Secondary electron transfer in reaction centers of Rhodopseudomonas sphaeroides. Out-of-phase periodicity of two for the formation of ubisemiquinone and fully reduced ubiquinone.

Electron transfer between purified reaction centers from Rhodopseudomonas sphaeroides and exogenous ubiquinone has been studied in the presence of electron donors by measurements of light-induced absorbance changes following a sequence of short actinic light flashes. Each odd flash promotes the formation of a molecule of ubisemiquinone; after each even flash the semiquinone disappears and a molecule of the fully reduced quinone appears. We interpret these results by means of a model where a specialized molecule of ubiquinone is reduced by the primary electron acceptor in a one-electron transfer reaction after each flash, and is reoxidized by a molecule of the ubiquinone pool in a two-electron transfer reaction every two flashes.     

Secondary electron transfer in reaction centers of Rhodopseudomonas sphaeroides. Out-of-phase periodicity of two for the formation of ubisemiquinone and fully reduced ubiquinone

Electron transfer between purified reaction centers from Rhodopseudomonas sphaeroides and exogenous ubiquinone has been studied in the presence of electron donors by measurements of light-induced absorbance changes following a sequence of short actinic light flashes. Each odd flash promotes the formation of a molecule of ubisemiquinone; after each even flash the semiquinone disappears and a molecule of the fully reduced quinone appears.We interpret these results by means of a model where a specialized molecule of ubiquinone is reduced by the primary electron acceptor in a one-electron transfer reaction after each flash, and is reoxidized by a molecule of the ubiquinone pool in a two-electron transfer reaction every two flashes.     

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.     

Absorption changes induced by the binding of triazines to the QB pocket in reaction centers of Rhodobacter capsulatus

Inhibitors which block electron transfer from the primary (Q(A)) to the secondary (Q(B)) quinone of the bacterial reaction center are competing with the pool ubiquinones for binding at the Q(B) pocket. Due to the much greater stability of the semiquinone state Q(B)(-) compared with fully oxidized or reduced quinone, a displacement of the inhibitors takes place after one flash from state Q(A)(-)I to state Q(A)Q(B)(-). This process can be monitored from near-IR absorption changes which reflect local absorption shifts specific to Q(A)(-) and Q(B)(-). An anomalous behavior was observed when using triazines in chromatophores of R. capsulatus: the IR absorption change reflecting the formation of Q(B)(-) after one flash was absent. A normal transient decay of this signal was, however, triggered by a second flash, followed by a rapid return to the baseline. We show that this phenomenon is due to an absorption change induced by inhibitor binding (thus present in the dark baseline), with a spectrum close to that of Q(B)(-), so that the Q(B)(-) changes are canceled out during the inhibitor displacement process. On the second flash, one monitors the destruction of the semiquinone, leading transiently to the Q(A)Q(B) state, followed by inhibitor rebinding. This allows a direct measurement of the binding kinetics. This behavior was observed both in chromatophores and in isolated reaction centers from R. capsulatus, but not in R. sphaeroides.     

Kinetic model of the operation of a 2-electron switch in the photosynthetic reaction center of bacteria

A mathematical model, describing the binary oscillation of the concentration of semiquinone form of the secondary acceptor (ubiquinone) in photosynthetic reaction center of purple bacteria is proposed. This model takes into account both the changes of the ubiquinone state when the chromatophores are subjected to short flashes of light, and the successive dark relaxation of the semiquinone form. The model allows to calculate such characteristics as the dependence of the flash number, the stationary level of semiquinone form which is being established, when the flash number increases, the velocity which the concentration of semiquinone form is aspirating towards this stationary level and other characteristics. The model shows that the quantum yield of primary charge separation on the reaction center is higher after odd-number flashes then after even-number flashes.     

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 ATP/P+X- 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 H+/P+x- 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.     

Cytochrome b oxidation and reduction reactions in the ubiquinone-cytochrome b/c2 oxidoreductase from Rhodopseudomonas sphaeroides

1. The kinetics of cytochrome b reduction and oxidation in the ubiquinone-cytochrome b/c2 oxidoreductase of chromatophores from Rhodopseudomonas sphaeroides Ga have been measured both in the presence and absence of antimycin, after subtraction of contributions due to absorption changes from cytochrome c2, the oxidized bacteriochlorophyll dimer of the reaction center, and a red shift of the antenna bacteriochlorophyll. 2. A small red shift of the antenna bacteriochlorophyll band centered at 589 nm has been identified and found to be kinetically similar to the carotenoid bandshift. 3. Antimycin inhibits the oxidation of ferrocytochrome b under all conditions; it also stimulates the amount of single flash activated cytochrome b reductions 3- to 4-fold under certain if not all conditions. 4. A maximum of approximately 0.6 cytochrome b-560 (Em(7) = 50 mV, n = 1, previously cytochrome b50) hemes per reaction center are reduced following activating flashes. This ratio suggests that there is one cytochrome b-560 heme functional per ubiquinone-cytochrome b/c2 oxidoreductase. 5. Under the experimental conditions used here, only cytochrome b-560 is observed functional in cyclic electron transfer. 6. We describe the existence of three distinct states of reduction of the ubiquinone-cytochrome b/c2 oxidoreductase which can be established before activation, and result in markedly different reaction sequences involving cytochrome b after the flash activation. Poising such that the special ubiquinone (Qz) is reduced and cytochrome b-560 is oxidized yields the conditions for optimal flash activated electron transfer rates through the ubiquinone-cytochrome b/c2 oxidoreductase. However when the ambient redox state is lowered to reduce cytochrome b-560 or raised to oxidize Qz, single turnover flash induced electron transfer through the ubiquinone-cytochrome b/c2 oxidoreductase appears impeded; the points of the impediment are tentatively identified with the electron transfer step from the reduced secondary quinone (QII) of the reaction center to ferricytochrome b-560 and from the ferrocytochrome b-560 to oxidized Qz, respectively.     

Modulation of the Primary Electron Transfer Rate in Photosynthetic Reaction Centers by Reduction of a Secondary Acceptor

Photosynthetic application of picosecond spectroscopic techniques to bacterial reaction centers has led to a much greater understanding of the chemical nature of the initial steps of photosynthesis. Within 10 ps after excitation, a charge transfer complex is formed between the primary donor, a “special pair” of bacteriochlorophyll molecules, and a transient acceptor involving bacteriopheophytin. This complex subsequently decays in about 120 ps by donating the electron to a metastable acceptor, a tightly bound quinone.

Recent experiments with conventional optical and ESR techniques have shown that when reaction centers are illuminated by a series of single turnover flashes in the presence of excess electron donors and acceptors, a stable, anionic ubisemiquinone is formed on odd flashes and destroyed on even flashes, suggesting that the acceptor region contains a second quinone that acts as a two-electron gate between the reaction center and subsequent electron transport events involving the quinone pool.

Utilizing standard picosecond techniques, we have examined the decay of the charge transfer complex in reaction centers in the presence of the stable semiquinone, formed by flash illumination with a dye laser 10 s before excitation by a picosecond pulse. In this state the decay rate for the charge transfer complex is considerably slower than when no electron is present in the quinone acceptor region. This indicates fairly strong coupling between constituents of the reaction center-quinone acceptor complex and may provide a probe into the relative positions of the various components.

     

Studies of Electron Transport Dynamics in Photosynthetic Reaction Centers Using Fast Temperature Changes

Rates of thermoinduced conformational transitions of reaction center (RC) complexes providing effective electron transport were studied in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria using methods of fast freezing and laser-induced temperature jump. Reactions of electron transfer from the primary to secondary quinone acceptors and from the multiheme cytochrome c subunit to photoactive bacteriochlorophyll dimer were used as probes of electron transport efficiency. The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied. It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds. In contrast to the quinone complex, the thermoinduced transition of the macromolecular RC complex to the state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220-280 K accounts for tens of seconds. This transition is thought to be mediated by large-scale conformational dynamics of the macromolecular RC complex.     

Redox reactions on the reducing side of photosystem II in chloroplasts with altered herbicide binding properties

Measurements of chlorophyll fluorescence have been used to monitor electron transfer from Q (the primary electron acceptor of photosystem II) to B (the bound quinone which serves as the secondary acceptor) in chloroplasts isolated from atrazine-susceptible and atrazine-resistant pigweed chloroplasts. The Q^? → B electron transfer was at least 10-fold slower in the plastids from resistant plants. Binary oscillations in the rate of Q^? decay after a series of flashes were of opposite phase in the two types. The data are interpreted to indicate that the apoprotein of B is altered in the photosytem II complex of the two types of plants—this is correlated to altered binding affinity of herbicides to this component and may be related to altered redox properties of the bound quinone cofactor.     

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)     

Primary and secondary electron transfer in hexane-solubilized proteolipid complexes of Rhodopseudomonas sphaeroides R-26

Primary electron transfer in hexane-solubilized reaction center proteolipid complexes is similar to that in detergent-solubilized reaction centers or chromatophores when diaminodurene is electron donor. Approximate values for the extinction coefficients of ubisemiquinone and the diaminodurene cation can be calculated. The primary and secondary quinone sites are dissimilar and results in only the transient formation of a semiquinone anion pair after two photochemical turnovers. One of the semiquinone anions decays rapidly, the remaining one and the diaminodurene cation have long lifetimes. Disproportionation between the semiquinone anions does not occur.     

Oxidation-reduction thermodynamics of the acceptor quinone complex in whole-membrane fragments from Chloroflexus aurantiacus

Oxidation-reduction thermodynamic equilibria involving the quinone-acceptor complex have been examined in whole-membrane fragments from Chloroflexus aurantiacus. The primary quinone acceptor was titrated by monitoring the amount of cytochrome c554 photooxidized by a flash of light as a function of the redox potential. In contrast to previous data obtained in purified plasma membranes, in which the primary quinone acceptor exhibited a midpoint potential equal to -50 mV at pH 8.2, in whole-membrane fragments it titrated at -210 mV (pH 8.0), with a pH dependence of -60 mV/pH up to a pK value of 9.3. o-Phenanthroline, an inhibitor of electron transfer from the primary to the secondary quinone acceptor, shifted the Em/pH curve of the primary acceptor to higher redox potentials. The midpoint potential of the secondary quinone acceptor and its dependence on pH has been determined by comparing the kinetics of the charge recombination processes within the reaction center complex in the presence and in the absence of o-phenanthroline. It is concluded that both the primary and the secondary quinone acceptors interact with a proton, with pK values of 9.3 and of approximately 10.2 respectively. At physiological pH the electron appears to be stabilized on the secondary with respect to the primary quinone acceptor by approximately 60 meV.     

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.     

Probing the primary quinone environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy

The photoreduction of the primary electron acceptor, QA, has been characterized by light-induced Fourier transform infrared difference spectroscopy for Rb. sphaeroides reaction centers and for Rsp. rubrum and Rp. viridis chromatophores. The samples were treated both with redox compounds, which rapidly reduce the photooxidized primary electron P+, and with inhibitors of electron transfer from QA- to the secondary quinone QB. This approach yields spectra free from P and P+ contributions which makes possible the study of the microenvironment of QA and QA-.     

The reaction between primary and secondary electron acceptors in bacterial photosynthesis

Following a 20-nsec actinic flash, which causes oxidation of P870 and cytochrome C422, Chromatium chromatophores enter a refractory state. While the chromatophores are in this state, a second flash does not cause further oxidation of P870 or cytochrome C422. The quanta of the second flash are wasted as fluorescence (and heat); apparently they do not energize an alternative photochemical reaction. The refractory state probably reflects the accumulation of the primary electron acceptor in a reduced form. By following the reappearance of the capacity for photochemistry, one can measure the kinetics of electron transfer between the primary electron acceptor and the secondary agent which reoxidizes it. In Chromatium chromatophores, this process requires about 60 μsec to proceed half-way to completion at pH 7, and 80 μsec at pH 8. The rate of the reaction increases with decreasing pH, but not in direct proportion to the proton concentration. It increases with temperature, with an E_a of about 8.3 kcal/mole. The kinetics are approximately second order in the concentration of the reduced acceptor.     

Herbicide-quinone competition in the acceptor complex of photosynthetic reaction centers from Rhodopseudomonas sphaeroides: a bacterial model for PS-II-herbicide activity in plants

A select group of herbicides that inhibit photosystem II also act at the acceptor side of the reaction center (RC) from the photosynthetic bacterium Rhodopseudomonas sphaeroides, with much the same relative specificity as in plants. These include the triazines and some phenolic compounds. The proposal that herbicides inhibit the electron transfer from the primary quinone (QA) to the secondary quinone (QB) by competing for the secondary quinone binding site--the B-site--[5], is tested here with terbutryn, the most potent of the triazines. Competition between terbutryn and ubiquinone (Q-10) was observed using the kinetics of the back-reaction as a measure of inhibition. The model includes binding equilibria before and after flash activation. The binding constants for the preflash (dark) equilibria, for reaction centers in 0.14% lauryl dimethylamine-N-oxide (LDAO), were KDi = 0.8 microM terbutryn, KDq = 2 microM Q-10; both are detergent-concentration dependent. After flash activation, binding equilibrium is not fully restored on the time scale of the back-reaction because terbutryn unbinds slowly. This gives rise to biphasic decay kinetics from which koff for terbutryn was estimated to be 3 sec-1. Titrations of the rate of the slow back reaction indicated that the post-flash equilibrium is less sensitive to inhibitor, in a manner that is independent of the much stronger binding of the semiquinone, Q-B, and indicative of a direct effect of the redox state of QA on the affinity of the B-site for ligands. However, the effects on KLi and KDq could not be separated: either KLi greater than KDi or KLq less than KDq. Some triazine-resistant mutants have been isolated and are described. All appear to be herbicide binding site mutants. Whole cells and photosynthetic membrane vesicles (chromatophores) exhibit a 10-50-fold increase in resistance to triazines due, in large part, to an increase in the rate of unbinding (koff). The modifications of the binding site appear to diminish the affinity of the B-site for ubiquinone as well as terbutryn. It is concluded that bacterial RCs are a useful model for the study of herbicide activity and specificity.     

Charge accumulation at the reducing side of system 2 of photosynthesis

A study was made of the reactions between the primary and secondary electron acceptors of Photosystem 2 by measurements of the increase of chlorophyll fluorescence induced in darkness by dithionite or by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The experiments were done either with chloroplasts to which hydroxylamine or carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) was added, or with chloroplasts treated with tris(hydroxymethyl)aminomethane (Tris) to which phenylenediamine and ascorbate were added as donor system. Under these conditions the fluorescence increase induced by dithionite or DCMU added after illumination with short light flashes was dependent on the flash number with a periodicity of two; it was large after an uneven number of flashes, and small after a long darktime or after an even number of flashes. The results are interpreted in terms of a model which involves a hypothetical electron carrier situated between Q and plastoquinone; this electron carrier is thought to equilibrate with plastoquinone in a two-electron transfer reaction; the results obtained with DCMU are explained by assuming that its midpoint potential is lowered by this inhibitor.     

Ubiquinone (coenzyme Q10) binding sites: low dielectric constant of the gate prevents the escape of the semiquinone

The photosynthetic reaction center (RC) from purple bacteria is frequently used as a model for the interaction of ubiquinones (coenzyme Q) with membrane proteins. Single-turnover flash activation of RC leads to formation of the semiquinone (SQ) of the secondary acceptor quinone after odd flashes and quinol after even flashes. The ubiquinol escapes the binding site in 1 ms, while the SQ does not leave the binding site for at least 5 min. Observed difference between these times suggests a large energetic barrier for the SQ. However, high apparent dielectric constant in the vicinity of the quinone ring (>or=25) results in a relatively small electrostatic energy of SQ stabilization. To resolve this apparent contradiction I suggest that a significant part of the kinetic stabilization of the SQ is achieved by the special topology of the binding site in which quinone can exit the binding site only by moving its headgroup toward the center of the membrane. The large energetic penalty of transferring the charged headgroup to the membrane dielectric can explain the observed kinetic stability of the SQ.     

The involvement of iron and ubiquinone in electron transfer reactions mediated by reaction centers from photosynthetic bacteria.

Reaction centers from Rhodopseudomonas sphaeroides strain R-26 were prepared with varying Fe and ubiquinone (Q) contents. The photooxidation of P-870 to P-870+ was found to occur with the same quantum yield in Fe-depleted reaction centers as in control samples. The kinetics of electron transfer from the initial electron acceptor (I) to Q also were unchanged upon Fe removal. We conclude that Fe has no measurable role in the primary photochemical reaction. The extent of secondary reaction from the first quinone acceptor (QA) to the second quinone acceptor (QB) was monitored by the decay kinetics of P-870+ after excitation of reaction centers with single flashes in the absence of electron donors, and by the amount of P-870 photooxidation that occurred on the second flash in the presence of electron donors. In reaction centers with nearly one iron and between 1 and 2 ubiquinones per reaction center, the amount of secondary electron transfer is proportional to the ubiquinone content above one per reaction center. In reaction centers treated with LiClO4 and o-phenanthroline to remove Fe, the amount of secondary reaction is decreased and is proportional to Fe content. Fe seems to be required for the secondary reaction. In reaction centers depleted of Fe by treatment with SDS and EDTA, the correlation between Fe content and secondary activity is not as good as that found using LiClO4. This is probably due in part to a loss of primary photochemical activity in samples treated with SDS; but the correlation is still not perfect after correction for this effect. The nature of the back reaction between P-870+ and Q-B was investigated using stopped flow techniques. Reaction centers in the P-870+ Q-B state decay with a 1-s half-time in both the presence and absence of o-phenanthroline, an inhibitor of electron transfer between Q-B and QB. This indicates that the back reaction between P-870+ and Q-A is direct, rather than proceeding via thermal repopulation of Q-A. The P-870+ Q-B state is calculated to lie at least 100 mV in free energy below the P-870+ Q-A state.