Cytochrome photooxidations in Chromatium chromatophores: Each P870 oxidizes two cytochrome C422 hemes

A single, 20-nsec actinic flash oxidizes all of the P870 in Chromatium chromatophores, but only about one half of the cytochrome C422. A second flash, 1–10 msec later, oxidizes most of the remaining cytochrome. The cytochromes which undergo oxidation on the first and second flashes are indistinguishable with respect to their absorption spectra, their kinetics of oxidation and reduction, and their response to N-methylphenazonium methosulfate (PMS) or continuous actinic illumination. The effect of PMS is to increase the total amount of cytochrome C422 which is in the reduced form in the dark, and which is available for photooxidation. The conclusion is that each P870 reaction center is responsible for the oxidation of two C422 hemes.     

The electrostatic interaction between the reaction-center bacteriochlorophyll derived from Rhodopseudomonas spheroides and mammalian cytochrome c and its effect on light-activated electron transport

1. By means of Q-switched ruby-laser flash excitation, the photooxidation of P870 in the reaction-center complex isolated from Rhodopseudomonas spheroides takes place within 1 μsec. The reduction of photooxidized P870 in the dark follows a first-order kinetics, with a pseudo first-order rate constant of 1.85×10⁸ l×mole^-1×sec^-1 and an activation energy of 6 kcal/mole.

2. Through an electrostatic interaction of the bacteriochlorophyll reaction-center complex and mammalian cytochrome c, an intimate contact between the two components resulted, and a collision-independent electron-transfer with a halftime of 25 μsec can be attained by laser-flash excitation. The absorbance changes at 870 and 550 nm indicated a good stoichiometry of the reaction. The oxidation of the c-type cytochrome in cells of Rps. spheroides (R-26 mutant) has a halftime of 12 μsec.

3. The portion of P870 which recovered rapidly was closely related to the mole ratio of cytochrome/P870. Complete recovery with a halftime of 25 μsec occurred when the cytochrome/P870 ratio was above approx. 10. At cytochrome/P870 ratios lower than 10, only the fraction of the reaction-center complex which have cytochromes bound at the active site can recover with the rapid decay time. Ultrafiltration measurements showed that each particle of the reaction-center complex can bind approx. 24 cytochrome molecules.

4. An electro static interaction is expected simply from the large difference between the isoelectric points of cytochrome c ( 10) and that of the reaction-center complex (4.1 measured by electro-focusing). The electro static interaction was further evidenced by the effects of pH, ionic strength, and by polylysine displacement of binding sites on the coupled oxidation of ferrocytochrome c by P870. From the limiting polylysine concentration giving complete blocking of cytochrome coupling, it was calculated that each reaction-center complex with a particle weight of 6.5×10⁵ contained approx. 500 negative charges.

5. Arrhenius plot of the first-order rate constants vs. the reciprocal absolute temperature yielded an activation energy of 12 kcal/mole for the cytochrome/P870 reaction, which is presumably the energy needed for cytochrome to achieve the most favorable orientation for the rapid electron transfer. Below the freezing temperature of the sample, the cytochrome reaction appeared to be uncoupled. The temperature dependence is consistent with the effect of viscosity on the reaction rate.

6. Double flash excitations spaced 200 μsec apart showed that at a cytochrome/P870 ratio of 24, the first flash caused maximum oxidation, indicating that all the reaction-center particles have at least one cytochrome attached to the active site. However, only 60% of the particles have a second cytochrome closely attached and capable of undergoing the rapid electron transport.     

Redistribution of electric charge accompanying photo-synthetic electron transport in Chromatium

The isoionic pH of Chromatium chromatophores is 5.2±0.1. At pH 7.7, the net charge on the chromatophore is approx. −1·10⁴. If a change in this charge accompanies the oxidation of an electron carrier, the midpoint redox potential (E_m) of that carrier should be a function of the solution ionic strength (I). of that carrier should be a function of the solution ionic strength (I).

The E_m values of P870 and cytochrome c-555 increase strongly with increasing I at low values of I. The E_m of cytochrome c-552 also increases with increasing I, though not so strongly. These effects probably cannot be attributed to an influence of I on the activity coefficient of a dissociable ion. We conclude that, when either P870 or cytochrome c-555 loses an electron, no specific ions (including protons) are bound or released in significant amounts, and the absolute value of the charge on the chromatophore decreases.

The E_m values of the primary and secondary electron acceptors, X and Y, do not depend on I. Because these E_m values have been shown previously to depend on pH, we conclude that the uptake of a proton keeps the charge on the chromatophore constant when either X or Y accepts an electron. This means that the primary and secondary electron transfer reactions in Chromatium result in a net decrease in the charge on the photosynthetic membrane. They do not result in the translocation of protons across the membrane.

The E_m of the soluble flavocytochrome c-552 from Chromatium depends only weakly on I, but depends strongly on the pH. The uptake of a proton appears to keep the net charge on this cytochrome constant upon reduction.     

Cytochrome b and photosynthetic sulfur bacteria

Chromatophores isolated from the purple sulfur bacterium Chromatium and the green sulfur bacterium Chlorobium exhibit absorbance changes in the cytochrome -band region consistent with the presence of a b-type cytochrome. Cytochrome content determined by reduced minus oxidized difference spectra and by heme analysis suggests that each bacterium contains one cytochrome b per molecule of photochemically active bacteriochlorophyll (reaction-center bacteriochlorophyll).

The b-type cytochrome in Chromatium has an -band maximum at 560 nm and a midpoint oxidation-reduction potential of −5 mV at pH 8.0. The b-type cytochrome in Chlorobium has an -band maximum at 564 nm and an apparent midpoint oxidation-reduction potential near −90 mV.

Chromatophores isolated from both Chromatium and Chlorobium cells catalyze a photoreduction of cytochrome b that is enhanced in the presence of antimycin A. Antimycin A and 2-n-heptyl-4-hydroxyquinoline-N-oxide inhibit endogenous (but not phenazine methosulfate-mediated) cyclic photophosphorylation in Chromatium chromatophores and non-cyclic electron flow from Na₂S to NADP in Chlorobium chromatophores. These observations suggest that b-type cytochromes may function in electron transport reactions in photosynthetic sulfur bacteria.     

Identification of ubiquinone as the secondary electron acceptor in the photosynthetic apparatus of Chromatium vinosum

Extracting Chromatium vinosum chromatophores with light petroleum destroys their ability to perform photochemistry on the second of two closely-spaced actinic flashes, without affecting photochemistry on the first flash. Extraction also increases the likelihood of a back-reaction in which an electron returns from the primary electron acceptor directly to P₈₇₀. These effects probably reflect the removal of a secondary electron acceptor. Extraction does not appear to interfere with the primary photochemical reaction. Reconstituting the extracted chromatophores with the lipid extract or with pure ubiquinone (Q) completely reverses the effects of the extraction. Chromatography of the lipid extract shows that Q is the only active material that it contains in detectable quantity. These observations support the conclusion that Q is the secondary electron acceptor.

Piericidin A, certain alkyl-substituted quinolinequinones, and a substituted 4,7-dioxobenzothiazole inhibit electron transfer between the primary and secondary acceptors. The sensitivity to these inhibitors, and the participation of Q and non-heme iron suggest that the secondary electron-transfer reaction resembles the reactions catalyzed by respiratory dehydrogenases.

The proton uptake that follows flash excitation does not seem to be tightly linked to the reduction of the secondary electron acceptor. It still occurs (though with decreased amplitude) in extracted chromatophores, and even in the presence of inhibitors of the secondary electron-transfer reaction.     

Shifts of bacteriochlorophyll and carotenoid absorption bands linked to cytochrome c-555 photooxidation in Chromatium

Shifts in the absorption bands of bacteriochlorophyll and carotenoids in Chromatium vinosum chromatophores were measured after short actinic flashes, under various conditions. The amplitude of the bacteriochlorophyll band shift correlated well with the amount of cytochrome c-555 that was oxidized by P₈₇₀⁺ after a flash. No bacteriochlorophyll band shift appeared to accompany the photooxidation of P₈₇₀ itself, nor the oxidation of cytochrome c-552 by P₈₇₀⁺. The carotenoid band shift also correlated with cytochrome c-555 photooxidation, although a comparatively small carotenoid shift did occur at high redox potentials that permitted only P₈₇₀ oxidation.

The results explain earlier observations on infrared absorbance changes that had suggested the existence of two different photochemical systems in Chromatium. A single photochemical system accounts for all of the absorbance changes.

Previous work has shown that the photooxidations of P₈₇₀ and cytochrome c-555 cause similar changes in the electrical charge on the chromatophore membrane. The specific association of the band shifts with cytochrome c-555 photooxidation therefore argues against interpretations of the band shifts based on a light-induced membrane potential.     

Fluorescence of bacteriochlorophyll as related to the photochemistry of chromatophores of photosynthetic bacteria

Time courses and the emission spectra of fluorescence and light-induced absorption changes of P890 in chromatophores of the photosynthetic bacteria Chromatium D, Rhodopseudomonas spheroides and Rhodospirillum rubrum were investigated.

The time course of fluorescence in chromatophores was separated into two phases, i.e. an initial rapid rise (ƒ_i) and a subsequent slow increase towards a steady level of emission (ƒ_v). The ƒ_i and the ƒ_v components showed different emission spectra having different peak position. The ƒ_v component was emitted from the longest wavelength-absorbing form of bulk bacteriochlorophyll (B890), the ƒ_i component from both B890 and B850.

The magnitude of the ƒ_v component depended on experimental conditions controlling the states of the cyclic electron transport in chromatophores, including changes in levels of redox potential of the medium, additions of electron donors and inhibitors. The magnitude of the ƒ_i component was not affected by these experimental conditions. It was, therefore, concluded that only the ƒ_v component is related to the cyclic electron transport, and that the magnitude of ƒ_v is controlled by the oxidation-reduction state of the primary electron acceptor for the photochemical reaction center in chromatophores.     

Laser-induced reactions of P700 and cytochrome f in a blue-green alga, Plectonema boryanum

Kinetics of the absorption change of P700 (blue band) and cytochrome f in whole cells of a blue-green alga, Plectonema boryanum, have been studied by Q-switched ruby-laser flash excitation (694 nm; approx. 20 nsec) to elucidate the sequential relationship of these two components in photosynthetic electron transport. “P700” was photooxidized within 2 μsec and recovered in two phases t_1/2 10 μsec and 200 μsec). Under the same conditions cytochrome f was oxidized with a half time of 15 μsec. The magnitude of the fast phase of “P700” recovery, however, diminished at lower laser intensity while the cytochrome f change remained unaffected. The result suggests that cytochrome f and P700 may not be on the same electron-transport chain.     

Nature of photochemical reactions in chromatophores of Chromatium D. II. Quantum yield of photooxidation of cytochromes in Chromatium chromatophores

Initial rates of the light-induced absorption decrease in Chromatium chromatophores due to the oxidation of cytochromes were measured under various experimental conditions. The initial rate in the presence of 10 mM potassium ferrocyanide and 50 μM potassium ferricyanide was about one-half to two-thirds of that in the presence of 30 mM ascorbate or in a medium with a redox potential (E_h) of − 78 mV.

Light-minus-dark difference spectrum indicated that, in the presence of 10 mM ferrocyanide and 50 μM ferricyanide, only cytochrome c-555 was photooxidized. In the presence of 30 mM ascorbate or at E_h values lower than about 0 mV, both cytochrome c-555 and cytochrome c-552 were photooxidized. The quantum yield of cytochrome c-555 photooxidation was calculated to be about 0.4.

The results obtained in the present study are compared with other investigators' and the possibility of the presence of two types of associations between the cytochromes and reaction-center bacteriochlorophyll is discussed.     

Kinetics of the generation of a photo-induced electric potential in chromatophores of photosynthetizing bacteria

Flash-induced formation of an electric potential difference (delta psi) was monitored by a direct method in chromatophores associated with the collodion phospholipid membrane. In Rhodospirillum rubrum and Rhodopseudomonas sphaeriodes chromatophores, the kinetics of delta psi generation exhibit fast (tau less than or equal to 0.3 microseconds) and slow (tau congruent to 200 microseconds) phases, the latter observed in the presence of exogenous quinones. Comparison of the kinetic and potentiometric characteristics of the process with those of electron transport reactions suggests that the fast phase of delta psi rise is due to charge separation between the primary electron donor, P870, and primary electron acceptor QIFe; the slow phase, which is inhibited by o-phenanthroline, is due to electron donation from QIFe to the secondary acceptor, quinone QII. The kinetics of delta psi decay include components arising form the recombination of primary separated charges (tau congruent to 30 ms) and from the passive discharge of the membrane (tau congruent to 400 ms; tau congruent to 1400 ms). From a redox titration of the photo-induced electric signal and the photo-induced absorption changes of P870 at different pH meanings, the value of pK for the primary acceptor FeQI was found to be 7.4 in Rps. sphaeroides chromatophores. In Chromatium minutissimum, a phase ( tau congruent to 20 microseconds) was observed in addition to those seen in Rps. sphaeroids and R. rubrum which was explained by the reduction of P890+ from the high potential cytochrome c555. Possible distribution of the electron transport components in the chromatophore membrane are discussed.     

The effect of pH on the reduction kinetics of P-680 in tris-treated chloroplasts

The primary donor of Photosystem II (PS II), P-680, was photo-oxidized by a short flash and its rate of reduction was measured at different pH values by following the recovery of the absorption change at 820 nm in chloroplasts pretreated with a high concentration of Tris. The re-reduction is biphasic with a fast phase (dominant after the first flash) attributed to the donation by a donor, D₁, and a slow phase (usually dominant after the second flash) attributed to a back-reaction with the primary acceptor.

It is found that pH has a strong influence on the donation from D₁ (τ = 2 μs at pH 9, 44 μs at pH 4), but no influence on the back reaction (τ ≈ 200 μs). pH also influences the stability of the charge separation since the contribution of donation from D₁ at the second flash increases at lower pH, getting close to 100% at pH 4.     

Photo-induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans.

Flash-induced redox changes of b-type and c-type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox-controlled conditions. The flash-oxidized primary donor P+ of the reaction center (RC) is rapidly re-reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low-potential hemes L1/L2 (Em,7 = 90 mV) of the RC-bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash-induced low-potential heme oxidation, a midpoint potential equal to -50 mV has been determined for the primary quinone acceptor QA. Only the photo-oxidized heme H2 is re-reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC-bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash-induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re-oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone-10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low-potential heme(s) rapidly re-reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q-cycle-type model.     

Spectral and photochemical properties of subchromatophore fractions derived from carotenoid-deficient Chromatium by Triton treatment

Triton treatment of chromatophores of carotenoid-deficient Chromatium followed by density-gradient centrifugation led to a separation into three subchromatophore fractions. Unlike the case with chromatophores of regular Chromatium, Triton releases about 1/3 of the total bulk bacteriochlorophyll into one fraction (designated G, for green) whose major absorption-band maximum is at 780 nm. One fraction (H, for heavy) absorbs at 805 and 885 nm, with an absorbance ratio A₈₈₅ nm/A₈₀₅ nm between 1.5 and 2; another fraction (L, for light) absorbs at 805 nm and has a shoulder at 825 nm. The absorption and fluorescence emission spectra of the three fractions at room temperature and 77°K indicate that the different bacteriochlorophyll forms are efficiently separated by Triton treatment.

The reaction center P890 is concentrated exclusively in the H-fraction, at a level of 5–7% of the bulk bacteriochlorophyll. The solubilized bacteriochlorophyll absorbing at 780 nm can be totally and irreversibly bleached by 5 mM ferricyanide. The other bacteriochlorophyll forms in the H- and L-fractions are also irreversibly bleached by ferricyanide to variable extents. P890 is the only component that can be re-reduced by ascorbate after ferricyanide oxidation. The P890 content estimated by reversible chemical bleaching agrees well with that obtained by reversible light bleaching. The different bacteriochlorophyll forms, with the exception of the 780-nm absorbing form, are relatively stable toward light bleaching. Again, only P890 is reversibly bleached by light.

Cytochromes-555 and -553 are distributed in both the H-and L-fractions, but not in the solubilized-bacteriochlorophyll G-fraction. However, only cytochromes in the H-fraction which contains all of the P890 can undergo coupled oxidation. Excitation with 20-nsec ruby-laser pulses shows that cytochrome-555 can be oxidized in 2–3 μsec by photooxidized P890, indicating that necessary conformation for rapid electron transport is retained in the subchromatophore particles.

The data on fractionation and redox reactions obtained here, together with direct kinetic measurements recently reported in the literature lend further support to the view that oxidation of these two cytochromes is mediated by the same reaction center, P890.     

Kinetic properties of the acceptor quinone complex in Rhodopseudomonas viridis

The kinetics of electron transfer from the primary (QA) to the secondary (QB) quinone acceptor in whole cells and chromatophores of Rhodopseudomonas viridis was studied as a function of the redox state of QB and of pH by using a photovoltage technique. Under conditions where QB was oxidized, the reoxidation of QA- was found to be essentially monophasic and independent of pH with a half-time of about 20 microseconds. When QB was reduced to the semiquinone form by a preflash, the reoxidation of QA- was slowed down showing a half-time between 40 and 80 microseconds at pH less than or equal to 9. Above pH 9, the rate of the second electron transfer decreased nearly one order of magnitude per pH unit. After a further preflash, the fast and pH-independent kinetics of QA- reoxidation was essentially restored. The concentration of QA still reduced 100 microseconds after its complete reduction by a flash showed distinct binary oscillations as a function of the number of preflashes, confirming the interpretation that the electron-transfer rate depends on the redox state of QB. After addition of o-phenanthroline, the reoxidation of QA- is slowed down to the time range of seconds as expected for a back-reaction with oxidized cytochrome. Under conditions where inhibitors of the electron transfer between the quinones fail to block this reaction in a fraction of the reaction centers due to the presence of the extremely stable and strongly bound semiquinone, QB-, these reaction centers show a slow electron transfer on the first flash and a fast one on the second, i.e., an out-of-phase oscillation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Flash-induced absorption changes in Photosystem I,Radical pair or triplet state formation?

Absorption changes induced in chlorophyll protein (CP 1) particles by short laser flashes have been analyzed in order to decide whether a state lasting for a few microseconds at 21°C or 800 μs at 10 K corresponds to the biradical P-700⁺ ... A⁻₁ (A₁ being a chlorophyll a) or to a triplet state produced in a submicrosecond recombination of the preceding state. At 21°C the spectrum of the flash-induced ΔA (720–870 nm) presents a flat-topped band from 740 to 820 nm, clearly different from that of P-700⁺. A saturation curve (ΔA vs. laser energy), obtained with a 2 or 10 ns laser pulse, indicates that ΔA saturates at a value 2- or 3-times smaller than that expected on the basis of the chemical oxidation of P-700. At 21°C the size of flash-induced ΔA is slightly decreased (5–15%) when the sample is subjected to a 400 G magnetic field. The kinetics of decay are not affected; they are not affected either by the oxygen concentration. At 10 K the spectrum of the flash-induced ΔA has been measured between 650 and 1700 nm. Between 650 and 720 nm, the spectrum presents only one major negative peak at 702 nm; it is quite different from that due to the chemical oxidation of P-700 (which has additional peaks at 688 and 677 nm). Between 720 and 870 nm, the spectrum is identical to that obtained at 21°C. Above 870 nm, the spectrum includes a broad band around 1250 nm, which is absent in P-700⁺. A saturation curve leads to a maximum ΔA greater than that at 21°C and which is also greater with a 1 μs dye laser flash than with a 10 ns ruby laser flash. An analysis of the spectral data indicates that these do not fit correctly with the hypothesis of a contribution of P-700⁺ and of a chlorophyll a anion radical. They fit more closely with the hypothesis of a triplet state of P-700, a hypothesis which is discussed in relation to other experimental data.     

Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides. I. Stimulation by secondary electron transport at low temperature

Light-induced absorbance changes were measured at temperatures between --30 and --55 degrees C in chromatophores of Rhodopseudomonas sphaeroides. Absorbance changes due to photooxidation of reaction center bacteriochlorophyll (P-870) were accompanied by a red shift of the absorption bands of a carotenoid. The red shift was inhibited by gramicidin D. The kinetics of P-870 indicated electron transport from the "primary" to a secondary electron acceptor. This electron transport was slowed down by lowering the temperature or increasing the pH of the suspension. Electron transport from soluble cytochrome c to P-870+ occurred in less purified chromatophore preparations. This electron transport was accompanied by a relatively large increase of the carotenoid absorbance change. This agrees with the hypothesis that P-870 is located inside the membrane, so that an additional membrane potential is generated upon transfer of an electron from cytochrome to P-870+. A strong stimulation of the carotenoid changes (more than 10-fold in some experiments) and pronounced band shifts of bacteriochlorophyll B-850 were observed upon illumination in the presence of artifical donor-acceptor systems. Reduced N-methylphenazonium methosulphate (PMS) and N,N,N',N'-tetramethyl-p-phenylene-diamine (TMPD) were fairly efficient donors, whereas endogenous ubiquinone and oxidized PMS acted as secondary acceptor. These results indicate the generation of large membrane potentials at low temperature, caused by sustained electron transport across the chromatophore membrane. The artificial probe, merocyanine MC-V did not show electrochromic band shifts at low temperature.     

Kinetics of the reduction and oxidation of cytochromes b6 and ƒ in isolated chloroplasts

Absorbance changes induced by flash illumination of a chloroplast suspension were monitored kinetically at selected wavelengths between 510 and 575 nm. Digital subtraction of the P518 component from the total transient signal permitted isolation of the responses due to cytochromes ƒ and b₆. The half rise time for cytochrome b₆ reduction (1.3±0.1 ms) was much greater than a previously reported value (< 10 μs); the half time for cytochrome b₆ oxidation was 35±4 ms. Cytochrome ƒ was oxidized with a half time of about 0.22 ms and the subsequent reduction occurred in two phases with half times of about 7.3 and 83 ms. These kinetic data show that cytochrome b₆ cannot be a primary electron acceptor in Photosystem 1. The rate of oxidation of cytochrome b₆ is consonant with this cytochrome being the source of electrons for the slower phase of cytochrome ƒ reduction.     

A kinetic completion of the cyclic photosynthetic electron pathway of Rhodopseudomonas sphaeroides: Cytochrome b-cytochrome c2 oxidation-reduction

In Rhodopseudomonas sphaeroides, following a single-turnover flash of light, cytochrome c₂ is oxidized by reaction center bacteriochlorophyll, and a cytochrome b is reduced by the primary electron acceptor, probably via ubiquinone. In this report we show that, in the uncoupled state, the rate of re-oxidation of the cytochrome b is identical to the rate of reduction of the cytochrome c₂, a kinetic completion of the cyclic photosynthetic electron transport system.     

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.