Electron-paramagnetic-resonance measurements of the electron-transfer components of the reaction centre of Rhodopseudomonas viridis. Oxidation–reduction potentials and interactions of the electron acceptors

Oxidation-reduction potentiometry was carried out on Rhodopseudomonas viridis chromatophores. Measurements of e.p.r. signals of the semiquinone-iron type at g=1.82 have revealed a more complex situation than previously reported. The presence of three different components is indicated. The midpoint potential (E(m)) of the primary acceptor quinone/semiquinone couple was found to be approx. -165mV at pH10, with a pK being reached at around pH7.5. The primary acceptor also accepts a second electron with an E(m) of -525mV, but this redox transition exhibits a hysteresis effect. Interaction effects indicate the presence of another component with E(m) values at pH10 of approx. -165mV (pK reached at around pH7.5) for single reduction and -350mV (pK at pH10 or greater) for double reduction. It is suggested that this component is the secondary acceptor. Another semiquinone-iron-type component which gives a g=1.82 signal is also present. This component is distinguishable from the primary acceptor by its e.p.r. spectrum, which shows a double peak at g=1.82 and a g(x) line at g=1.76. This component has E(m) values at pH10 for single and double reduction of -15mV and approx. -150mV respectively. Both of these E(m) values are pH-dependent. The presence of an interaction between this component and the photoreduced primary acceptor indicates the close proximity of these components. However, the midpoint potential of this component indicates a function as a secondary electron-transport component rather than an electron acceptor in the reaction centre. The dependence of the bacteriopheophytin intermediate (I) doublet e.p.r. signal on the presence of the semiquinone-iron form of the primary acceptor is demonstrated. The midpoint potential of the I/I(-) couple is estimated to be lower than -600mV.     

The effect of o-phenanthroline on the midpoint potential of the primary electron acceptor of photosystem II.

The primary electron acceptor of Photosystem II has a midpoint oxidation-reduction potential of +95 mV at pH 7.0 in Photosystem II chloroplast fragments prepared by digitonin treatment. The midpoint potential of the acceptor has a pH dependence of -60 mV/pH unit. At concentrations that inhibit oxygen evolution, o-phenanthroline shifts the midpoint potential of the primary acceptor by +70 mV. The shifted potential retains the same dependence on pH. The effect of o-phenanthroline suggests that it interacts directly with the primary electron acceptor of photosystem II in a manner similar to that reported previously for the primary electron acceptor in purple photosynthetic bacteria.     

The effect of o-phenanthroline on the midpoint potential of the primary electron acceptor of Photosystem II

The primary electron acceptor of Photosystem II has a midpoint oxidation-reduction potential of +95 mV at pH 7.0 in Photosystem II chloroplast fragments prepared by digitonin treatment. The midpoint potential of the acceptor has a pH dependence of −60 mV/pH unit. At concentrations that inhibit oxygen evolution, o-phenanthroline shifts the midpoint potential of the primary acceptor by +70 mV. The shifted potential retains the same dependence on pH. The effect of o-phenanthroline suggests that it interacts directly with the primary electron acceptor of Photosystem II in a manner similar to that reported previously for the primary electron acceptor in purple photosynthetic bacteria.     

Characterization of photosystem ii electron acceptors in Phormidium laminosum

Chlorophyll a fluorescence has been used to monitor the redox state of the primary electron acceptor of photosystem II (PS II) in the blue-green alga Phormidium laminosum during equilibrium titrations. The shape of induction curves measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) have been analyzed. The induction curves were very similar in unfractionated thylakoid membranes and PS II particles. In both, the fast (alpha) phase was sigmoidal, and was followed by a slow (beta) exponential tail. Thus, the structural organization and complexity of the particles (J. M. Bowes and P. Horton, Biochim, Biophys. Acta 680, 127-133 (1982), as indicated by the occurrence of energy transfer between alpha centers and presence of beta centers, must preexist in the membranes. Redox titration of the initial level of fluorescence indicated the presence of a single quencher QH in the unfractionated thylakoids, midpoint potential: Em7.0 approximately -35 mV (n = 1). Thus, the occurrence of a single acceptor is characteristic for P. laminosum and the absence of a low potential acceptor in PS II particles (J.M. Bowes, P. Horton, and D.S. Bendall, FEBS Lett. 135, 261-264 (1981] was not the result of its removal during their preparation. The midpoint potential of Q varied by -60 mV/pH unit in PS II particles and membrane fragments, with a pK at pH greater than 8.5 (particles) and at pH 7.5 (fragments). In PS II particles, DCMU raised the pK by approximately 0.5 pH units. It is argued that the pH dependence of Q is conferred by protonation of a protein which accompanies its reduction rather than protonation of the semiquinone Q X itself.     

Electrostatic interactions between charged amino acid residues and the bacteriochlorophyll dimer in reaction centers from Rhodobacter sphaeroides.

The extent of electrostatic contributions from the protein environment was assessed by the introduction of ionizable residues near the bacteriochlorophyll dimer in reaction centers from Rhodobacter sphaeroides. Two mutations at symmetry-related sites, M199 Asn to Asp and L170 Asn to Asp, resulted in a 48 and 44 mV lowering of the midpoint potential, respectively, compared to the wild type at pH 8, while a 75 mV decrease in the midpoint potential was observed for the mutation L168 His to Glu. The decrease relative to wild type was found to be approximately additive, up to 147 mV, for various combinations of the mutations. As the pH was lowered from 9.5 to 6.0, the relative decrease in the midpoint potential became smaller for each of these three mutations. Titration of the pH dependence of the change in midpoint potential of the M199 Asn to Asp mutant compared to wild type yielded a pK(a) value of 7.9 and a change in midpoint potential from low to high pH of 59 mV. The major effect of the mutation on the midpoint potential of the dimer is interpreted as stemming from a negative charge on the residue. An average dielectric constant of approximately 20 was estimated for the local protein environment, consistent with a relatively hydrophobic environment for residue M199. The rate of charge recombination between the primary quinone acceptor and the bacteriochlorophyll dimer decreased in the M199 Asn to Asp mutant at high pH, reflecting the decrease in midpoint potential.     

Thermodynamic properties of the semiquinone and its binding site in the ubiquinol-cytochrome c (c2) oxidoreductase of respiratory and photosynthetic systems

The antimycin-sensitive ubisemiquinone radical (QC) of the ubiquinol-cytochrome c oxidoreductase of submitochondrial particles and chromatophores of Rhodopseudomonas sphaeroides Ga has been studied by a combination of redox potentiometry and EPR spectroscopy. This g = 2.005 radical signal appears at physiological pH values and increases in intensity with increasing pH up to pH 7.6 in submitochondrial particles and pH 9.0 in R. sphaeroides after which its intensity remains unchanged. The Em7 (ubiquinone/quinol) of the signal, estimated from redox titration data is 80 mV for submitochondrial particles, and 150 mV in chromatophores. Each of these values is higher than that of the quinone pool by 20 mV in submitochondrial particles and 60 mV in R. sphaeroides. This indicates that the quinone at the binding site is out of equilibrium with the pool, and that binding site preferentially binds quinol over quinone. Analysis of the shapes of the semiquinone titration curves, taken together with the midpoint elevation, indicates a quinone-binding site: cytochrome c1 stoichiometry of 1:1 in both submitochondrial particles and chromatophores. At its maximal intensity, the semiquinone concentration at the binding site is 0.26 in submitochondrial particles (greater than pH 7.6) and 0.4 in chromatophores (greater than pH 9.0). In both systems, the midpoint of the ubiquinone/ubisemiquinone couple is constant as the pH is raised up to the pH of maximal semiquinone formation whereafter it becomes more negative at the rate of -60 mV/pH unit. The midpoint of the ubisemiquinone/quinol couple, on the other hand, varies by -120 mV/pH unit at pH values up to the transition pH, after which it, too, changes by -60 mV/pH unit. This seemingly anomalous behavior may be explained by invoking a protonated group at or near the quinone-binding site whose pK corresponds to the pH transition point in the quinone/semiquinone/quinol redox chemistry when the site is free or when quinone or quinol occupies the site. This pK is elevated to at least pH 9.0 in submitochondrial particles and 10.5 in R. sphaeroides when semiquinone is bound to the site.     

Some effects of o-phenanthroline on electron transport in chromatophores from photosynthetic bacteria

The midpoint potentials of the primary electron acceptors in chromatophores from Rhodopseudomonas spheroides and Chromatium have been studied by titrating the laser-induced P605 and cytochrome c oxidations, respectively. Both midpoint potentials are pH dependent (60 mV/pH unit).o-Phenanthroline shifts the midpoint potentials of the primary acceptors, by +40 mV in Rps spheroides and +135 mV in Chromatium. A similar though less extensive change in midpoint potential was observed in the presence of batho-phenanthroline, but not with 8-hydroxyquinoline. The shifted midpoints retain the same dependence on pH.Some of the effects of o-phenanthroline can be explained by assuming that it chelates the reduced form of the primary electron acceptor. This suggests the presence in the primary electron acceptor of a metal chelated by o- and batho-phenanthroline.In Rps spheroides chromatophores o-phenanthroline inhibits the laser- and flash-induced carotenoid shift at all redox potentials, stimulates the laser-induced P605 oxidation at redox potentials between +350 and +420 mV and slows the decay of the laser-induced cytochrome c oxidation below +180 mV. These effects show that o-phenanthroline may have more than one site of action.     

Some thermodynamic and kinetic properties of the primary photochemical reactants in a complex from a green photosynthetic bacterium.

We have examined the bacteriochlorophyll reaction-center complex of Chlorobium limicola f. thiosulfatophilum, strain Tassajara. Our results indicate that the midpoint potential of the primary electron donor bacteriochlorophyll of the reaction center is +250 mV at pH 6.8, while that of cytochrome c-553 is +165 mV. There are two cytochrome c-553 hemes per reaction center, and the light-induced oxidation of each is biphasic (t1/2 of less than 5 mus and approximately 50 mus). We belive that this indicates a two state equilibrium with each cytochrome heme being either close to, or a little removed from, the reaction-center bacteriochlorophyll. We have also titrated the primary electron acceptor of the reaction center. Its equilibrium midpoint potential at pH 6.8 is below -450 mV. This is very much lower than the previous estimate for green bacteria, and also substantially lower than values obtained for purple bacteria. Such a low-potential primary acceptor would be thermodynamically capable of direct reduction of NAD+ via ferredoxin in a manner analagous to photosystem I in chloroplasts and blue-green algae.     

Determination of the redox properties of the Rieske [2Fe-2S] cluster of bovine heart bc1 complex by direct electrochemistry of a water-soluble fragment.

The redox potential of the Rieske [2Fe-2S] cluster of the bc1 complex from bovine heart mitochondria was determined by cyclic voltammetry of a water-soluble fragment of the iron/sulfur protein. At the nitric-acid-treated bare glassy-carbon electrode, the fragment gave an immediate and stable quasireversible response. The midpoint potential at pH 7.2, 25 degrees C and I of 0.01 M was Em = +312 +/- 3 mV. This value corresponds within 20 mV to results of an EPR-monitored dye-mediated redox titration. With increasing ionic strength, the midpoint potential decreased linearly with square root of I up to I = 2.5 M. From the cathodic-to-anodic peak separation, the heterogeneous rate constant, k degrees, was calculated to be approximately 2 x 10(-3) cm/s at low ionic strength; the rate constant increased with increasing ionic strength. From the temperature dependence of the midpoint potential, the standard reaction entropy was calculated as delta S degrees = -155 J.K-1.mol-1. The pH dependence of the midpoint potential was followed over pH 5.5-10. Above pH 7, redox-state-dependent pK changes were observed. The slope of the curve, -120 mV/pH above pH9, indicated two deprotonations of the oxidized protein. The pKa values of the oxidized protein, obtained by curve fitting, were 7.6 and 9.2, respectively. A group with a pKa,ox of approximately 7.5 could also be observed in the optical spectrum of the oxidized protein. Redox-dependent pK values of the iron/sulfur protein are considered to be essential for semiquinone oxidation at the Qo center of the bc1 complex.     

Redox titration of electron acceptor Q and the plastoquinone pool in Photosystem II

The primary photochemical quencher Q and the secondary electron acceptor pool in Photosystem II have been titrated. We used particles of Scenedesmus mutant No. 8 that lack System I and allowed the system to equilibrate with external redox mediators in darkness prior to measurement of the fluorescence rise curve.The titration of Q, as indicated by the dark level of F_i, occurs in two discrete steps. The high-potential component (Q_h) has a midpoint potential of +68 mV (pH 7.2) and accounts for ～67% of Q. The pH sensitivity of the midpoint potential is ?60 mV, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The low-potential component (Q₁) accounts for the remaining 33% of Q and shows a midpoint potential near?300 mV (pH 7.2).The plastoquinone pool, assayed as the half-time of the fluorescence rise curve, titrates as a single component with a midpoint potential 30–40 mV more oxidizing than that of Q_h, i.e., at 106 mV (pH 7.2). The E_m shows a pH sensitivity of ?60 mV/pH unit, indicating the involvement of 1 $\text{H}{}^{\mathrm{+}}\text{e}$. The observation that all 12–14 electron equivalents in the pool titrate as a single component indicates that the heterogeneity otherwise observed in the secondary acceptor system is a kinetic rather than a thermodynamic property.Illumination causes peculiar, and as yet unclarified, changes of both Q and the secondary pool under anaerobic conditions that are reversed by oxygen.     

Redox study of electron donation to P-680 in Photosystem II

Flash-induced absorption changes at 820 nm were studied as a function of redox potential in Tris-extracted Photosystem II oxygen-evolving particles and Triton subchloroplast fraction II particles. The rereduction kinetics of P-680+ in both preparations showed biphasic recovery phases with half-times of 42 and 625 microseconds at pH 4.5. The magnitude of the 42 microseconds phase of P-680+ rereduction was strongly dependent on the redox potential of the medium. This absorption transient, attributed to electron donation from D1 (the secondary electron donor in oxygen-inhibited chloroplasts), titrated as a single redox component with a midpoint potential of +240 +/- 35 mV. The experimentally determined midpoint potential was found to be independent of pH over the tested range 4.5-6.0. In contrast, the magnitude of the 625 microseconds phase of P-680+ rereduction was independent of redox potential between +350 and +100 mV. These results are interpreted in terms of a model in which an alternate electron donor with Em approximately equal to 240 mV, termed D0, serves as a rapid donor (t 1/2 less than or equal to 2 microseconds) to P-680+ in Tris-extracted and Triton-treated Photosystem-II preparations. According to this model, the slower electron donor, D1, is functional only when D0 becomes oxidized.     

The Rieske FeS center from the gram-positive bacterium PS3 and its interaction with the menaquinone pool studied by EPR.

The Rieske 2Fe2S center from Bacillus PS3, a Gram-positive thermophilic eubacterium, has been studied by EPR spectroscopy. Its redox midpoint potential at pH 7.0 was determined to be +165 +/- 10 mV and was found to decrease with an apparent slope of -80 mV/pH unit above pH 7.9. The Qo-site inhibitor stigmatellin induced spectral changes analogous to those reported for Rieske centers from mitochondria and chloroplasts. The redox midpoint potential of the PS3 Rieske cluster was not affected by stigmatellin. The orientation of the g tensor was similar to other Rieske centers (gz and gy are oriented parallel, gx is oriented perpendicular to the membrane plane). The shape of the EPR spectrum of the Rieske cluster from PS3 changed as a function of the redox state of the menaquinone (MK) pool. This permitted the redox midpoint potential of the MK pool to be determined in the membrane. Values of -60 +/- 20 mV at pH 7.0 and of -130 +/- 20 mV at pH 8.0 were obtained. The results are compared with already published data from other Rieske centers. It is proposed that all Rieske centers that function in electron transport chains using MK as pool quinone show common features that distinguish them from Rieske centers operating in ubiquinone- or plastoquinone-based electron transfer chains.     

Purification and characterization of reaction centers from the obligate aerobic phototrophic bacteria Erythrobacter litoralis,Erythromonas ursincola and Sandaracinobacter sibiricus

Reaction centers (RC) from the species Erythrobacter (Eb.) litoralis, Erythromonas (Em.) ursincola and Sandaracinobacter (S.) sibiricus have been purified by LDAO treatment of light-harvesting-reaction center complexes and DEAE chromatography. The content and overall organisation of the RCs' chromophores, determined by linear dichroism (LD) and absorption spectroscopy, are similar to those isolated from anaerobic photosynthetic bacteria. The redox properties of the primary electron donor are pH-independent and very similar to those determined for anaerobic photosynthetic bacteria with midpoint potential values equal to 445 (± 10), 475 and 510 mV for Eb. litoralis, S. sibiricus and Em. ursincola, respectively. The RC purified from Eb. litoralis does not contain bound cytochrome (cyt), whereas RCs isolated from S. sibiricus and Em. ursincola possess a tetraheme cyt c. Each of these tetraheme cyts contains two high potential hemes and two low potential hemes. Their redox properties are very similar, with midpoint potentials equal to 385 (± 10), 305, 40, -40 mV for Em. ursincola and 355, 285, 30, -48 mV for S. sibiricus. At physiological pH, the midpoint potential of the primary electron acceptor (Q_A) varies with a slope of -60 mV/pH unit. The reduced form of Q_A presents pK values of 9, 9.8, 10.5 for S. sibiricus, Em. ursincola and Eb. litoralis, respectively. The main difference observed between RCs isolated from anaerobic photosynthetic and from obligate aerobic bacteria is the _Emvalues of Q_A which are 65 to 120 mV higher in the last case. This difference is proposed to be a major reason for the inability of these species to grow under anaerobic photosynthetic conditions.     

Redox potential of plastoquinone A in spinach chloroplasts.

The redox potential of plastoquinone A in spinach chloroplasts was determined. The midpoint potential of the quinone is about +80 mV at pH 7.0 with an n value of 2. The pH-dependence of the potential is -30 mV per pH between pH 4.0 and 5.7, and -60 mV per pH between pH 5.7 and 8.0. The change of the slope at pH 5.7 is interpreted as the protonation of the oxidized plastoquinone A.     

The redox midpoint potential of the primary quinone of reaction centers in chromatophores of Rhodobacter sphaeroides is pH independent

The redox midpoint potential (E (m)) of the primary quinone of bacterial reaction centers, Q(A), in native membranes (chromatophores) measured by redox potentiometry is reported to be pH dependent (-60 mV/pH) up to a highly distinctive pK ( a ) (9.8 in Rba. sphaeroides) for the reduced state. In contrast, the E (m) of Q(A) in isolated RCs of Rba. sphaeroides, although more variable, has been found to be essentially pH-independent by both redox potentiometry and by delayed fluorescence, which determines the free energy (DeltaG (P*A)) of the P(+)Q (A) (-) state relative to P*. Delayed fluorescence was used here to determine the free energy of P(+)Q (A) (-) in chromatophores. The emission intensity in chromatophores is two orders of magnitude greater than from isolated RCs largely due to the entropic effect of antenna pigments "drawing out" the excitation from the RC. The pH dependence of DeltaG (P*A) was almost identical to that of isolated RCs, in stark contrast with potentiometric redox titrations of Q(A). We considered that Q(A) might be reduced by disproportionation with QH(2) through the Q(B) site, so the titration actually reflects the quinone pool, giving the -60 mV/pH unit dependence expected for the Q/QH(2) couple. However, the parameters necessary to achieve a strong pH-dependence are not in good agreement with expected properties of Q(A) and Q(B). We also consider the possibility that the time scale of potentiometric titrations allows the reduced state (Q (A) (-) ) to relax to a different conformation that is accompanied by stoichiometric H(+) binding. Finally, we discuss the choice of parameters necessary for determining the free energy level of P(+)Q (A) (-) from delayed fluorescence emission from chromatophores of Rba. sphaeroides.     

Redox titration of the primary electron acceptor of Photosystem I in spinach chloroplasts

The midpoint potential of the primary electron acceptor of Photosystem I in spinach chloroplasts was titrated using the photooxidation of P₇₀₀ at −196 °C as an index of the amount of primary acceptor present in the oxidized state. The redox potential of the chloroplast suspension was established by the reducing power of hydrogen gas (mediated by clostridial hydrogenase and 1,1′-trimethylene-2,2′-dipyridylium dibromide) at specific pH values at 25 °C. Samples were frozen to −196 °C and the extent of the photooxidation of P₇₀₀ was determined from light-minus-dark difference spectra. This titration indicated a midpoint potential of −0.53 V for the primary electron acceptor of Photosystem I.     

NMR studies of electron transfer mechanisms in a protein with interacting redox centres: Desulfovibrio gigas cytochrome c3

The proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio gigas were examined while varying the pH and the redox potential. The analysis of the NMR reoxidation pattern was based on a model for the electron distribution between the four haems that takes into account haem-haem redox interactions. The intramolecular electron exchange is fast on the NMR time scale (larger than 10(5) s-1). The NMR data concerning the pH dependence of the chemical shift of haem methyl resonances in different oxidation steps and resonance intensities are not compatible with a non-interacting model and can be explained assuming a redox interaction between the haems. A complete analysis at pH* = 7.2 and 9.6, shows that the haem-haem interacting potentials cover a range from -50 mV to +60 mV. The midpoint redox potentials of some of the haems, as well as some of their interacting potentials, are pH-dependent. The physiological relevance of the modulation of the haem midpoint redox potentials by both the pH and the redox potential of the solution is discussed.     

Energetics for oxidation of a bound manganese cofactor in modified bacterial reaction centers

The energetics of a Mn cofactor bound to modified reaction centers were determined, including the oxidation/reduction midpoint potential and free energy differences for electron transfer. To determine these properties, a series of mutants of Rhodobacter sphaeroides were designed that have a metal-ion binding site that binds Mn2+ with a dissociation constant of 1 μM at pH 9.0 (Thielges et al. (2005) Biochemistry 44, 7389-7394). In addition to the Mn binding site, each mutant had changes near the bacteriochlorophyll dimer, P, that resulted in altered P/P+ oxidation/reduction midpoint potentials, which ranged from 480 mV to above 800 mV compared to 505 mV for wild type. The bound Mn2+ is redox active and after light excitation can rapidly reduce the oxidized primary electron donor, P+. The extent of P+ reduction was found to systematically range from a full reduction in the mutants with high P/P+ midpoint potentials to no reduction in the mutant with a potential comparable to wild type. This dependence of the extent of Mn2+ oxidation on the P/P+ midpoint potential can be understood using an equilibrium model and the Nernst equation, yielding a Mn2+/Mn3+ oxidation/reduction midpoint potential of 625 mV at pH 9. In the presence of bicarbonate, the Mn2+/Mn3+ potential was found to be 90 mV lower with a value of 535 mV suggesting that the bicarbonate serves as a ligand to the bound Mn. Measurement of the electron transfer rates yielded rate constants for Mn2+ oxidation ranging from 30 to 120 s(-1) as the P/P+ midpoint potentials increased from 670 mV to approximately 805 mV in the absence of bicarbonate. In the presence of bicarbonate, the rates increased for each mutant with values ranging from 65 to 165 s(-1), reflecting an increase in the free energy difference due to the lower Mn2+/Mn3+ midpoint potential. This dependence of the rate constant on the P/P+ midpoint potential can be understood using a Marcus relationship that yielded limits of at least 150 s(-1) and 290 meV for the maximal rate constant and reorganization energy, respectively. The implications of these results are discussed in terms of the energetics of proteins with redox active Mn cofactors, in particular, the Mn4Ca cofactor of photosystem II.     

Kinetics of ubisemiquinone redox changes in primary reactions of bacterial photosynthesis

Absorbance changes at 450 nm of the semiquinone form of the secondary electron acceptor were studied in chromatophores of Rhodospirillum rubrum. When chromatophores are illuminated by a series of single turnover flashes ubisemiquinone is formed and destroyed on alternate flashes at ambient redox potential from 100 to 250 mV. A simple kinetic model of the binary oscillations is suggested. On the base of the model it is shown that the rate constant of electron transfer from primary to secondary quinone after the first flash is larger that after the second flash. Cooperativity in electron transfer from primary to secondary quinone can be explained by electrostatic interactions of charged carriers.     

Electrogenic Protonation of the Secondary Quinone Acceptor Q<Subscript>B</Subscript> in Spinach Photosystem II Complexes Incorporated into Lipid Vesicles

The generation of transmembrane electric potential difference (delta psi) in quinone acceptor complex of proteoliposomes containing core complexes of photosystem II from spinach was studied using for the measurements a direct electrometric technique. Besides the fast increase in the membrane potential associated with the electron transfer between the redox-active tyrosine 161 residue (Y(Z)) in D1 polypeptide and the primary quinone acceptor Q(A), an additional electrogenic phase with tau approximately 0.85 msec at pH 7.3 and the maximal relative amplitude of approximately 11% of the Y(Z)ox Q(A)- phase was observed after the second light flash. The sensitivity of this phase to diuron (an inhibitor of electron transfer between Q(A) and the secondary quinone acceptor Q(B)), the dependence of its amplitude on the light flash parity, and also a decrease in its rate constant with increase in pH indicated that it was due to dismutation of Q(A)- and Q(B)- with the subsequent protonation of a doubly reduced plastoquinone molecule: Q(A)- Q(B)- + 2H+ --> Q(A)Q(B)H2.