can give a functional isolation of at least two sites coupled to phosphorylation. Only one of these sites, located between the oxidation of plastoquinone and the reduction of cytochrome f, is sensitive to mercuric chloride. The energy conservation site located before plastoquinone and close to photosystem II is unaffected by HgCl₂ concentrations up to 10-fold those required to inhibit phosphorylation by the coupling site after plastoquinone. This site-specific inhibition may reflect a mechanistic difference in the mode of energy coupling at the two coupling sites or a variable accessibility of HgCl₂ to these sites.     

Plastocyanin as the possible site of photosynthetic electron transport inhibition by glutaraldehyde

Treatment of spinach chloroplasts with glutaraldehyde causes an inhibition in the electron transport chain between the two photosystems. Measurements of O₂ flash yields, pH exchange, and fluorescence induction show that the O₂ evolving apparatus, photosystem II and its electron acceptor pool are not affected. The behavior of P700 indicates that its reduction but not its oxidation, is severely inhibited. Cytochrome f is still reducible by photosystem II but also slowly oxidizable by photosystem I. The sensitivity of isolated plastocyanin to glutaraldehyde further supports the conclusion that glutaraldehyde inhibits at the plastocyanin level and thereby induces a break between P700 and cytochrome f.     

Role of the Twin Arginine Protein Transport Pathway in the Assembly of the Streptomyces coelicolor Cytochrome bc1 Complex

The cytochrome bc₁-cytochrome aa₃ complexes together comprise one of the major branches of the bacterial aerobic respiratory chain. In actinobacteria, the cytochrome bc₁ complex shows a number of unusual features in comparison to other cytochrome bc₁ complexes. In particular, the Rieske iron-sulfur protein component of this complex, QcrA, is a polytopic rather than a monotopic membrane protein. Bacterial Rieske proteins are usually integrated into the membrane in a folded conformation by the twin arginine protein transport (Tat) pathway. In this study, we show that the activity of the Streptomyces coelicolor M145 cytochrome bc₁ complex is dependent upon an active Tat pathway. However, the polytopic Rieske protein is still integrated into the membrane in a ΔtatC mutant strain, indicating that a second protein translocation machinery also participates in its assembly. Difference spectroscopy indicated that the cytochrome c component of the complex was correctly assembled in the absence of the Tat machinery. We show that the intact cytochrome bc₁ complex can be isolated from S. coelicolor M145 membranes by affinity chromatography. Surprisingly, a stable cytochrome bc₁ complex containing the Rieske protein can be isolated from membranes even when the Tat system is inactive. These findings strongly suggest that the additional transmembrane segments of the S. coelicolor Rieske protein mediate hydrophobic interactions with one or both of the cytochrome subunits.     

Cytochrome function in the cyclic electron transport pathway of chloroplasts

Flash excitation of isolated intact chloroplasts promoted absorbance transients corresponding to the electrochromic effect (P-518) and the α-bands of cytochrome b₆ and cytochrome f. Under conditions supporting coupled cyclic electron flow, the oxidation of cytochrome b₆ and the reduction of cytochrome f had relaxation half-times of 15 and 17 ms, respectively. Optimal poising of cyclic electron flow, achieved by addition of 0.1 μM 3-(3,4-dichlorophenyl)-1,1-dimethylurea, increased phosphorylation of endogenous ADP and prolonged these relaxation times. The presence of NH₄Cl, or monensin plus NaCl, decreased the half-times for cytochrome relaxation to approximately 2 ms. Uncouplers also revealed the presence of a slow rise component in the electrochromic absorption shift, with formation half-time of about 2 ms. The inhibitors of cyclic phosphorylation antimycin and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone abolished the slow rise in the electrochromic shift and prolonged the uncoupled relaxation times of cytochromes b₆ and f by factors of ten or more.These observations indicate that cytochrome b₆, plastoquinone and cytochrome f participate in a coupled electron transport process responsible for cyclic phosphorylation in intact chloroplasts. Estimations of cyclic phosphorylation rates from 40 to 120 μmol ATP/mg chlorophyll per h suggest that this process can provide a substantial fraction of the ATP needed for CO₂ fixation.     

Inhibition of photosynthetic electron transport and formation of inactive chlorophyll in winter stressed Pinus silvestris

Kinetics of fluorescence at room temperature, electron transport and photooxidation of P700 and cytochrome f have been studied in chloroplasts isolated from active and winter stressed Pinus silvestris. The winter stress induced block in the electron transport chain between the two photosystems is close to the site of plastoquinone, since winter stress and DCMU caused the same type of inhibition of the reoxidation of the primary electron acceptor Q of photosystem II. No winter inhibition of the electron transport between cytochrome f and P700 was observed. Time course studies of P700 photooxidation in chloroplasts of active and winter stressed pine have shown that the photosynthetic unit size must be about equal in the two types of chloroplasts. An apparent increase of the photosynthetic unit size was induced by winter stress, as revealed by the high chlorophyll/P700 ratio of winter stressed pine. The phenomenon is explained by the formation of photosynthetically inactive chlorophyll. Low-temperature fluorescence emission spectra were recorded when either chlorophyll a (433 nm) or chlorophyll b (477 nm) were preferentially excited. Winter stress induced the formation of a chlorophyll a fraction emitting at 673 nm. This chlorophyll is most likely derived from the chlorophyll a antennae of the two photosystems, and it probably contributes to the photosynthetically inactive pool of chlorophyll in winter stressed pine. The light harvesting chlorophyll a/b complex is relatively resistant to winter stress.     

The role of the Rieske iron-sulfur center as the electron donor to ferricytochrome c2 in Rhodopseudomonas sphaeroides

The Rieske iron-sulfur center in the photosynthetic bacterium Rhodopseudomonas sphaeroides appears to be the direct electron donor to ferricytochrome c₂, reducing the cytochrome on a submillisecond timescale which is slower than the rapid phase of cytochrome oxidation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{3–5 μ}\text{s}$). The reduction of the ferricytochrome by the Rieske center is inhibited by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) but not by antimycin. The slower (1–2 ms) antimycin-sensitive phase of ferricytochrome c₂ reduction, attributed to a specific ubiquinone-10 molecule (Q_z), and the associated carotenoid spectral response to membrane potential formation are also inhibited by UHDBT. Since the light-induced oxidation of the Rieske center is only observed in the presence of antimycin, it seems likely that the reduced form of Q_z (Q_zH₂) reduces the Rieske center in an antimycin-sensitive reaction. From the extent of the UHDBT-sensitive ferricytochrome c₂ reduction we estimate that there are 0.7 Rieske iron-sulfur centers per reaction center.UHDBT shifts the EPR derivative absorption spectrum of the Rieske center from g_y 1.90 to g_y 1.89, and shifts the E_m,7 from 280 to 350 mV. While this latter shift may account for the subsequent failure of the iron-sulfur center to reduce ferricytochrome c₂, it is not clear how this can explain the other effects of the inhibitor, such as the prevention of cytochrome b reduction and the elimination of the uptake of H⁺_II; these may reflect additional sites of action of the inhibitor.     

Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803

The cytochrome b₆f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b₆f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane.Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.     

Lipid enrichment of thylakoid membranes. I. Using soybean phospholipids

(1) Using asolectin (mixed soybean phospholipids) liposomes, extra lipid, with or without additional plastoquinone, has been introduced into isolated thylakoid membranes of pea chloroplasts. (2) Evidence for this lipid enrichment was obtained from freeze-fracture which indicated that a decrease in the numbers of EF and PF particles per unit area of membrane occurred with increasing lipid incorporation. The decrease was not due to loss of integral membrane polypeptides as judged by assay of cytochrome present or SDS-polyacrylamide gel electrophoresis of lipid-enriched membrane fractions. Moreover, the enrichment procedure did not lead to extraction of low molecular weight lipophilic membrane components or of thylakoid membrane lipids. (3) The introduction of phospholipids into the membrane affected steady-state electron transport. Inhibition of electron transport was observed when either water (Photosystem (PS) II + PS I) or duroquinol (PS I) was used as electron donor with methyl viologen as electron acceptor, and the degree of inhibition increased with higher enrichment levels. Introduction of exogenous plastoquinone with the additional lipid had little effect on whole-chain electron transport, but caused an increase in the 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB)-sensitive rate of PS I electron transport. The inhibition was also detected by flash-induced oxidation-reduction changes of cytochrome f.