Photoinduced electron transfer in a molecular dyad by nanosecond pump-pump-probe spectroscopy.

The design of robust and inexpensive molecular photocatalysts for the conversion of abundant stable molecules like H2O and CO2 into an energetic carrier is one of the major fundamental questions for scientists nowadays. The outstanding challenge is to couple single photoinduced charge separation events with the sequential accumulation of redox equivalents at the catalytic unit for performing multielectronic catalytic reactions. Herein, double excitation by nanosecond pump-pump-probe experiments was used to interrogate the photoinduced charge transfer and charge accumulation on a molecular dyad composed of a porphyrin chromophore and a ruthenium-based catalyst in the presence of a reversible electron acceptor. An accumulative charge transfer state is unattainable because of rapid reverse electron transfer to the photosensitizer upon the second excitation and the low driving force of the forward photodriven electron transfer reaction. Such a method allows the fundamental understanding of the relaxation mechanism after two sequential photon absorptions, deciphering the undesired electron transfer reactions that limit the charge accumulation efficiency. This study is a step toward the improvement of synthetic strategies of molecular photocatalysts for light-induced charge accumulation and more generally, for solar energy conversion.

The effect of myoglobin crowding on the dynamics of water: an infrared study.

Solutions containing 8 and 32 wt% myoglobin are studied by means of infrared spectroscopy, as a function of temperature (290 K and lower temperatures), in the mid- and far-infrared spectral range. Moreover, ultrafast time-resolved infrared measurements are performed at ambient temperature in the O-D stretching region. The results evidence that the vibrational properties of water remain the same in these myoglobin solutions (anharmonicity, vibrational relaxation lifetime…) and in neat water. However, the collective properties of the water molecules are significantly affected by the presence of the protein: the orientational time increases, the solid-liquid transition is affected in the most concentrated solution and the dynamical transition of the protein is observed, from the point of view of water, even in the least concentrated solution, proving that the water and myoglobin dynamics are coupled.

Electron transfer in photosystem I.

This mini-review focuses on recent experimental results and questions, which came up since the last more comprehensive reviews on the subject. We include a brief discussion of the different techniques used for time-resolved studies of electron transfer in photosystem I (PS I) and relate the kinetic results to new structural data of the PS I reaction centre.

Modulation of primary radical pair kinetics and energetics in photosystem II by the redox state of the quinone electron acceptor Q(A).

Time-resolved photovoltage measurements on destacked photosystem II membranes from spinach with the primary quinone electron acceptor Q(A) either singly or doubly reduced have been performed to monitor the time evolution of the primary radical pair P680(+)Pheo(-). The maximum transient concentration of the primary radical pair is about five times larger and its decay is about seven times slower with doubly reduced compared with singly reduced Q(A). The possible biological significance of these differences is discussed. On the basis of a simple reversible reaction scheme, the measured apparent rate constants and relative amplitudes allow determination of sets of molecular rate constants and energetic parameters for primary reactions in the reaction centers with doubly reduced Q(A) as well as with oxidized or singly reduced Q(A). The standard free energy difference DeltaG degrees between the charge-separated state P680(+)Pheo(-) and the equilibrated excited state (Chl(N)P680)* was found to be similar when Q(A) was oxidized or doubly reduced before the flash (approximately -50 meV). In contrast, single reduction of Q(A) led to a large change in DeltaG degrees (approximately +40 meV), demonstrating the importance of electrostatic interaction between the charge on Q(A) and the primary radical pair, and providing direct evidence that the doubly reduced Q(A) is an electrically neutral species, i.e., is doubly protonated. A comparison of the molecular rate constants shows that the rate of charge recombination is much more sensitive to the change in DeltaG degrees than the rate of primary charge separation.

Effect of metal binding on electrogenic proton transfer associated with reduction of the secondary electron acceptor (QB) in Rhodobacter sphaeroides chromatophores.

The influence of metal ion (Cd(2+), Zn(2+), Ni(2+)) binding on the electrogenic phases of proton transfer connected with reduction of quinone Q(B) in chromatophores from Rhodobacter sphaeroides was studied by time-resolved electric potential changes. In the presence of metals, the electrogenic transients associated with proton transfer on first and second flash at pH 8 were found to be slower by factors of 3-6. This is essentially the same effect of metal binding that was observed on optical transients in isolated reaction centers (RC), where the metal ion was shown to inhibit proton transfer [Paddock, M. L., Graige, M. S., Feher, G., and Okamura, M. Y. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 6183-6188]. The effect of metal binding on the kinetics in chromatophores is, therefore, similarly attributed to inhibition of proton uptake, which becomes rate-limiting. A striking observation was an increase in the amplitude of the electrogenic proton-uptake phase after the first flash with bound metal ion. We attribute this to a loss of internal proton rearrangement, requiring that the protons that stabilize Q(B)(-) come from solution. In mutant RCs, in which His-H126 and His-H128 are replaced with Ala, the apparent binding of Cd(2+) and Ni(2+) was decreased, showing that the binding site of these metal ions is the same as found in RC crystals [Axelrod, H. L., Abresch, E. C., Paddock, M. L., Okamura, M. Y., and Feher, G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1542-1547]. Therefore, the unique proton entry point near His-H126, His-H128, and Asp-M17 that was identified in isolated RCs is also the entry point in chromatophores.

Fusion of chromatophores from photosynthetic bacteria with a supported lipid layer: characterization of the electric units.

Direct electrometric measurements of membrane potential changes are a valuable tool for study of vectorial transfer of electrons, protons, and ions. Commonly model membrane systems are created by fusion of lipid/protein vesicles with lipid-coated thin films. We characterized the electric units resulting from this process using chromatophores from the purple bacterium Rhodobacter sphaeroides and either a Mylar film or a planar modified gold electrode as support. Investigation of the shunting activity of the ionophore gramicidin on the flash-induced potential change demonstrates fusion of individual chromatophores to form independent 'blisters', which preserve an interior aqueous compartment. Under current-clamp conditions the photovoltage follows the change of the membrane potential of the individual blisters.

FTIR study of the primary electron donor of photosystem I (P700) revealing delocalization of the charge in P700(+) and localization of the triplet character in (3)P700.

The effect of global (15)N or (2)H labeling on the light-induced P700(+)/P700 FTIR difference spectra has been investigated in photosystem I samples from Synechocystis at 90 K. The small isotope-induced frequency shifts of the carbonyl modes observed in the P700(+)/P700 spectra are compared to those of isolated chlorophyll a. This comparison shows that bands at 1749 and 1733 cm(-)(1) and at 1697 and 1637 cm(-)(1), which upshift upon formation of P700(+), are candidates for the 10a-ester and 9-keto C=O groups of P700, respectively. A broad and relatively weak band peaking at 3300 cm(-)(1), which does not shift upon global labeling or (1)H-(2)H exchange, is ascribed to an electronic transition of P700(+), indicating that at least two chlorophyll a molecules (denoted P(1) and P(2)) participate in P700(+). Comparisons of the (3)P700/P700 FTIR difference spectrum at 90 K with spectra of triplet formation in isolated chlorophyll a or in RCs from photosystem II or purple bacteria identify the bands at 1733 and 1637 cm(-)(1), which downshift upon formation of (3)P700, as the 10a-ester and 9-keto C=O modes, respectively, of the half of P700 that bears the triplet (P(1)). Thus, while the P(2) carbonyls are free from interaction, both the 10a-ester and the 9-keto C=O of P(1) are hydrogen bonded and the latter group is drastically perturbed compared to chlorophyll a in solution. The Mg atoms of P(1) and P(2) appear to be five-coordinated. No localization of the triplet on the P(2) half of P700 is observed in the temperature range of 90-200 K. Upon P700 photooxidation, the 9-keto C=O bands of P(1) and P(2) upshift by almost the same amount, giving rise to the 1656(+)/1637(-) and 1717(+)/1697(-) cm(-)(1) differential signals, respectively. The relative amplitudes of these differential signals, as well as of those of the 10a-ester C=O modes, appear to be slightly dependent on sample orientation and temperature and on the organism used to generate the P700(+)/P700 spectrum. If it is assumed that the charge density on ring V of chlorophyll a, as measured by the perturbation of the 10a-ester or 9-keto C=O IR vibrations, mainly reflects the spin density on the two halves of the oxidized P700 special pair, a charge distribution ranging from 1:1 to 2:1 (in favor of P(2)) is deduced from the measurements presented here. The extreme downshift of the 9-keto C=O group of P(1), indicative of an unusually strong hydrogen bond, is discussed in relation with the models previously proposed for the PSI special pair.

Transient electron paramagnetic resonance spectroscopy on green-sulfur bacteria and heliobacteria at two microwave frequencies.

Spin polarized transient EPR spectra taken at X-band (9 GHz) and K-band (24 GHz) of membrane fragments of Chlorobium tepidum and Heliobacillus mobilis are presented along with the spectra of two fractions obtained in the purification of reaction centers (RC) from C. tepidum. The lifetime of P+. is determined by measuring the decay of the EPR signals following relaxation of the initial spin polarization. All samples except one of the RC fractions show evidence of light induced charge separation and formation of chlorophyll triplet states. The lifetime of P+. is found to be biexponential with components of 1.5 ms and 30 ms for C. tepidum and 1.0 and 4.5 ms for Hc. mobilis at 100 K. In both cases, the rates are assigned to recombination from F-X. The spin polarized radical pair spectra for both species are similar and those from Hc. mobilis at room temperature and 100 K are identical. In all cases, an emission/absorption polarization pattern with a net absorption is observed. A slight narrowing of the spectra and a larger absorptive net polarization is found at K-band. No out-of-phase echo modulation is observed. Taken together, the recombination kinetics, the frequency dependence of the spin polarization and the absence of an out-of-phase echo signal lead to the assignment of the spectra to the contribution from P+. to the state P+.F-X. The origin of the net polarization and its frequency dependence are discussed in terms of singlet-triplet mixing in the precursor. It is shown that the field-dependent polarization expected to develop during the 600-700 ps lifetime of P+.A-.0 is in qualitative agreement with the observed spectra. The identity that the acceptor preceding FX and the conflicting evidence from EPR, optical methods and chemical analyses of the samples are discussed.

Light gradients in spherical photosynthetic vesicles.

Light-gradient photovoltage measurements were performed on EDTA-treated thylakoids and on osmotically swollen thylakoids (blebs), both of spherical symmetry but of different sizes. In the case of EDTA vesicles, a negative polarity (due to the normal light gradient) was observed in the blue range of the absorption spectrum, and a positive polarity, corresponding to an inverse light gradient, was observed at lambda = 530 and lambda = 682 nm. The sign of the photovoltage polarity measured in large blebs (swollen thylakoids) is the same as that obtained for whole chloroplasts, although differences in the amplitudes are observed. An approach based on the use of polar coordinates was adapted for a theoretical description of these membrane systems of spherical symmetry. The light intensity distribution and the photovoltage in such systems were calculated. Fits to the photovoltage amplitudes, measured as a function of light wavelength, made it possible to derive the values of the dielectric constant of the protein, epsilons = 3, and the refractive index of the photosynthetic membrane for light propagating perpendicular and parallel to the membrane surface, nt = 1.42 and nn = 1.60, respectively. The latter two values determine the birefringence of the biological membrane, Deltan = nn - nt = 0.18.

Electron transfer in photosystem I reaction centers follows a linear pathway in which iron-sulfur cluster FB is the immediate electron donor to soluble ferredoxin.

Reaction centers of photosystem I contain three different [4Fe-4S] clusters named FX, FA, and FB. The terminal photosystem I acceptors (FA, FB) are distributed asymmetrically along the membrane normal, with one of them (FA or FB) being reduced from FX and the other one (FB or FA) reducing soluble ferredoxin. In the present work, kinetics of electron transfer has been measured in PSI from the cyanobacterium Synechocystis sp. PCC 6803 after inactivation of FB by treatment with HgCl2. Photovoltage measurements indicate that, in the absence of FB, reduction of FA by FX is still faster than the rate of FX reduction [(210 ns)-1]. Flash-absorption measurements show that the affinity of ferredoxin for HgCl2-treated PSI is only decreased by a factor of 3-4 compared to untreated photosystem I. The first-order rate of ferredoxin reduction by FA-, within the photosystem I/ferredoxin complex, has been calculated from measurements of P700+ decay. Compared to control PSI, this rate is several orders of magnitude smaller (6 s-1 versus 10(4)-10(6) s-1). Moreover, it is smaller than the rate of recombination from FA-, resulting in inefficient ferredoxin reduction (yield of 25%). After reconstitution of FB, about half of the reconstituted photosystem I reaction centers recover fast reduction of ferredoxin with kinetics similar to that of untreated photosystem I. These results support FB as the direct partner of ferredoxin and as the more distal cluster of photosystem I with respect to the thylakoid membrane, in accordance with a linear electron-transfer pathway FX-->FA-->FB-->ferredoxin.

Electron transfer in the heliobacterial reaction center: evidence against a quinone-type electron acceptor functioning analogous to A1 in photosystem I.

Membrane fragments from Heliobacillus mobilis were characterized using time resolved optical spectroscopy and photovoltage measurements in order to detect a possible participation of menaquinone (MQ), functioning analogous to the phylloquinone A1 in photosystem I, as intermediate in electron transfer from the primary acceptor A0 to the iron-sulfur cluster FX in the photosynthetic reaction center. The spectroscopic data obtained exclude that electron transfer from a semiquinone anion MQ- to FX occurred in the time window from 2 ns to 4 micros, where it would be expected in analogy to photosystem I. In the case of a prereduction of FX, only the primary pair P798+A0- was formed. The photovoltage data yielded a single kinetic phase with a time constant of 700 ps for the transmembrane electron transfer beyond A0; the relative amplitude of this phase suggests that it reflects electron transfer from A0- to FX.

A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700.

The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site-directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA-His676 and PsaB-His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls.

Energy and electron transfer upon selective femtosecond excitation of pigments in membranes of Heliobacillus mobilis.

Excitation energy transfer steps in membranes of Heliobacillus mobilis were directly monitored by transient absorption spectroscopy with a time resolution of 30 fs under selective excitation within the inhomogeneously broadened bacteriochlorophyll g QY band. The initial anisotropy was found to be > 0.4, indicating that the pigments are excitonically coupled. After initial decay of this anisotropy in < 50 fs, major sub-picosecond components associated with spectral equilibration were identified, corresponding to uphill energy transfer with a 300 fs time constant (812 nm excitation) and downhill energy transfer with 100 and 500 fs components (770 nm excitation). These equilibrations are ascribed predominantly to single excitation transfer steps, as anisotropy measurements showed that equilibration within spectrally similar pigments occurs on the same time scale as spectral equilibration, a situation which contrasts with that in photosystem I. Downhill energy transfer occurs to a significant extent directly to an energetically heterogeneous population of excited states as well as in a sequential way via gradually lower-lying pools of bacteriochlorophyll g. This finding supports a description in which all pigments, including the bluemost absorbing, are spatially organized in a random way rather than in clusters of spectrally similar species. Spectral equilibration is not entirely completed prior to formation of the primary radical pair P798 + A0-, which was found to proceed in a multiexponential way (time constants of 5 and 30 ps). No indication for the formation of radical species other than P798 + A0- on the time scale up to 100 ps was found.

FTIR spectroscopy of primary donor photooxidation in Photosystem I, Heliobacillus mobilis, and Chlorobium limicola. Comparison with purple bacteria.

The photooxidation of the primary electron donor in several Photosystem I-related organisms (Synechocystis sp. PCC 6803, Heliobacillus mobilis, and Chlorobium limicola f. sp. thiosulphatophilum) has been studied by light-induced FTIR difference spectroscopy at 100 K in the 4000 to 1200 cm(-1) spectral range. The data are compared to the well-characterized FTIR difference spectra of the photooxidation of the primary donor P in Rhodobacter sphaeroides (both wild type and the heterodimer mutant HL M202) in order to get information on the charge localization and the extent of coupling within the (bacterio)chlorophylls constituting the oxidized primary donors. In Rb. sphaeroides RC, four marker bands mostly related to the dimeric nature of the oxidized primary donor have been previously observed at ≈2600, 1550, 1480, and 1295 cm(-1). The high-frequency band has been shown to correspond to an electronic transition (Breton et al. (1992) Biochemistry 31: 7503-7510) while the three other marker bands have been described as phase-phonon bands (Reimers and Hush (1995) Chem Phys 197: 323-332). The absence of these bands in PS I as well as in the heterodimer HL M202 demonstrates that in P700(+) the charge is essentially localized on a single chlorophyll molecule. For both H. mobilis and C. limicola, the presence of a high-frequency band at ≈ 2050 and 2450 cm(-1), respectively, and of phase-phonon bands (at ≈ 1535 and 1300 cm(-1) in H. mobilis, at ≈ 1465 and 1280 cm(-1) in C. limicola) indicate that the positive charge in the photooxidized primary donor is shared between two coupled BChls. The structure of P840(+) in C. limicola, in terms of the resonance interactions between the two BChl a molecules constituting the oxidized primary donor, is close to that of P(+) in purple bacteria reaction centers while for H. mobilis the FTIR data are interpreted in terms of a weaker coupling of the two bacteriochlorophylls.

Photoelectric characterization of forward electron transfer to iron-sulfur centers in photosystem I.

The photoelectric response of oriented PS I membranes from the cyanobacterium Synechocystis 6803 has been investigated in the nanosecond time range. Besides an unresolved rapidly rising phase, there is a further positive electrogenic phase with a rise time constant of 220 +/- 20 ns. The amplitude of the 220-ns phase is 66 +/- 10% that of the subnanosecond phase. The fast phase contains two kinetic components faster than 100 ps, which have recently been resolved and attributed to primary charge separation (P+Ao-formation) and subsequent electron transfer to A1, respectively (Hecks, B., Wulf, K., Breton, J., Leibl, W., & Trissl, H.-W. (1994) Biochemistry 33, 8619-8624). The 220-ns phase is lost under conditions where iron-sulfur centers FA, FB, and Fx are prereduced, and its kinetics match the reoxidation kinetics of A1- as verified by absorbance change measurements at 380 nm. Therefore, this electrogenic phase is attributed to electron transfer to the iron-sulfur centers that function as further electron acceptors in the PS I reaction center. Gradual removal of FA and FB by urea treatment reveals that the amplitude of the 220-ns phase is linearly correlated with the fraction of FA,B present. However, complete removal of FA,B does not lead to a complete loss of the nanosecond phase but reduces its amplitude by more than a factor of 2 to yield an amplitude of 25-30% relative to the initial picosecond rise, with only a slight change in kinetics. The residual amplitude is further reduced when a large fraction of Fx is removed.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural and functional consequences of a Glu L212-->Lys mutation in the QB binding site of the photosynthetic reaction center of Rhodopseudomonas viridis.

The properties of the quinone acceptor complex in the photosynthetic reaction center of the atrazine-resistant Rhodopseudomonas viridis mutant A2 (Glu L212-->Lys) were studied by EPR spectroscopy and by photoelectric measurements. The EPR signal attributed to the semiquinone-iron (QB-Fe2+) was significantly different from wild type and resembled that found in PS II. Essentially normal oscillations of QB-Fe2+ were observed upon flash illumination. The kinetics of the first and the second electron transfer from QA to QB were characterized by a photoelectric double-flash method. Compared to wild type, the rate of the first electron transfer in the large majority of reaction centers was decreased drastically from k1 = (18 microseconds)-1 in the wild type to (70 ms)-1 in the mutant, whereas the second electron transfer was only slightly slowed down with a rate of k2 = (260 microseconds)-1 compared to (65 microseconds)-1 in wild type (pH 7). When the pH was raised above 10, in a major fraction of the reaction centers a fast kinetics of the first electron transfer, like that in wild type, reappeared. The experimental results are interpreted as an effect of the positive charge on the lysine causing a significant structural change of the QB binding pocket and a strongly diminished affinity for ubiquinone. The slow QA(-)-->QB electron transfer kinetics are thus attributed to ubiquinone binding, which is rate limiting. The possible role of the residue Glu L212, which is conserved in all purple bacteria, in electron and proton transfer to QB is discussed.

Primary charge separation in photosystem I: a two-step electrogenic charge separation connected with P700+A0- and P700+A1- formation.

A PS I membrane preparation from a PS II deficient mutant of Synechocystis sp. PCC 6803 (psb DI/DII/C) was investigated by picosecond photovoltage and fluorescence measurements. The photovoltage kinetics show two distinct phases. At low excitation energies the fast phase correlates with the fluorescence decay time constant of 22 +/- 4 ps. This phase is ascribed to the trapping of excitons as described by the reaction (AntiP700)* A0-->AntiP700+A0-. In addition to this phase, the photovoltage displays a second rising phase of smaller amplitude with a time constant of 50 +/- 15 ps. We assign the latter phase to further electron transfer from A0 to the secondary acceptor, A1. Assuming the protein as a homogeneous dielectric, our results suggest that the transmembrane distance A0-A1 spans only a small part (20 +/- 8%) of the distance P700-A1.

Why does the light-gradient photovoltage from photosynthetic organelles show a wavelength-dependent polarity?

The light-gradient photovoltage from photosynthetic organisms and organelles is thought to arise from the primary charge separation in the reaction centers. The current explanation of the effect is the stronger excitation of the membrane side of a vesicle facing the light source than the one on the opposite side. Together with the known orientation of reaction centers, this explanation predicts unequivocally the polarity of the photovoltage. However, a polarity opposite to the one expected has often been reported. A dependence of the polarity on the wavelength has been published but no explanation was given (Gräber, P., and H.-W. Trissl. 1981. FEBS Lett. 123:95-99). Here we report on a theoretical treatment of light propagation and interference in pigmented and nonpigmented multilayers. A model calculation is carried out for a pair of membranes, demonstrating the wavelength-dependent light distribution as well as the relative photovoltage and its polarity. When the membranes contain no chromophores or when the absorption coefficient is low, the predicted polarity to that expected from a simple macroscopic absorption behavior. The model is tested by comparing new photovoltage data obtained at 532 nm as well as in the blue and red absorption bands of chlorophyll in chloroplasts. It is concluded that outside the main absorption bands the amplitude and polarity of the photovoltage is determined by the ratio of the refractive indices of the membrane and the medium.

Evidence that serine L223 is involved in the proton transfer pathway to QB in the photosynthetic reaction center of Rhodopseudomonas viridis.

In the reaction center of purple photosynthetic bacteria, the reducing equivalents produced by primary charge separation are exported via an ubiquinone molecule working as a two-electron shuttle. This loosely-bound quinone, called QB, accepts in successive flashes two electrons from the tightly bound primary quinone acceptor QA, along with two protons from the external medium. The surrounding protein plays an important role in stabilizing the semiquinone anion and in providing a pathway for protons from the cytoplasmic phase to QB. Herbicides of the triazine type compete with QB for the binding pocket and their binding is controlled by nearby amino acid residues. We have studied the kinetics of the first and second electron transfer from QA to QB in two herbicide-resistant mutants from Rhodopseudomonas viridis, T1 (ArgL217-->His,Ser L223-->Ala) and MAV5 (Arg L217-->His, Val L220-->Leu), in order to determine whether these residues are involved in proton transfer to the reduced QB. The main effect of the mutant T1 was a drastic (600-fold at pH 7) decrease in the rate of the second electron transfer to QB compared to the wild type. In contrast, the rate of the second electron transfer in the mutant MAV5 was decreased only slightly (10-fold) in the pH range from 7 to 11. We attribute the inhibition of the second electron transfer in the Ser L223-->Ala mutation to an essential role of Ser L223 in the donation of the first proton to the reduced QB.(ABSTRACT TRUNCATED AT 250 WORDS)

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)