Geometry for the Primary Electron Donor and the Bacteriopheophytin Acceptor in Rhodopseudomonas viridis Photosynthetic Reaction Centers

The tetrapyrrole electron donors and acceptors (bacteriochlorophyll, BCh; bacteriopheophytin, BPh) within the bacterial photosynthetic reaction center (RC) are arranged with a specific geometry that permits rapid (picosecond time scale) electron tunneling to occur between them. Here we have measured the angle between the molecular planes of the bacteriochlorophyll dimer (primary donor), B₂, and the acceptor bacteriopheophytin, H, by analyzing the dichroism of the absorption change associated with H reduction, formed by photoselection with RCs of Rhodopseudomonas viridis. This angle between molecular planes is found to be 60° ± 2. This means that the ultrafast electron tunneling must occur between donors and acceptors that are fixed by the protein to have a noncoplanar alignment. Nearly perpendicular alignments have been determined for other electron tunneling complexes involving RCs. These geometries can be contrasted with models proposed for heme-heme electron transfer complexes, which have emphasized that mutually parallel orientations should permit the most kinetically facile transfers.     

Reaction of Reconstituted Acceptor Quinone and Dynamic Equilibration of Electron Transfer in the Photosystem I Reaction Center

Electron transfer mechanism in the spinach photosystem I reactioncenter that contains artificial quinones in place of phylloquinone(2-methyl-3-phytyl-1,4-naphthoquinone, vitamin K₁) as the secondaryelectron acceptor, Q_ø (or A₁) was discussed. (1) Mostof the reconstituted quinones oxidized the primary acceptorchlorophyll a, A⁰, at a rate rapid enough to compete againstthe charge recombination between A₀^– and the oxidizeddonor chlorophyll P700⁺. (2) The pathway of electron transferfrom the semiquinone^– varied depending on the redox potentialvalue of each semiquinone^– /quinone couple. Low potentialquinones reduced the tertiary acceptor iron-sulfur center, F_x,while the high potential ones reduced P700⁺ directly with a200-µs halftime. (3) The E_m value of each semiquinone^–/quinone couple in situ in the reaction center was estimatedto be shifted by about 0.3 volt to the negative side from theirhalf wave redox potential values that were measured polarographicallyin dimethylformamide. The shift seems to represent the acceptorproperty of the protein environment at the Q_ø site. (4)The E_m of reconstituted phylloquinone was estimated to be 50–80mV more negative than that of F_x. (5) The mechanism of efficientelectron transfer in the reaction center was discussed basedon the dynamic equilibria between the electron transfer componentsand on the estimated E_m values. (Received April 9, 1994; Accepted July 7, 1994)     

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.     

Charge Separation in Photosynthetic Reaction Centers under Femtosecond Excitation

The process of electron transfer from the primary electron donor P* to the primary electron acceptor B_A in the reaction center of Rhodobacter sphaeroides R-26 under 30 fsec pulse excitation was studied in this work with the aim of establishing a relationship between the nuclear subsystem motion and charge transfer. For this purpose the fsec and psec oscillations in the bands of stimulated emission of P* and in the band of reaction product B _A ^- at 1020 nm were investigated. It was established that the reversible formation of the P⁺B _A ^- state is characterized by two vibration modes (130 and 320 cm^-1) and connected with an arrival of the wavepacket induced by fsec excitation to the intersection of potential surfaces P*B_A and P⁺B _A ^- . The irreversible formation of the P⁺B _A ^- state with the time constant of 3 psec is followed by oscillations with frequencies of 9 and 33 cm^-1. These results show that the irreversibility of electron transfer is determined by two factors: 1) by a difference between the energy width of the wavepacket and the gap between the named surfaces; 2) by a difference between the duration of wavepacket residence near the intersection of the surfaces and the relaxation time of the P⁺B _A ^- state.     

Protein Dielectric Environment Modulates the Electron-Transfer Pathway in Photosynthetic Reaction Centers

The replacement of tyrosine by aspartic acid at position M210 in the photosynthetic reaction center of Rhodobacter sphaeroides results in the generation of a fast charge recombination pathway that is not observed in the wild-type. Apparently, the initially formed charge-separated state (cation of the special pair, P, and anion of the A-side bacteriopheophytin, H_A) can decay rapidly via recombination through the neighboring bacteriochlorophyll (B_A) soon after formation. The charge-separated state then relaxes over tens of picoseconds and recombination slows to the hundreds-of-picoseconds or nanosecond timescale. This dielectric relaxation results in a time-dependent blue shift of B_A^– absorption, which can be monitored using transient absorbance measurements. Protein dynamics also appear to modulate the electron transfer between H_A and the next electron carrier, Q_A (a ubiquinone). The kinetics of this reaction are complex in the mutant, requiring two kinetic terms, and the spectra associated with the two terms are distinct; a red shift of the H_A ground-state bleaching is observed between the shorter and longer H_A-to-Q_A electron-transfer phases. The kinetics appears to be pH-independent, suggesting a negligible contribution of static heterogeneity originating from protonation/deprotonation in the ground state. A dynamic model based on the energy levels of the two early charge-separated states, P⁺B_A^– and P⁺H_A^–, has been developed in which the energetics of these states is modulated by fast protein dielectric relaxations and this in turn alters both the kinetic complexity of the reaction and the reaction pathway.     

Bacteriochlorophyll and Photosynthetic Reaction Centers in Rhizobium Strain BTAi 1

Rhizobium strain BTAi 1, which nodulates both stems and roots of Aeschynomene indica L., formed bacteriochlorophyll and photosynthetic reaction centers resembling those of purple photosynthetic bacteria when grown aerobically ex planta under a light-dark cycle. Bacteriochlorophyll formation was not observed under continuous dark or light growth conditions. The amount of pigment formed was similar to that previously found in aerobic photosynthetic bacteria. Stem nodules appear to fix nitrogen photosynthetically, as illumination of A. indica stem nodules with near-infrared light resulted in an enhanced rate of acetylene reduction. Near-infrared light did not enhance acetylene reduction when either A. indica or soybean root nodules were illuminated. The BTAi 1 isolate can be differentiated from members of the family Rhodospirillaceae by several criteria.     

Photosynthetic Electron Transfer in Preparations of the Cyanobacterium Spirulina platensis

Electron transfer activity in intact trichomes of Spirulina platensis (Nordst.) Geitl. can be observed with either CO₂ or methylviologen as the Hill acceptor. Ferricyanide cannot penetrate the intact trichomes, but photoreduction of this oxidant can be observed when mediated by lipophilic oxidants such as p-phenylenediamine or 2,5-dimethyl-p-benzoquinone. The insensitivity of these reactions to dibromothymoquinone indicates that they are due largely to the activity of photosystem II. Direct photoreduction of ferricyanide can be observed in spheroplasts of Spirulina, indicating that such preparations have altered permeability properties when compared with intact trichomes. Preparation of these spheroplasts, which are osmotically fragile, requires that intact trichomes be washed with KCl and EDTA to induce lysozyme sensitivity and thereby allow digestion of the cell wall. The KCl/EDTA washing procedure used for spheroplast preparation alters the permeability of Spirulina trichomes, as evidenced by the ability of these preparations to photoreduce ferricyanide. This photoreduction reaction is insensitive to dibromothymoquinone, and is stimulated by high concentrations of divalent cations. During assays, the reaction is inhibited by the inclusion of polyethyleneglycol as an osmotic protectant. Photoreduction of methylviologen and NADP⁺ is also observed in the washed trichomes, along with an endogenously catalyzed photoreduction of O₂ to H₂O₂. Photophosphorylation cannot be observed in the washed preparations, but cyclic photophosphorylation with phenazinemethosulfate is observed after mild sonication. These results indicate that KCl/EDTA-washed trichomes of S. platensis retain the full range of energy transducing capacities associated with thylakoid membranes of the intact trichomes; the washing procedure facilitates spheroplast formation and alters, but does not abolish, permeability barriers in these preparations.     

A Nonadiabatic Theory for Ultrafast Catalytic Electron Transfer: A Model for the Photosynthetic Reaction Center

A non-adiabatic theory of Electron Transfer (ET), which improves the standard theory near the inversion point and becomes equivalent to it far from the inversion point, is presented. The complex amplitudes of the electronic wavefunctions at different sites are used as Kramers variables for describing the quantum tunneling of the electron in the deformable potential generated by its environment (nonadiabaticity) which is modeled as a harmonic classical thermal bath. After exact elimination of the bath, the effective electron dynamics is described by a discrete nonlinear Schrödinger equation with norm preserving dissipative terms and a Langevin random force, with a frequency cut-off, due to the thermalized phonons. This theory reveals the existence of a specially interesting marginal case when the linear and nonlinear coefficients of a two electronic states system are appropriately tuned for forming a Coherent Electron-Phonon Oscillator (CEPO). An electron injected on one of the electronic states of a CEPO generates large amplitude charge oscillations (even at zero temperature) associated with coherent phonon oscillations and electronic level oscillations. This fluctuating electronic level may resonate with a third site which captures the electron so that Ultrafast Electron Transfer (UFET) becomes possible. Numerical results are shown where two weakly interacting sites, a donor and a catalyst, form a CEPO that triggers an UFET to an acceptor. Without a catalytic site, a very large energy barrier prevents any direct ET. This UFET is shown to have many qualitative features similar to those observed in the primary charge separation in photosynthetic reaction centers. We suggest that more generally, CEPO could be a paradigm for understanding many selective chemical reactions involving electron transfer in biosystems.     

Effects of Oxygen,Heavy Water,and Glycerol on Electron Transfer in the Acceptor Part of <Emphasis Type=Italic>Rhodobacter sphaeroides</Emphasis> Reaction Centers

The kinetics of electron transfer between primary and secondary quinone acceptors of the photosynthetic reaction center (RC) of the purple bacterium Rhodobacter sphaeroides wild type was studied at the wavelengths 400 and 450 nm. It was shown that removing of molecular oxygen from RC preparations slowed down the fast phase of the process from 4–4.5 μsec to tens of microseconds. Similar effects were observed after the incubation of RC in heavy water for 72 h or glycerol addition (90% v/v) to RC preparations. The observed effects are interpreted in terms of the influence of these agents on the hydrogen bond system of the RC. The state of this system can determine the formation of different RC conformations that are characterized by different rates of electron transfer between quinone acceptors. __________ Translated from Biokhimiya, Vol. 70, No. 11, 2005, pp. 1541–1547. Original Russian Text Copyright ? 2005 by Knox, Baptista, Uchoa, Zakharova.     

Photochemical Electron Transport in Photosynthetic Reaction Centers from Rhodopseudomonas spheroides: III. Effects of Orthophenanthroline and Other Chemicals

Reaction centers from Rhodopseudomonas spheroides mediate the photochemical oxidation of cytochrome c (cyt c), and show a time-varying fluorescence of P870. Analyses of these effects indicate that the reaction centers contain a primary photochemical electron acceptor capable of holding one electron. Native or added ubiquinone (UQ) can act as a secondary electron acceptor. Orthophenanthroline (o-phen) blocks electron transfer from primary to secondary acceptors, and allows the primary acceptor to be exhibited in the foregoing experiments. Other chelators (with the possible exception of 8-hydroxyquinoline) and dichlorophenyldimethylurea (DCMU) are without apparent effect on reaction centers. o-Phen also inhibits the primary photochemical act in reaction centers; this effect is prevented by the presence of UQ. 2-n-Nonyl-4-hydroxyquinoline-N-oxide (NQNO) inhibits the primary photochemistry in reaction centers but does not affect secondary electron transfer.     

Electron Transfer in the Photosynthetic Membrane: Influence of PH and Surface Potential on the P-680 Reduction Kinetics

The primary electron donor P-680 of the Photosystem-II reaction center was photoxidized by a short flash given after dark adaptation of photosynthetic membranes in which oxygen evolution was inhibited. The P-680⁺ reduction rate was measured under different conditions of pH and salt concentration by following the recovery of the absorption change at 820 nm. As previously reported for Tris-washed chloroplasts (Conjeaud, H., and P. Mathis, 1980, Biochim. Biophys. Acta, 590:353-359) a fast phase of P-680⁺ reduction slows down as the bulk pH decreases. When salt concentration increases, this fast phase becomes faster for pH above 4.5-5 and slower below. A quantitative interpretation is proposed in which the P-680⁺ reduction kinetics by the secondary electron donor Z are controlled by the local pH. This pH, at the membrane level, can be calculated using the Gouy-Chapman theory. A good fit of the results requires to assume that the surface charge density of the inside of the membrane, near the Photosystem-II reaction center, is positive at low pH values and becomes negative as the pH increases, with a local isoelectric point ~4.8. These results lead us to propose a functional scheme in which a pH-dependent proton release is coupled to the electron transfer between secondary and primary donors of Photosystem-II. The H⁺/e ratio varies from 1 at low pH to 0 at high pH, with a real pK ~6.5 for the protonatable species.     

Chlorophyll Chemistry Before and After Crystals of Photosynthetic Reaction Centers

The experimental, theoretical and structural research leading to the identification and characterization of the (bacterio) chlorophyll species that mediate the primary events of solar energy transduction and dynamics is reviewed and examined from the author's perspective.     

Mathematical Simulation of Electron Transport in the Primary Photosynthetic Processes

Biochemistry (Moscow) - Summarized results of investigation of regulation of electron transport and associated processes in the photosynthetic membrane using methods of mathematical and computer...     

On the Ratio of Photosynthetic Reaction Centers RC2/RC1 in Chlorella

Chlorella pyrenoidosa (Emerson strain) was grown under high and under low irradiance. Measurements of the O₂ flash yield and the P700 absorption change gave estimates for the ratio of photoreaction centers RC2/RC1 at 1.2 to 1.5. A background light 1 did not increase flash yield.     

The Effect of Polyene Antibiotics on Photosynthetic Electron Transfer

The effect of four polyene antibiotics and digitonin on photosyntheticelectron transfer by maize mesophyll chloroplasts has been investigated.All five compounds, at concentrations between 0.1 mM and 20mM, inhibited photosystem 2 activity as measured by the photo-reductionof ferricyanide and 2,6-dichlorophenolindophenol from water.Etruscomycin, amphotericin B, and digitonin were more inhibitorythan filipin and nystatin. Photosystem 1 activity was inhibitedby 1 mM concentrations of etruscomycin, amphotericin B, anddigitonin but not by filipin and nystatin. In all cases whereinhibition occurred, it was temperature dependent. The inhibition of photosystem 1 activity could be relieved byplastocyanin. Etruscomycin and digitonin, at concentrationsof 0.5 mM and above, caused disintegration of the chloroplasts,and this disintegration was accompanied by a two- to three-foldincrease in photosystem 1 activity in the presence of plastocyanin.It is concluded that the action of polyene antibiotics resultsin the release of plastocyanin from its site in the photosyntheticelectron transfer chain. The results are discussed in termsof the abilities of polyene antibiotics and digitonin to formcomplexes with sterols.     

Photosynthetic Unit Organization in a Red Alga : Relationships between Light-Harvesting Pigments and Reaction Centers

The relative concentration of biliproteins, phycobilisomes, chlorophyll a, and reaction centers I and II are reported for Neoagardhiella bailyei, a macrophytic red alga collected in the field and compared with Anacystis nidulans, a cyanobacterium cultured in the laboratory. The ratios of chlorophyll to reaction center I, of chlorophyll to reaction center II, and the mass of phycobiliprotein per reaction center II are quite similar in Neoagardhiella and Anacystis, supporting the concept that the red algal chloroplast is derived from a cyanobacterial progenitor. The ratios of reaction center I to reaction center II are about 2.3 in both species. The Anacystis phycobilisome has about 40% of the mass of the Neoagardhiella phycobilisome, 4.9 and 14.9 × 10⁶ daltons, respectively. The reaction center II/phycobilisome ratio is about 1.7 in Anacystis and 4.1 in Neoagardhiella. Phycobilisome size and physical restrictions on phycobilisome packing may be a major constraint on the reaction center II-phycobilisome association and the assembly of the photosynthetic membrane in both the red algae and cyanobacteria.     

Early Bacteriopheophytin Reduction in Charge Separation in Reaction Centers of Rhodobacter sphaeroides

A question at the forefront of biophysical sciences is, to what extent do quantum effects and protein conformational changes play a role in processes such as biological sensing and energy conversion? At the heart of photosynthetic energy transduction lie processes involving ultrafast energy and electron transfers among a small number of tetrapyrrole pigments embedded in the interior of a protein. In the purple bacterial reaction center (RC), a highly efficient ultrafast charge separation takes place between a pair of bacteriochlorophylls: an accessory bacteriochlorophyll (B) and bacteriopheophytin (H). In this work, we applied ultrafast spectroscopy in the visible and near-infrared spectral region to Rhodobacter sphaeroides RCs to accurately track the timing of the electron on B_A and H_A via the appearance of the B_A and H_A anion bands. We observed an unexpectedly early rise of the H_A⁻ band that challenges the accepted simple picture of stepwise electron transfer with 3 ps and 1 ps time constants. The implications for the mechanism of initial charge separation in bacterial RCs are discussed in terms of a possible adiabatic electron transfer step between B_A and H_A, and the effect of protein conformation on the electron transfer rate.