Cyclic electron transport in chloroplasts. The Q-cycle and the site of action of antimycin

Cyclic electron transport systems have been set up in broken chloroplasts, with photochemically reduced ferredoxin or 9,10-anthraquinone-2-sulphonate as cofactor. In good agreement with the literature, only the ferredoxin-catalyzed pathway was found to be inhibited by antimycin; but both pathways were found to have a slow electrogenic reaction, both were inhibited by the cytochrome b-563 oxidation inhibitor 2-heptyl-4-hydroxyquinoline N-oxide (the inhibition being strongest at limiting light intensity), and the two pathways had the same proton/electron stoichiometry at limiting light intensity. It is concluded that a Q-cycle can occur in cyclic electron transport with either cofactor; and therefore that the site of action of antimycin in chloroplasts is not within the Q-cycle, as it is believed to be in mitochondria and bacteria. Instead, a ferredoxin-quinone reductase is proposed as the site of action of antimycin in the ferredoxin-catalyzed cyclic pathway. It is also concluded that the data presented here are consistent with the suggestion that the Q-cycle in photosynthetic electron transport is a facultative one, its degree of engagement depending on competition between the Rieske centre and cytochrome b-563 for reducing equivalents from plastosemiquinone.     

Effects of dibromothymoquinone on oxidation-reduction reactions and the midpoint potential of the Rieske iron-sulfur center in photosynthetic electron transport of Synechococcus Sp.

The effects of 2,5-dibromo-3-methyl-p-benzoquinone (DBMIB) on the reduction kinetics of flash-oxidized P-700 and cytochrome c-553 were studied in the thermophilic cyanobacterium Synechococcus sp. (1) The reduction kinetics of P-700 showed two exponential phases with half-times of 0.2 and 2 ms at the recording time used (Nanba, M. and Katoh, S. (1983) Biochim. Biophys. Acta 725, 272–279). DBMIB strongly slowed down the 2 ms reduction phase but not the 0.2 ms phase. (2) The content of an electron donor which transfers its electrons to P-700 with the half time of 0.2 ms was estimated to be comparable to that of cytochrome f. (3) The magnitudes of the 0.2 ms reduction phase and cytochrome c-553 oxidation decreased as the flash interval was shortened below 2 s in the poisoned cells. Assuming a rapid equilibrium of electrons in the electron donor pool of Photosystem I, the midpoint potential of the 0.2 ms donor was estimated as 280 mV by comparing its percent reduction with that of cytochrome c-553 at three different flash intervals. (4) A similar value was obtained for the midpoint potential of the 0.2 ms donor in the cells in which the plastoquinone pool had been oxidized by dark starvation. It is concluded that the 0.2 ms reduction phase of P-700 is due to the electron donation from the Rieske iron-sulfur center and that DBMIB inhibits strongly but incompletely the reduction of the iron-sulfur center with electrons from the plastoquinone pool, whereas the inhibitor has no effect on the midpoint potential and Photosystem-I-dependent oxidation of the iron-sulfur center.     

Reactions of b-cytochromes in the red alga Porphyridium aerugineum

1. 1. The kinetics of light-induced absorbance changes due to oxidation and reduction of cytochromes were measured in a suspension of intact cells of the unicellular red alga Porphyridium aerugineum. Absorbance changes in the region 540–570 nm upon alternating far-red light and darkness indicated the oxidation of cytochrome ƒ and reduction of cytochrome b₅₆₃ upon illumination. The relative efficiencies of far-red and orange light indicated that both reactions were driven by Photosystem I.

2. 2. Experiments with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), with anaerobic cells and in alternating far-red and orange light indicated that cytochrome b₅₆₃ reacts in a cyclic chain around Photosystem I, and that the reduced cytochrome does not react with oxygen or with another oxidized product of Photosystem II. The quantum requirement for the photoreduction was about 6 quanta/equiv at 700 nm. A low concentration of N-methylphenazonium methosulphate (PMS) enhanced the rate of reoxidation of cytochrome b₅₆₃ in the dark. In the presence of higher concentrations of PMS a photooxidation, driven by Photosystem I, instead of reduction was observed. These observations suggest that PMS enhances the rate of reactions between reduced cytochrome b₅₆₃ and oxidized products of Photosystem I.

3. 3. In the presence of carbonylcyanide m-chlorophenylhydrazone (CCCP) a light-induced decrease of absorption at 560 nm occurred. Spectral evidence suggested the photooxidation of cytochrome b₅₅₉ under these conditions. Inhibition by DCMU and a relatively efficient action of orange light suggested that this photooxidation is driven by Photosystem II.

Evidence for complexed plastocyanin as the immediate electron donor of P-700

The reduction of P-700 by its electron donors shows two fast phases with half-times of 20 and 200 μs in isolated spinach chloroplasts. We have studied this electron transfer and the oxidation kinetics of cytochrome f.

Incubation of chloroplasts with KCN or HgCl₂ decreased the amplitude of the 20 μs phase. This provides evidence for a function of plastocyanin as the immediate electron donor of P-700.

At low concentrations of salt and sugar the fast phases of P-700⁺ reduction were largely inhibited. Increasing concentrations of MgCl₂, KCl and sorbitol (up to 5, 150 and 200 mM, respectively) were found to increase the relative amplitudes of the fast phases to about one-third of the total P-700 signal. Addition of both 3 mM MgCl₂ and 200 mM sorbitol increased the relative amplitude of the 20 μs phase to 70%. The interaction between P-700 and plastocyanin is concluded to be favoured by a low internal volume of the thylakoids and compensation of surface charges of the membrane.

The half-time of 20 μs was not changed when the amplitude of this phase was altered either by salt and sorbitol, or by inhibition of plastocyanin. This is evidence for the existence of a complex between plastocyanin and P-700 with a lifetime long compared to the measuring time. The 200 μs phase exhibited changes in its half-time that indicated the participation of a more mobile pool of plastocyanin.

Cytochrome f was oxidized with a biphasic time course with half-times of 70–130 μs and 440–860 μs at different salt and sorbitol concentrations. The half-time of the faster phase and a short lag of 30–50 μs in the beginning of the kinetics indicate an oxidation of cytochrome f via the 20 μs electron transfer to P-700. An inhibition of this oxidation by MgCl₂ suggests that the electron transfer from cytochrome f to complexed plastocyanin is not controlled by negative charges in contrast to that from plastocyanin to P-700.     

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.     

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.     

The b-type cytochromes of bovine heart mitochondria: Absorption spectra, enzymatic properties, and distribution in the electron transfer complexes

1. 1. Three b-type cytochromes (b_557.5, b₅₆₀, and b_562.5), plus a chromophore with an absorption peak at 558 nm at 77 °K, have been found to be associated with the electron transport system of bovine heart mitochondria. The reduced minus oxidized spectra of these components at 77 °K, as well as that of cytochrome c₁, have been recorded with a wavelength accuracy of ± 0.1 nm and presented to the nearest 0.5 nm. All the major and β absorption peaks of cytochromes b_557.5, b₅₆₀, b_562.5, c₁ and c have been shown by fourth derivative analysis to be present in the dithionite-reduced minus oxidized spectra of mitochondria and submitochondrial particles.

2. 2. The distribution of the above components has been studied in the four electron transfer complexes of the respiratory chain. Cytochromes b₅₆₀, b_562.5 and c₁, as well as chromophore-558, were found to fractionate into Complex III (reduced ubiquinone-cytochrome c reductase), whereas cytochrome b_557.5 was found in Complex II (succinate-ubiquinone reductase).

3. 3. Cytochrome b₅₆₀ was readily reduced by NADH or succinate, but b_562.5 was not reduced by substrates unless the preparation was treated with antimycin A. In antimycin-treated preparations pre-reduction of c₁ with ascorbate inhibited the subsequent reduction of b_562.5 by substrates. These results indicate that b₅₆₀ and b_562.5 correspond, respectively, to b_K and b_T previously described by Chance et al.¹⁴ (1970, Proc. Natl. Acad. Sci. U.S. 66, 1175–1182).

4. 4. Similar to b₅₆₀, chromophore-558 can be reduced by substrates in the absence or presence of antimycin A. However, in antimycin-treated preparations, pre-reduction of c₁ inhibits its subsequent reduction by substrates. This property is similar to that of b_562.5.

5. 5. Cytochrome b_557.5, which occurs in Complex II, appears to have a low mid-point potential. It can be reduced with dithionite and oxidized by fumarate or ubiquinone. CO treatment of dithionite-reduced b_557.5 neither modified the spectrum of this cytochrome nor diminished the extent of b_557.5 reoxidation by fumarate.

6. 6. Antimycin A treatment does not appear to alter the spectra of the above cytochromes. However, small amounts (< 4%) of ethanol or methanol, which are usually added to particles as solvent for antimycin A, have a pronounced effect on the peaks of cytochrome c₁. The spectrum of cytochrome c₁ at 77 °K as modified by 3% (v/v) ethanol is shown.

The reaction of antimycin with a cytochrome b preparation active in reconstitution of the respiratory chain

1. Succinate-cytochrome c reductase activity was reconstituted by incubating a mixture of succinate dehydrogenase, cytochrome c₁, ubiquinone-10, phospholipid and a preparation of cytochrome b, made by the method of .

2. Preparations of cytochrome b active in reconstitution contained 5–28% native cytochrome b, as adjudged by reducibility with succinate in the reconstituted preparation and by lack of reaction with CO. Preparations of cytochrome b containing no native cytochrome b according to this criterion were inactive in reconstitution.

3. With a fixed amount of cytochrome b, the activity of the reconstituted preparation increased with increasing amounts of cytochrome c₁ until a ratio of about 2b (total): 1c₁ (allowing for the cytochrome c₁ present in the cytochrome b preparation) was reached.

4. The amount of antimycin necessary for maximal inhibition of the reconstituted enzyme is a function of the amount of the cytochrome b and is independent of the amount of cytochrome c₁. It is equal to about one half the amount of native cytochrome b.

5. Preparations of intact or reconstituted succinate-cytochrome c reductase or of cytochrome b completely quench the fluorescence of added antimycin, until an amount of antimycin equal to onehalf the amount of native cytochrome b present was added. Antimycin added in excess of this amount fluoresces with normal intensity. The quenching is only partial in the presence of Na₂S₂O₄. Denatured cytochrome b does not quench the fluorescence.

6. Since preparations of cytochrome b active in reconstitution contained cytochrome c₁ in an amount exceeding one half the amount of native cytochrome b present in the preparation, there is no evidence that native cytochrome b has been resolved from cytochrome c₁. The stimulatory action of cytochrome c₁ may be due to the restoration of a damaged membrane conformation.

7. Based on the assumption that the bc₁ segment of the respiratory chain contains 2b:1c₁:1 antimycin-binding sites, the specific quenching of antimycin fluorescence by binding to cytochrome b enables an accurate determination of the absorbance coefficients of cytochromes b and c₁. These are 25.6 and 20.1 mM⁻¹×cm⁻¹ for the wavelength pairs 563–577 nm and 553–539 nm, respectively, in the difference spectrum reduced minus oxidized.     

Photooxidation of cytochrome b-559 in leaves and chloroplasts at room temperature

1. Light-induced absorbance changes of cytochrome b-559 and cytochrome f in the -band region were examined in leaves and in isolated chloroplasts.

2. Absorbance changes of cytochrome b-559 were not detected in untreated leaves or in most preparations of isolated chloroplasts. After treatment of leaves or chloroplasts with carbonyl cyanide m-chlorophenylhydrazone, high rates of photooxidation of cytochrome b-559 were obtained, both in far-red (>700 nm) and red actinic light. Cytochrome f was photooxidized in far-red light, but in red light it remained mainly in the reduced state. The initial rates of photooxidation of cytochrome b-559 in leaves or chloroplasts treated with carbonyl cyanide m-chlorophenylhydrazone were considerably decreased by 3-(3′,4′-dichlorophenyl)-1,1-dimethyl urea.

3. A slow photoreduction of cytochrome b-559 was observed in aged mutant pea chloroplasts in red light.

4. The results do not support the view that cytochrome b-559 is a component of the electron transport chain between the light reactions. It is suggested that cytochrome b-559 is located on a side path from Photosystem II, but with a possible additional link to Photosystem I.     

The fractionation of plant photoactive pigment-protein complexes I and II

The pigment-protein complexes enriched with Photosystem I (PPC-I) and Photosystem II (PPC-II) were obtained using sievorptive chromatography on DEAE-Sephadex column. Both types of complexes contain Chlorophyll a, β-carotene and minor quantities of Chl b. Red absorbance maxima are located at 676 nm and 673 nm for PPC-I and PPC-II, respectively. The degrees of reaction centre enrichment were measured by the method of differential spectrophotometry: PPC-I has one P-700 per 35 bulk Chl a molecules, PPC-II contains one P-680 per 18 bulk Chl a molecules. The yield of PPC-II is 7–10 times lower than that of PPC-I. After one chromatographic procedure the amount of P-680 in PPC-I preparation does not exceed 7% of that of P-700, the amount of P-700 in PPC-II preparation 2% of that of P-680. The product of PPC-II degradation was studied.     

Oxidoreduction of cytochrome b in the presence of antimycin

1. The effect of oxidizing equivalents on the redox state of cytochrome b in the presence of antimycin has been studied in the presence and absence of various redox mediators.

2. The antimycin-induced extra reduction of cytochrome b is always dependent on the initial presence of an oxidant such as oxygen. After removal of the oxidant this effect remains or is partially (under some conditions even completely) abolished depending on the redox potential of the substrate used and the leak through the antimycin-inhibited site.

3. The increased reduction of cytochrome b induced by oxidant in the presence of antimycin involves all three spectroscopically resolvable b components (b-562, b-566 and b-558.

4. Redox mediators with an actual redox potential of less than 100–170 mV cause the oxidation of cytochrome b reduced under the influence of antimycin and oxidant.

5. Redox titrations of cytochrome b with the succinate/fumarate couple were performed aerobically in the presence of cyanide. In the presence of antimycin two b components are separated potentiometrically, one with an apparent midpoint potential above 80 mV (at pH 7.0), outside the range of the succinate/fumurate couple, and one with an apparent midpoint potential of 40 mV and an n value of 2. In the absence of antimycin cytochrome b titrates essentially as one species with a midpoint potential of 39 mV (at pH 7.0) and n = 1.14.

6. The increased reducibility of cytochrome b induced by antimycin plus oxidant is considered to be the result of two effects: inhibition of oxidation of ferrocytochrome b by ferricytochrome c₁ (the effect of antimycin), and oxidation of the semiquinone form of a two-equivalent redox couple such as ubiquinone/ubiquinol by the added oxidant, leading to a decreased redox potential of the QH₂/QH^• couple and reduction of cytochrome b.     

Antimycin-insensitive mutants of Candida utilis. II. The effects of antimycin on cytochrome b

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutant. A similar reoxidation is observed in the wild type in the presence of low concentrations of antimycin.

2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steadystate reduction; reduction in the presence of substrate, cyanide and oxygen; the ‘red shift’ and lowering of E′₀ of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable.

3. The red shift in the mutant is more extensive than in the wild type.

4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes.

5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant.

6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH₂-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycinbinding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.     

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.     

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.     

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.     

Study of electrogenic electron transfer steps in chromatophore membrane of Chromatium vinosum by the response of merocyanin dye

1. Electrogenic steps in photosynthetic cyclic electron transport in chromatophore membrane of Chromatium vinosum were studied by measuring absorption changes of added merocyanin dye and of intrinsic carotenoid.

2. The change in dye absorbance was linear with the membrane potential change induced either by light excitation or by application of diffusion potential by adding valinomycin in the presence of K⁺ concentration gradient.

3. It was estimated that chromatophore membrane became 40–60 mV and 110–170 mV inside positive upon single and multiple excitations with single-turnover flashes, respectively, from the responses of the dye and the carotenoid.

4. Electron transfers between cytochrome c-555 or c-552 and reaction center bacteriochlorophyll dimer (BChl₂) and between BChl₂ and the primary electron acceptor were concluded to be electrogenic from the redox titration of the dye response.

5. No dye response which corresponded to the change of redox level of cytochrome b was observed in the titration curve. Addition of antimycin A slightly decreased the dye response.

6. The dye response was decreased under phosphorylating conditions.

7. From the results obtained localization of the electron transfer components in chromatophore membrane is discussed.     

The effect of ethylenediamine chemical modification of plastocyanin on the rate of cytochrome f oxidation and P-700 reduction

Chemical modification of plastocyanin was carried out using ethylenediamine plus a water-soluble carbodiimide, which has the effect of replacing a negatively charged carboxylate group with a positively charged amino group at pH 6–8. The conditions were adjusted to produce a series of singly and doubly modified forms of plastocyanin. Differences in charge configuration allowed separation of these forms on a Pharmacia fast protein liquid chromatograph using a Mono Q anion exchange column. These forms were used to study the interaction of plastocyanin with its reaction partner cytochrome f. The rate of cytochrome f oxidation was progressively inhibited upon incorporation of increasing numbers of ethylenediamine moieties indicating a positively charged binding site on cytochrome f. However, differential inhibition was obtained for the various singly modified forms allowing mapping of the binding site on plastocyanin. The greatest inhibition was found for forms modified at negatively charged residues Nos. 42–45 and Nos. 59–61 which comprise a negative patch surrounding Tyr-83. In contrast, the form modified at residue No. 68, on the opposite side of the globular plastocyanin molecule, showed the least inhibition. It can be concluded that the binding site for cytochrome f is located in the vicinity of residues Nos. 42–45 and Nos. 59–61. Modification of plastocyanin at residues Nos. 42–45 showed no effect on the rate of P-700⁺ reduction, suggesting that these residues are not involved in the binding of Photosystem I. However, an increase in the rate of P-700⁺ reduction was observed for plastocyanins modified at residue No. 68 or Nos. 59–61, which is consistent with the idea that the reaction domain of Photosystem I is negatively charged and Photosystem I binds at the top of the molecule and accepts electrons via His-87 in plastocyanin. These results raise the possibility that plastocyanin can bind both cytochrome f and Photosystem I simultaneously. The effect of ethylenediamine modification on the formal potential of plastocyanin was also examined. The formal potential of control plastocyanin was found to be +372 ± 5 mV vs. normal hydrogen electrode at pH 7. All modified forms showed a positive shift in formal potential. Singly modified forms showed increases in formal potentials between +8 and +18 mV with the largest increases being observed for plastocyanins modified at residues Nos. 42–45 or Nos. 59–61.     

Respiratory cytochromes of Euglena gracilis

1. 1. Difference spectra of whole cells and of a particulate fraction of a streptomycin-bleached strain of Euglena gracilis showed the presence of a b-type cytochrome, cytochrome b (561 Euglena), and an a-type cytochrome, cytochrome a-type (609 Euglena). The cytochromes were characterized by pyridine hemochromogen formation and were found associated with a particulate fraction enriched with mitochondria.

2. 2. Both b-type and a-type cytochromes were reduced by succinate, oxidized by oxygen and reacted with a soluble c-type cytochrome, cytochrome c-type (556 Euglena), in reversible oxidation-reduction reactions. The steady-state level of reduction for each cytochrome was 92, 22 and 5% of the anaerobic level for the b-type, c-type and a-type cytochrome, respectively.

3. 3. Oxidation of c-type and a-type cytochromes was completely inhibited by cyanide, although respiration of a particulate fraction was only 60% inhibited by the same concentration of cyanide. Antimycin A inhibited respiration by up to 70% but completely inhibited reduction of the c-type cytochrome.

4. 4. The data suggest that electron transfer in the respiratory pathway of Euglena involves the b-, c- and a-type cytochrome in a direct sequence. The cyanide and antimycin A-insensitive oxidation pathway is considered to involve a more direct oxidation of the b-type cytochrome.

The kinetics of reactions around the cytochrome bf complex studied in an isolated system

The kinetics of oxidation and reduction of P700, plastocyanin, cytochrome f and cytochrome b-563 were studied in a reconstituted system consisting of Photosystem I particles, cytochrome bf complex and plastocyanin, all derived from pea leaf chloroplasts. Decyl plastoquinol was the reductant of the bf complex. Turnovers of the system were initiated by laser flashes. The reaction between oxidised P700 and plastocyanin was non-homogeneous in that a second-order rate coefficient of c. 5×10^–7 M^–1 s^–1 applied to 80% of the P700⁺ and c. 0.7×10⁷ M^–1 s^–1 to the remainder. In the presence of bf complex, but without quinol, the electron transfer between cytochrome f and oxidised plastocyanin could be described by a second-order rate coefficient of c. 4×10⁷ M^–1 s^–1 (forward), and c. 1.6×10⁷ M^–1 s^–1 (reverse). The equilibrium coefficient was thus 2.5. Unexpectedly, there was little reduction of cytochrome f ⁺ or plastocyanin⁺ by electrons from the Rieske centre. With added quinol, reduction of cytochrome b-563 occurred. Concomitantly, electrons appeared in the oxidised species. It was inferred that either the Rieske centre was not involved in the high-potential chain of electron transfer events, or that, only in the presence of quinol, electrons were quickly passed from the Rieske centre to cytochrome f ⁺. Additionally, the presence of quinol altered the equilibrium coefficient for the cyt f/PC interaction from 2.5 to c. 5. The reaction between quinol and the bf complex was describable by a second-order rate coefficient of about 3×10⁶ M^–1 s^–1. The pattern of the redox reactions around the bf complex could be simulated in detail with a Q-cycle model as previously found for chloroplasts.Abbreviations AQS anthraquinone sulphonate - cyt cytochrome - cyt b-563(H) high-potential cyt b-563 - cyt b-563(L) low potential cyt b-563 - FeS(R) the Rieske protein of the cyt bf complex, containing an Fe₂S₂ centre - PC plastocyanin - PS photosystem - P700 reaction centre in PS I     

The orientation of membrane bound radicals. An EPR investigation of magnetically ordered spinach chloroplasts

The orientation of membrane-bound radicals in spinach chloroplasts is examined by electron paramagnetic resonance (EPR) spectroscopy of chloroplasts oriented by magnetic fields. Several of the membrane-bound radicals which possess g-tensor anisotropy display EPR signals with a marked dependence on the orientation of the membranes relative to the applied EPR field. The fraction of oxidized and reduced plastocyanin, P-700, iron-sulfur proteins A and B, and the X center, an early acceptor of Photosystem I, can be controlled by the light intensity during steady-state illumination and can be trapped by cooling. The X center can be photoreduced and trapped in the absence of strong reductants and high pH, conditions previously found necessary for its detection. These results confirm its role as an early electron acceptor in P-700 photo-oxidation. X is oriented with its smallest principal g-tensor axis (g_x) predominantly parallel to the normal to the thylakoid membrane, the same orientation as was found for an early electron acceptor based on time-resolved electron spin polarization studies. We propose that the X center is the first example of a high potential iron-sulfur protein which functions in electron transfer in its ‘;superreduced’; state. We present evidence which suggests that iron-sulfur proteins A and B are 4Fe-4S clusters in an 8Fe-8S protein. Center B is oriented with g_y predominantly normal to the membrane plane. The spectra of center A and plastocyanin do not show significant changes with sample orientation. In the case of plastocyanin, this may indicate a lack of molecular orientation. The absence of an orientation effect for reduced center A is reconcilable with a 4Fe-4S geometry, provided that the electron obtained upon reduction can be shared between any pair of Fe atoms in the center. Orientation of the ‘;Rieske’; iron-sulfur protein is also observed. It has axial symmetry with g close to the plane of the membrane. A model is proposed for the organization of these proteins in the thylakoid membrane.

A new EPR signal was observed in oriented chloroplasts. This broad unresolved resonance displays a g value of 3.2 when the membrane normal is parallel to the field. It shifts to g = 1.9 when the membrane normal is perpendicular to the field. The signal is sensitive to illumination and to washing of the thylakoid membranes of broken chloroplasts. We suggest that there is a relation between this signal and the water-oxidizing enzyme system.