Flash-induced photophosphorylation in Rhodospirillum rubrum chromatophores I. The relationship between cytochrome c-420 content and photophosphorylation

The content of cytochrome c-420 in Rhodospirillum rubrum chromatophores prepared by grinding with alumina is 5–10% of that in whole cells, and 20–40% in chromatophores by ‘French’ pressing.Flash-induced phosphorylation of various chromatophores which varied in cytochrome content from 7 to 40% is proportional to the cytochrome content. Extrapolating the cytochrome c-420 content to that observed in whole cells, a ratio $\text{ATP}\text{P}{}^{\mathrm{+}}\text{X}{}^{\mathrm{?}}$ near 1 is calculated. At low flash intensity the phosphorylation per flash is proportional to flash energy.Photophosphorylation in flashes given after a time of several minutes is only slightly dependent on the number of flashes. If the flashes are spaced from 0.1 to 10 s, relative phosphorylation in the first flash is about 70% and in the second 90% of that observed in the following flashes. Proton binding is not affected by the cytochrome c-420 content and a ratio of $\text{H}{}^{\mathrm{+}}\text{P}{}^{\mathrm{+}}\text{X}{}^{\mathrm{?}}$ of 2.3 was found.These results can be explained by a working hypothesis in which charge separation occurring at one reaction centre and the resulting electron transport mediated amongst others by c-420, results in the injection of two protons into an ATPase, this in contrast to a chemiosmotic mechanism, where the protons are released in the chromatophore inner space.     

Function of three cytochromes in photosynthesis of whole cells of Rhodospirillum rubrum as studied by flash spectroscopy. Evidence for two types of reaction center.

1. Changes in the absorption spectrum induced by 10-mus flashes and continuous light of various intensities were studied in whole cells of Rhodospirillum rubrum in the presence and absence of 2-n-heptyl-4-hydroxyquinoline-N-oxide(HOQNO) and antimycin A. 2. Three cytochromes, c-420 (cytochrome c2), c-560 (cytochrome b) and c-428 were photoactive and gamma and alpha peaks at 420 and 550, 428 and 560, and 428 and 551 nm, respectively; they were photooxidized following the flash with half times of 0.3, 0.6 and 7 ms in the approximate ratios of 1/100, 1/300 and 1/1000 (cytochrome oxidized/antenna chlorophyll) and became reduced with half times of 12 ms, 60 ms and 0.7 s, respectively. c-428 and c-560 have not been distinguished before. 3. From a detailed analysis of the kinetics of P+ (oxidized reaction center chlorophyll) and the cytochromes, we conclude that 5% of the P+ (P2+) oxidizes c-428, whereas the remaining 95% of P+ (P1+) oxidizes c-420. At actinic light intensities low enough to keep c-420 fully reduced, approx. 4-5% of P becomes oxidized, accompanied by all c-428. The P2+ -P2 difference spectrum induced by this weak light is, when corrected for a shift to longer wavelengths of the bacteriochlorophyll absorption band at 878 nm, identical to the difference spectrum caused by the photooxidation of the remaining P1. At low flash intensity, c-428 becomes preferentially photooxidized, which suggests that the reaction centers where c-428 functions as a secondary donor contain much more antenna pigments compared to the centers where c-420 serves this purpose. 4. c+-420 is reduced in a competitive way by reduced c-560 (t 1/2=7 ms), and by an electron donor pool, (t 1/2=15 ms). HOQNO inhibits both pathways; antimycin A only the first. In the presence of HOQNO, c-560 is in the oxidized state in the dark, and is reduced in a light flash (t 1/2=100 ms), indicating that c-560 acts in a cyclic electron transport chain connected to P1. 5. The ratio of numbers of molecules P1 and antenna bacteriochlorophyll, transferring excitation energy to P1, is P1/bacteriochlorophyll1=1/30 P2: bacteriochlorophyll2=1/300; c-420/P1=1:2; c-560/P1=1/6; C-428/P2=1/1; bacteriochlorophyll2=7:3. If P2 is oxidized, excitation energy is transferred from bacteriochlorophyll2 to bacteriochlorophyll1.     

The mechanism of reduction of the ubiquinone pool in photosynthetic bacteria at different redox potentials.

(1) A flash number dependency of flash-induced absorbance changes was observed with whole cells of Rhodospirillum rubrum and chromatophores of R. rubrum and Rhodopseudomonas sphaeroides wild type and the G1C mutant. The oscillatory behavior was dependent on the redox potential; it was observed under oxidizing conditions only. Absorbance difference spectra measured after each flash in the 275--500 nm wavelength region showed that a molecule of ubiquinone, R, is reduced to the semiquinone (R-) after odd-numbered flashes and reoxidized after even-numbered flashes. The amount of R reduced was approximately one molecule per reaction center. (2) The flash number dependency of the electrochromic shift of the carotenoid spectrum was studied with chromatophores of Rps. sphaeroides wild type and the G1C mutant. At higher values of the ambient redox potential a relatively slow phase with a rise time of 30 ms was observed after even-numbered flashes, in addition to the fast phase (completed within 0.2 ms) occurring after each flash. Evidence was obtained that the slow phase represents the formation of an additional membrane potential during a dark reaction that occurs after flashes with an even number. This reaction is inhibited by antimycin A, whereas the oscillations of the R/R- absorbance changes remain unaffected. At low potentials (E = 100 mV) no oscillations of the carotenoid shift were observed: a fast phase was followed by a slow phase (antimycin-sensitive) with a half-time of 3 ms after each flash. (3) The results are discussed in terms of a model for the cyclic electron flow as described by Prince and Dutton (Prince, R.C. and Dutton, P.L. (1976) Bacterial Photosynthesis Conference, Brussels, Belgium, September 6--9, Abstr. TB4) employing the so-called Q-cycle.     

ATP-synthase of Rhodobacter capsulatus: coupling of proton flow through F0 to reactions in F1 under the ATP synthesis and slip conditions

A stepwise increasing membrane potential was generated in chromatophores of the phototrophic bacterium Rhodobacter capsulatus by illumination with short flashes of light. Proton transfer through ATP-synthase (measured by electrochromic carotenoid bandshift and by pH-indicators) and ATP release (measured by luminescence of luciferin-luciferase) were monitored. The ratio between the amount of protons translocated by F0F1 and the ATP yield decreased with the flash number from an apparent value of 13 after the first flash to about 5 when averaged over three flashes. In the absence of ADP, protons slipped through F0F1. The proton transfer through F0F1 after the first flash contained two kinetic components, of about 6 ms and 20 ms both under the ATP synthesis conditions and under slip. The slower component of proton transfer was substantially suppressed in the absence of ADP. We attribute our observations to the mechanism of energy storage in the ATP-synthase needed to couple the transfer of four protons with the synthesis of one molecule of ATP. Most probably, the transfer of initial protons of each tetrad creates a strain in the enzyme that slows the translocation of the following protons.     

The influence of energy-transfer inhibitors on proton permeability and photophosphorylation in normal and preilluminated Rhodospirillum rubrum chromatophores.

(1) Chromatophores were preilluminated in the presence of phenazine methosulphate or diaminodurene, and without phosphorylation substrates; next they were transferred to fresh medium and assayed for light-induced proton uptake, light-induced 9-aminoacridin fluorescence quenching, and photophosphorylation. (2) Preillumination in the presence of phenazine methosulphate or diaminodurene causes an inhibition of the photophosphorylation rate. The presence of ADP + MgCl2 + phosphate, or ADP + MgCl2 + arsenate during preillumination provides full protection against this effect. (3) Preilluminated chromatophores are leaky for protons. The leak is expressed as an accelerated dark decay, and a diminished extent of succinate-supported, light-induced proton uptake. The extent of light-induced 9-aminoacridin fluorescence quenching is also diminished. (4) The proton leak can be closed by oligomycin and by dicyclohexyl carbodiimide (at concentrations similar to those used to inhibit photophosphorylation), but not by aurovertin. Closure of the proton leak results in partial restoration of the photophosphorylation rate. (5) The inhibition of phosphorylation by oligomycin or dicyclohexyl carbodiimide is time-dependent. In untreated chromatophores, the time-dependence is determined by the extent of membrane energization. In preilluminated chromatophores, the time-dependence is determined in addition by the extent to which the proton leaks have been closed. The reasons for this are briefly discussed.     

The extent of the stimulated electrical potential decay under phosphorylating conditions and the H+/ATP ratio in Rhodopseudomonas sphaeroides chromatophores following short flash excitation.

1. In chromatophores from Rps. sphaeroides, the stimulation by ADP and Pi of the electric potential decay indicated by the carotenoid shift is greater than the stimulation of the decay of pH change indicated by the colour change of added cresol red under similar conditions. This difference is attributed to H+ consumption during the synthesis of ATP. The ratio of H+ translocated across the membrane to ATP synthesized was estimated to be approximately 1.7 H+/ATP. 2. The stimulation of the electrical potential decay by ADP and Pi was found to be a constant fraction (10%) of the total decay when the flash intensity was varied. No 'critical' or 'threshold' potential was observed. 3. The stimulated electrical potential decay after a second flash, given within a few seconds of the first, was related to the amplitude of the electrical potential produced by the second flash (10%) but neither to the dark time between the flashes, nor to the total extent of the electrical potential above the dark level. These results are consistent with two hypotheses (a) the chromatophores are a mixed population of vesicles, only a small fraction (10%) of which possess an active ATP synthesizing system (b) the activity of the ATP synthesizing system, though driven by a proton motive force, is controlled by electron transport processess. If alternative (a) is correct then the overall single turnover flash yield of 1 ATP per 1470 bacteriochlorophyll measured in (1) would mean that the yield of the active vesicles is approximately 10 ATP per 1470 bacteriochlorophyll or 30 ATP per vesicle. 4. The stimulation of the electrical potential decay by ADP and Pi is approximately 40% less in antimycin-treated chromatophores. It is shown that this is probably a consequence of antimycin-inhibited H+-release on the inside of the chromatophore vesicles following a flash.     

Effects of pH on reactions on the donor side of photosystem II.

The effects of pH on the increase of fluorescence yield measured in the microsecond range, and on the microsecond delayed fluorescence have been studied in dark adapted chloroplasts as a function of flash number. (1) At pH 7, the amplitude of the fast-phase of the microsecond fluorescence yield rise oscillated as a function of flash number with period 4 and with maxima on flashes 1 and 5, and minima on flashes 3 and 7. The damped oscillations were apparent over the range between 6 and 8, although the absolute amplitude of the fast phase was diminished at the lower end of the range. At pH 4, there was no fast phase in the rise and, at pH 9, an enhanced fast-phase occurred only for the first flash. (2) The decay of microsecond delayed fluorescence was described by the sum of exponentials with half-times of 10--15 mus and 40--50 mus. Over the pH range 6- less than 8, the extrapolated initial amplitude and the proportion of the change due to the faster component showed oscillations which were opposite in phase to those observed for the prompt fluorescence yield rise; the slower component showed weaker oscillations of the same phase. At pH 4, there were no oscillations and the slow phase predominated. At pH 9, the delayed fluorescence intensity was diminished on the first flash, and high on subsequent flashes. (3) The results are interpreted in terms of a model in which protons are released during all transitions of the S-states with the exception of S1 leads to S2, and in which ther are two sites of inhibition on the donor side of the photo-system at extreme pH values. At pH 4, electron donation to P+ occurs with a half-time approx. 135 mus, either by a back reaction from Q-, or from D; electron transport is interrupted between Z1 and P. At pH 9, electron transport is inhibited between Z1 and Z2; rapid re-reduction of P+ by Z1 occurs after 1 flash, and on subsequent flashes electrons from D, an alternative donor reduce P+. The location of the positive charge on states S2 and S3 is discussed.     

A simple, quantitative approach to the coupling of photophosphorylation to electron flow in terms of proton fluxes.

A simple relationship between observed phosphorylation efficiencies (P/e ratios) and internal proton concentration in spinach chloroplast thylakoids has been derived. P/e ratios, varked by either changing the light intensity or by adding the energy transfer inhibitor, 4'-deoxyphlorizin, were found to change with internal proton concentration in accordance with this relationship. A quantitative prediction of the effect of uncouplers on the P/e ratio can probably also be made. By extrapolation of plots of observed P/e ratios against internal proton concentration divided by the overall rate of electron flow, a maximum intrinsic P/e of about 0.66 is obtained. Assuming that two protons appear inside thylakoids per electron transferred, a P/e ratio of 0.66 suggests that three internal protons are consumed for each ATP formed. Internal protons may be considered to be substrates for the phosphorylation reaction. Hill plots of phosphorylation rate vs. internal proton concentration also indicate that three protons are consumed for each ATP synthesized. Thus, the H+ concentration gradient behaves quantitatively, as well as qualitatively, as if it is the connecting link between electron flow and phosphorylation in illuminated thylakoids.     

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.     

Delayed fluorescence from Rhodopseudomonas sphaeroides following single flashes.

Delayed fluorescence from Rhodopseudomonas sphaeroides chromatophores was studied with the use of short flashes for excitation. Although the delayed fluorescence probably arises from a back-reaction between the oxidized reaction center bacteriochlorophyll complex (P+) and the reduced electron acceptor (X-), the decay of delayed fluorescence after a flash is much faster (tau1/2 approximately 120 mus) than the decay of P+X-. The rapid decay of delayed fluorescence is not due to the uptake of a proton from the solution, nor to a change in membrane potential. It correlates with small optical absorbance changes at 450 and 770 nm which could reflect a change in the state of X-. The intensity of the delayed fluorescence is 11-18-fold greater if the excitation flashes are spaced 2 s apart than it is if they are 30 s apart. The enhancement of delayed fluorescence at high flash repetition rates occurs only at redox potentials which are low enough (less than +240 mV) so that electron donors are available to reduce P+X- to PX- in part of the reaction center population. The enhancement decays between flashes as PX- is reoxidized to PX, as measured by the recovery of photochemical activity. Evidently, the reduction of P+X- to PX- leads to the storage of free energy that can be used on a subsequent flash to promote delayed fluorescence. The reduction of P+X- also is associated with a carotenoid spectral shift which decays as PX- is reoxidized to PX. Although this suggests that the free energy which supports the delayed fluorescence might be stored as a membrane potential, the ionophore gramicidin D only partially inhibits the enhancement of delayed fluorescence. With widely separated flashes, gramicidin has no effect on delayed fluorescence. At redox potentials low enough to keep X fully reduced, delayed fluorescence of the type described above does not occur, but one can detect weak luminescence which probably is due to phosphorescence of a protoporphyrin.     

The influence of energy-transfer inhibitors on proton permeability and photophosphorylation in normal and preilluminated Rhodospirillum rubrum chromatophores

(1) Chromatophores were preilluminated in the presence of phenazine methosulphate or diaminodurene, and without phosphorylation substrates; next they were transferred to fresh medium and assayed for light-induced proton uptake, light-induced 9-aminoacridin fluorescence quenching, and photophosphorylation.(2) Preillumination in the presence of phenazine methosulphate or diaminodurene causes an inhibition of the photophosphorylation rate. The presence of ADP + MgCl₂ + phosphate, or ADP + MgCl₂ + arsenate during preillumination provides full protection against this effect.(3) Preilluminated chromatophores are leaky for protons. The leak is expressed as an accelerated dark decay, and a diminished extent of succinate-supported, light-induced proton uptake. The extent of light-induced 9-aminoacridin fluorescence quenching is also diminished.(4) The proton leak can be closed by oligomycin and by dicyclohexyl carbodiimide (at concentrations similar to those used to inhibit photophosphorylation), but not by aurovertin. Closure of the proton leak results in partial restoration of the photophosphorylation rate.(5) The inhibition of phosphorylation by oligomycin or dicyclohexyl carbodiimide is time-dependent. In untreated chromatophores, the time-dependence is determined by the extent of membrane energization. In preilluminated chromatophores, the time-dependence is determined in addition by the extent to which the proton leaks have been closed. The reasons for this are briefly discussed.     

Proton translocation in chloroplasts and its relationship to electron transport between the photosystems.

Using dark adapted isolated spinach chloroplasts and sequences of brief saturating flashes the correlation of the uptake and release of protons with electron transport from Photosystem II to Photosystem I were studied. The following observations and conclusions are reported: (1) Flash-induced proton uptake shows a weak, damped binary oscillation, with maxima occurring after the 2nd, 4th, etc. flashes. The damping factor is comparable to that observed in the O2 flash yield oscillation and therefore explained by misses in Photosystem II. (2) On the average and after a steady state is reached, each flash (i.e. each reduction of Q) induces the uptake of 2H+ from outside the chloroplasts. (3) Flash induced proton release inside the chloroplast membrane shows a strong damped binary oscillation with maximum release occurring also after the 2nd, 4th, etc. flashes. (4) This phenomenon is correlated with the earlier reported binary oscillations of electron transport [2] and shows that both electrons and protons are transported in pairs between the photosystems. (5) In two sequential flashes 4H+ from the outside of the thylakoid and 2e- from water are accumulated at a binding site B. Subsequently, the two electrons are transferred to non-protonated acceptors in Photosystem I (probably plastocyanin and cytochrome f) and the 4H+ are released inside the thylakoid. (6) It is concluded that a primary proton transporting site and/or energy conserving step located between the photosystems is being observed.     

The effect of glutaraldehyde fixation on the primary photochemical processes in bacterial photosynthesis

In Chromatium D the half-time for laser-induced (20–30-nsec flash) cytochrome C553 oxidation in redox poised chromatophores (1 μsec) and cytochrome C555 oxidation in whole cells (2.5μsec) is not affected by glutaraldehyde fixation. The reduction half-times for both cytochromes, however, increase as different functions of the glutaraldehyde concentration during the whole cell fixation process. At a cell-fixing concentration of 0.8%, cytochrome C555 but not C553 is observed after a laser flash. Steady light-induced spectra using similar preparations suggest the possibility of four components observable in the 500–620-nm range. These are cytochrome C555, P600, a species peaking at 560 nm and a component displaying a light-induced blue shift in the 500–540-nm region which may be a carotenoid response. The wavelength expected for the α-peak (reduced-minus-oxidized) of cytochrome cc′ is 560 nm, but the lack of a corresponding Soret peak makes identification uncertain and raises the possibility that we are observing a totally new component. Comparison of the amount of cytochrome oxidized by steady illumination and by a laser flash shows that on the average there are three cytochrome C555 molecules per reaction center in both whole cells and chromatophores. If the glutaraldehyde acts directly on the reaction center cytochromes then it is clear that cytochrome reduction requires large amplitude motion, but that oxidation does not. However, glutaraldehyde fixation may simply block the path of reducing electrons before they reach reaction center bound cytochromes.     

The induction kinetics of bacterial photophosphorylation. Threshold effects by the phosphate potential and correlation with the amplitude of the carotenoid absorption band shift

1. ATP synthesis (monitored by luciferin-luciferase) can be elicited by a single turnover flash of saturating intensity in chromatophores from Rhodopseudomonas capsulata, Kb1. The ATP yield from the first to the fourth turnover is strongly influenced by the phosphate potential: at high phosphate potential (?11.5 kcal/mol) no ATP is formed in the first three turnovers while at lower phosphate potential (?8.2 kcal/mol) the yield in the first flash is already one half of the maximum, which is reached after 2–3 turnovers.2. The response to ionophores indicates that the driving force for ATP synthesis in the first 20 turnovers is mainly given by a membrane potential. The amplitude of the carotenoid band shift shows that during a train of flashes an increasing ΔΨ is built up, which reaches a stationary level after a few turnovers; at high phosphate potential, therefore, more turnovers of the same photosynthetic unit are required to overcome an energetic threshold.3. After several (six to seven) flashes the ATP yield becomes constant, independently from the phosphate potential; the yield varies, however, as a function of dark time (t_d) between flashes, with an optimum for t_d = 160–320 ms.4. The decay kinetics of the high energy state generated by a long (125 ms) flash have been studied directly measuring the ATP yield produced in post-illumination by one single turnover flash, under conditions of phosphate potential (?10 kcal/mol), which will not allow ATP formation by one single turnover. The high energy state decays within 20 s after the illumination. The decay rate is strongly accelerated by 10^?8 M valinomycin.5. Under all the experimental conditions described, the amplitude of the carotenoid signal correlates univocally with the ATP yield per flash, demonstrating that this signal monitores accurately an energetic state of the membrane directly involved in ATP synthesis.6. Although values of the carotenoid signal much larger than the minimal threshold are present, relax slowly, and contribute to the energy input for phosphorylation, no ATP is formed unless electron flow is induced by a single turnover flash.7. The conclusions drawn are independent from the assumption that a ΔΨ between bulk phases is evaluable from the carotenoid signal.     

Kinetic factors limiting the synthesis of ATP by chromatophores exposed to short flash excitation.

ATP synthesis was measured after chromatophores from Rhodopseudomonas capsulata had been subjected to illumination by single turnover flashes fired at variable frequencies. Three processes were examined, which under different conditions can limit the net yield of ATP. (1) A process with an apparent relaxation time of 10-20 ms. This reaction probably limits the rate of ATP synthesis in continuous illumination. It has similar time dependence to the stimulation of the carotenoid shift decay by ADP after a single flash. (2) An active state of the ATPase only persists when the chromatophores are excited more often than once in 10 s. This state decays with similar kinetics to the entire carotenoid shift decay. Full activation is achieved after two flashes. (1) and (2) are not significantly affected by concentrations of antimycin A sufficient to block electron flow through the cytochrome b/c2 oxidoreductase and abolish phase III in the generation of the carotenoid shift. (3) In the presence of antimycin A, after the third, fourth and subsequent flashes ATP synthesis is limited by the quantity of reducing equivalents transported through the reaction centre rather than by the level of the electrochemical proton gradient.     

Electron transfer kinetics in purified reaction centers from the green sulfur bacterium Chlorobium tepidum studied by multiple-flash excitation.

Reaction center preparations from the green sulfur bacterium Chlorobium tepidum, which contain monoheme cytochrome c, were studied by flash-absorption spectroscopy in the near-UV, visible, and near-infrared regions. The decay kinetics of the photooxidized primary donor P840(+), together with the amount of photooxidized cytochrome c, were analyzed along a series of four flashes spaced by 1 ms: 95% of the P840(+) was reduced by cytochrome c with a t(1/2) of approximately 65 micros after the first flash, 80% with a t(1/2) of approximately 100 micros after the second flash, and 23% with a t(1/2) of approximately 100 micros after the third flash; after the fourth flash, almost no cytochrome c oxidation occurred. The observed rates, the establishment of redox equilibrium after each flash, and the total amount of photooxidizable cytochrome c are consistent with the presence of two equivalent cytochrome c molecules per photooxidizable P840. The data are well fitted assuming a standard free energy change DeltaG degrees of -53 meV for electron transfer from one cytochrome c to P840(+), DeltaG degrees being independent of the oxidation state of the other cytochrome c. These observations support a model with two monoheme cytochromes c which are symmetrically arranged around the reaction center core. From the ratio of menaquinone-7 to the bacteriochlorophyll pigment absorbing at 663 nm, it was estimated that our preparations contain 0.6-1.2 menaquinone-7 molecules per reaction center. However, no transient signal due to menaquinone could be observed between 360 and 450 nm in the time window from 10 ns to 4 micros. No recombination reaction between the primary partners P840(+) and A(0)(-) could be detected under normal conditions. Such a recombination was observed (t(1/2) approximately 19 ns) under highly reducing conditions or after accumulation of three electrons on the acceptor side during a series of flashes, showing that the secondary acceptors can stabilize three electrons. From our data, there is no evidence for involvement of menaquinone in charge separation in the reaction center of green sulfur bacteria.     

Chlorophyll radical cation in photosystem II of chloroplasts. Millisecond decay at low temperature.

We compare the absorption changes, in the near infrared and in the green part of the spectrum, induced in spinach chloroplasts suspensions, at -- 170 degrees C, by continuous light and by flashes. (1) Following flash excitation, an absorption increase peaking at 825 nm which reverses rapidly (t 1/2 = 3.0 ms) is not affected by ferricyanide; it is suppressed when chloroplasts are preilluminated in the presence of 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea (DCMU) and hydroxylamine. The reversion of that signal is simultaneous with a partial back reoxidation of C-550 (fully reduced by the flash) and with partial (about 25%) oxidation of cytochrome b559. The magnitude of the signal peaking at 825 nm (that we attribute to the radical cation of the trap chlorophyll of Photosystem II, acting as a primary electron donor) decreases progressively within a series of successive flashes. (2) An absorption increase (40% of which is slowly reversible) with a broad peak around 810 nm is induced by continuous light or by a flash. It is suppressed by pretreatment with ferricyanide, but it is little affected by the treatment with 3-(3',4'-dichlorophenyl)-1,1'-dimethylurea and hydroxylamine. We attribute it to oxidized P700. (3) With chloroplasts pretreated with 10 mM ferricyanide, an absorption increase, whose magnitude is nearly independent of wavelength between 790 and 870 nm, can be induced by continuous light. One saturating flash produces only 20% of the signal. This absorption change (20% of which is reversible in 30 s) might be due to a secondary donor of Photosystem II.     

Flash-induced proton release in Rhodopseudomonas sphaeroides spheroplasts

Proton release by flash excitations was measured with right-side-out vesicles prepared from Rhodopseudomonas sphaeroides by lysozyme-EDTA treatment followed by hypotonic treatment. Absorbance change at 586 nm in the presence of bromcresol purple was measured to monitor the pH change. In the presence of horse heart cytochrome c, which catalyzes the electron transfer from the cytochrome b-c1 complex to the primary electron donor, the single-turnover flash elicited release of about two protons per primary electron donor, which was rereduced rapidly by the added cytochrome c. The halftime of the proton release was about 70 ms at pH 6.3 and at a redox potential of about 150 mV. The rate was considerably lower than that of the electron transfer from the cytochrome b-c1 complex to cytochrome c. However, multiple flashes with intervals of 60 ms caused release of the same amount of protons as that by flashes with longer intervals. This indicated that the proton release itself was rapid, but delocalization was slower. Antimycin A inhibited the proton release, and myxothiazol almost completely abolished it.     

The localized coupling of bacterial photophosphorylation: Effect of antimycin a and N,N-dicyclohexylcarbodiimide) in chromatophores from Rhodopseudomonas sphaeroides,Ga, studied by single turnover event analysis

1. The inhibition by antimycin A of the cyclic electron transfer has been studied in chromatophores from Rhodopseudomonas sphaeroides Ga following an approach based on the analysis of the relaxation kinetics of the reaction center optical changes in pulsed light. The recovery kinetics of the bacteriochlorophyll redox state have been found to be clearly biphasic. The half-times of the fast phase (13 ms) and slow phase (about 400 ms) were not modified by antimycin in a range of concentrations from 0.1 to 9 μM. On the other hand the percentage extent of the fast phase, which reflects the rate of the cyclic electron transfer, was monotonically decreased by increasing concentrations of the inhibitor. This indicates that antimycin decreases progressively the fraction of the photosynthetic units, active in cyclic electron transfer. 2. The ATP yield per flash observed under conditions of controlled inhibition of electron flow was strongly dependent upon the amount of active redox cycles. On the other hand, the amplitude of the carotenoid band shift, which has been demonstrated unequivocally to be correlated to the ATP yield per flash in uninhibited chromatophores, was not affected by antimycin up to a 40% inhibition of electron flow. 3. The effect of a progressive limitation by DCCD in the number of active ATP synthetase complexes on flash-induced phosphorylation has been examined. The decrease in ATP yield observed over a wide range of flash frequencies is related simply to the ATPase activity and to phosphorylation in continuous light, irrespective of the value of the membrane potential, which appears to be stabilized by this inhibitor. 4. As a whole, the results obtained at low concentrations of antimycin and under conditions of partial inhibition by DCCD evidence a localized coupling between the redox reactions and phosphorylation.