Andrzej Dobek

Modulation of Primary Radical Pair Kinetics and Energetics in Photosystem II by the Redox State of the Quinone Electron Acceptor QA

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.

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.

Mechanism of Recombination of the P(+)H(A)(-) Radical Pair in Mutant Rhodobacter Sphaeroides Reaction Centers with Modified Free Energy Gaps between P(+)B(A)(-) and P(+)H(A)(-).

The kinetics of recombination of the P(+)H(A)(-) radical pair were compared in wild-type reaction centers from Rhodobacter sphaeroides and in seven mutants in which the free energy gap, ΔG, between the charge separated states P(+)B(A)(-) and P(+)H(A)(-) was either increased or decreased. Five of the mutant RCs had been described previously, and X-ray crystal structures of two newly constructed complexes were determined by X-ray crystallography. The charge recombination reaction was accelerated in all mutants with a smaller ΔG than in the wild-type, and was slowed in a mutant having a larger ΔG. The free energy difference between the state P(+)H(A)(-) and the PH(A) ground state was unaffected by most of these mutations. These observations were consistent with a model in which the P(+)H(A)(-) → PH(A) charge recombination is thermally activated and occurs via the intermediate state P(+)B(A)(-), with a mean rate related to the size of the ΔG between the states P(+)B(A)(-) and P(+)H(A)(-) and not the ΔG between P(+)H(A)(-) and the ground state. A more detailed analysis of charge recombination in the mutants showed that the kinetics of the reaction were multiexponential, and characterized by ~0.5, ~1-3, and 7-17 ns lifetimes, similar to those measured for wild-type reaction centers. The exact lifetimes and relative amplitudes of the three components were strongly modulated by the mutations. Two models were considered in order to explain the observed multiexponentiality and modulation, involving heterogeneity or relaxation of P(+)H(A)(-) states, with the latter model giving a better fit to the experimental results.

Internal electrostatic control of the primary charge separation and recombination in reaction centers from Rhodobacter sphaeroides revealed by femtosecond transient absorption.

We report the observation of two conformational states of closed RCs from Rhodobacter sphaeroides characterized by different P(+)H(A)(-) --> PH(A) charge recombination lifetimes, one of which is of subnanosecond value (700 +/- 200 ps). These states are also characterized by different primary charge separation lifetimes. It is proposed that the distinct conformations are related to two protonation states either of reduced secondary electron acceptor, Q(A)(-), or of a titratable amino acid residue localized near Q(A). The reaction centers in the protonated state are characterized by faster charge separation and slower charge recombination when compared to those in the unprotonated state. Both effects are explained in terms of the model assuming modulation of the free energy level of the state P(+)H(A)(-) by the charges on or near Q(A) and decay of the P(+)H(A)(-) state via the thermally activated P(+)B(A)(-) state.

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.

Analysis of the temperature-dependence of P(+)HA(-) charge recombination in the Rhodobacter sphaeroides reaction center suggests nanosecond temperature-independent protein relaxation.

The temperature dependence of charge recombination of the pair P(+)HA(-) in isolated reaction centers from the purple bacterium Rhodobacter sphaeroides with prereduced quinone QA was studied by sub-nanosecond to microsecond time-scale transient absorption. Overall, the kinetics slowed down substantially upon cooling from room temperature to ∼200 K, and then remained virtually unchanged down to 77 K, indicating the coexistence of two competitive pathways of charge recombination, a thermally-activated pathway appearing only above ~200 K and a temperature-independent pathway. In our modelling, the thermally activated pathway includes an uphill electron transfer from HA(-) to BA(-) leading to transient formation of the state P(+)BA(-), whereas the temperature-independent pathway is due to direct downhill electron transfer from HA(-) to P(+). At all temperatures studied, the kinetics could be approximated by a four-component decay. Detailed analysis of the lifetimes and amplitudes of particular phases over the range of temperatures suggests that the kinetically resolved phases reveal the consecutive appearance of three conformational states characterized by an increasing free energy gap between the states P(+)BA(-) and P(+)HA(-). The initial gap between these states was estimated to be only ~8 meV, the intermediate gap being ~92 meV, and the final gap ~135 meV, with no dependence on temperature. It was also calculated through a very straightforward approach that the relaxation process from the initial to the intermediate state occurs within 0.6 ± 0.1 ns, whereas the second step of relaxation from the intermediate to the final state takes 11 ± 2 ns. Both phases of the protein relaxation process are essentially temperature-independent. Possible alternative models to describe the experimental data that cannot be definitely excluded are also discussed.

Excitation and electron transfer in reaction centers from Rhodobacter sphaeroides probed and analyzed globally in the 1-nanosecond temporal window from 330 to 700 nm

Global analysis of a set of room temperature transient absorption spectra of Rhodobacter sphaeroides reaction centers, recorded in wide temporal and spectral ranges and triggered by femtosecond excitation of accessory bacteriochlorophylls at 800 nm, is presented. The data give a comprehensive review of all spectral dynamics features in the visible and near UV, from 330 to 700 nm, related to the primary events in the Rb. sphaeroides reaction center: excitation energy transfer from the accessory bacteriochlorophylls (B) to the primary donor (P), primary charge separation between the primary donor and primary acceptor (bacteriopheophytin, H), and electron transfer from the primary to the secondary electron acceptor (ubiquinone). In particular, engagement of the accessory bacteriochlorophyll in primary charge separation is shown as an intermediate electron acceptor, and the initial free energy gap of approximately 40 meV, between the states P(+)B(A)(-) and P(+)H(A)(-) is estimated. The size of this gap is shown to be constant for the whole 230 ps lifetime of the P(+)H(A)(-) state. The ultrafast spectral dynamics features recorded in the visible range are presented against a background of results from similar studies performed for the last two decades.

Re-examination of primary radical pair recombination in Rp. viridis with QA reduced

Charge recombination in the primary radical pair P+H− of the purple photosynthetic bacterium Rp. viridis with pre-reduced secondary acceptor QA, has been studied by time-resolved photovoltage measurements and transient absorption spectroscopy with a time resolution of 1 ns. The lifetime of P+H− was found to be 2.4–3 ns in intact membranes and ∼5 ns in isolated reaction centers, in contrast to a lifetime of ∼15 ns which has been widely accepted in the literature for reaction centers. The origin of the difference between lifetimes of P+H− in membranes and isolated reaction centers is discussed.

Sodium chloride-induced conformational change in tRNA as measured by circular dichroism

Abstract The effect of 0.01-1 M sodium ions on the conformation of the folded brewer’s yeast tRNAPhe was examined by circular dichroism method in the region 200-350 nm. The minimum peak at about 210 nm for tRNA solution with 50 mM sodium chloride showed a decrease in magnitude by 26-30% in comparison to that recorded for the solution of higher NaCl content. The depths of the peaks at 225 nm and 233 nm for two solutions with the lowest sodium chloride concentrations (cNaCl = 10mM, cNaCl = 50mM) were changed by 3-10% relative to the those in the spectra of other samples, for the 260 nm maximum peak a decrease in height was 21-25%. In the region 300-350 nm no significant difference was observed. The results point to a strong relationship between concentration of sodium ions and stabilization process of secondary and tertiary tRNA structure, which indicates the influence of sodium ions on stacking and base-pairing interactions.

A0 → A1 Electron Transfer in Chlamydomonas reinhardtii PS I with Replaced A0 Axial Ligand

Replacement of methionine, the natural axial ligand to the primary electron acceptor (A0) in Photosystem I, with a series of different amino acids results in dramatic increase of the A0 − lifetime from 20 ps in wild type to a few nanoseconds in the mutants in the case of Chlamydomonas reinhardtii (Ramesh et al. 2004, 2007). This effect is similar independently if the mutation affects A-side or B-side A0. This observation confirms an existence of two equivalent primary electron acceptors in both symmetric branches of Photosystem I in Chlamydomonas reinhardtii, which makes this photosystem unusual among other photosystems (from purple bacteria, PS II), which are essentially unidirectional. However, it is still not clear if the bidirectionality of electron transfer in Photosystem I is complete, i.e. if the electron from A0 − reaches A1 in both branches or takes another route in the “non-active” branch. In order to solve this issue, in this contribution we attempted to compare kinetics of A0 − reoxidation to the kinetics of A1 − formation in the case of B-side A0 mutant with methionine replaced by serine.