Primary processes of charge separation in reaction centers of YM210L/FM197Y and YM210L mutants of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Difference femtosecond absorption spectroscopy with 20-fsec temporal resolution was applied to study a primary stage of charge separation and transfer processes in reaction centers of YM210L and YM210L/FM197Y site-directed mutants of the purple bacterium Rhodobacter sphaeroides at 90 K. Photoexcitation was tuned to the absorption band of the primary electron donor P at 880 nm. Coherent oscillations in the kinetics of stimulated emission of P* excited state at 940 nm and of anion absorption of monomeric bacteriochlorophyll B_A⁻ at 1020 nm were monitored. The absence of tyrosine YM210 in RCs of both mutants leads to strong slowing of the primary reaction P* → P⁺B_A⁻ and to the absence of stabilization of separated charges in the state P⁺B_A⁻. Mutation FM197Y increases effective mass of an acetyl group of pyrrole ring I in the bacteriochlorophyll molecule P_B of the double mutant YM210L/FM197Y by a hydrogen bond with OH-TyrM197 group that leads to a decrease in the frequency of coherent nuclear motions from 150 cm⁻¹ in the single mutant YM210L to ∼100 cm⁻¹ in the double mutant. Oscillations with 100–150 cm⁻¹ frequencies in the dynamics of the P* stimulated emission and in the kinetics of the reversible formation of P⁺B_A⁻ state of both mutants reflect a motion of the P_B molecule relatively to P_A in the area of mutual overlapping of their pyrrole rings I. In the double mutant YM210L/FM197Y the oscillations in the P* emission band and the B_A⁻ absorption band are conserved within a shorter time ∼0.5 psec (1.5 psec in the YM210L mutant), which may be a consequence of an increase in the number of nuclei forming a wave packet by adding a supplementary mass to the dimer P.     

Picosecond spectroscopy of the charge separation in reaction centers of Chloroflexus aurantiacus with selective excitation of the primary electron donor

Absorbance changes in the picosecond region were studied in isolated reaction centers of the green photosynthetic bacterium Chloroflexus aurantiacus upon selective excitation of the primary electron donor, P, at 870 nm. The results indicate that the first observed state is an excited state of P (P1) which appears to transfer an electron to a bacteriochlorophyll a molecule absorbing at 812 nm (B₁) in 10 ± 2 ps as indicated by a bleaching at this wavelength. This reaction is followed by a rapid electron transfer (3 ± 1 ps) from B₁⁻ to bacteriopheophytin a, so that the fraction of reaction centers in the state P⁺B₁⁻ remains small during the experiment. An apparent bleaching at 925 nm is ascribed to stimulated emission from excited P, which emission disappears upon formation of P⁺. The difference between these time constants for electron transfer and those observed for the same reactions in reaction centers of the purple photosynthetic bacterium Rhodopseudomonas (Rhodobacter) sphaeroides is discussed in terms of the energy difference between P1 and P⁺B₁⁻, which appears to be larger for C. aurantiacus.     

Electron transfer in deuterated reaction centers of Rhodobacter sphaeroides at 90 K according to femtosecond spectroscopy data

The primary act of charge separation was studied in P⁺B_A ^– and P⁺H_A ^– states (P, primary electron donor; B_A and H_A, primary and secondary electron acceptor) of native reaction centers (RCs) of Rhodobacter sphaeroides R-26 using femtosecond absorption spectroscopy at low (90 K) and room temperature. Coherent oscillations were studied in the kinetics of the stimulated emission band of P* (935 nm), of absorption band of B_A ^– (1020 nm) and of absorption band of H_A (760 nm). It was found that in native RCs kept in heavy water (D₂O) buffer the isotopic decreasing of basic oscillation frequency 32 cm ^–1 and its overtones takes place by the same factor 1.3 in the 935, 1020, and 760 nm bands in comparison with the samples in ordinary water H₂O. This suggests that the femtosecond oscillations in RC kinetics with 32 cm ^–1 frequency may be caused by rotation of hydrogen-containing groups, in particular the water molecule which may be placed between primary electron donor P_B and primary electron acceptor B_A. This rotation may appear also as high harmonics up to sixth in the stimulated emission of P*. The rotation of the water molecule may modulate electron transfer from P* to B_A. The results allow for tracing of the possible pathway of electron transfer from P* to B_A along a chain consisting of polar atoms according to the Brookhaven Protein Data Bank (1PRC): Mg(P_B)-N-C-N(His M200)-HOH-O = B_A. We assume that the role of 32-cm ^–1 modulation in electron transfer along this chain consists of a fixation of electron density at B_A ^– during a reversible electron transfer, when populations of P* and P⁺B_A ^– states are approximately equal.     

Charge separation in Rhodobacter sphaeroides mutant reaction centers with increased midpoint potential of the primary electron donor

Primary charge separation dynamics in four mutant reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides with increased midpoint potential of the primary electron donor P (M160LH, L131LH, M197FH, and M160LH + L131LH + M197FH) have been studied by femtosecond transient absorption spectroscopy at room temperature. The decay of the excited singlet state in the wild-type and mutant RCs is complex and has two main exponential components, which indicates heterogeneity of electron transfer rates or the presence of reverse electron transfer reactions. The radical anion band of monomeric bacteriochlorophyll B_A at 1020 nm was first observed in transient absorbance difference spectra of single mutants. This band remains visible, although with somewhat reduced amplitude, even at delays up to tens of picoseconds when stimulated emission is absent and the reaction centers are in the P⁺H _A ^? state. The presence of this band in this time period indicates the existence of thermodynamic equilibrium between the P⁺B _A ^? H_A and P⁺B_AH _A ^? states. The data give grounds for assuming that the value of the energy difference between the states P*, P⁺B _A ^? H_A, and P⁺B_AH _A ^? at early times is of the same order of magnitude as the energy kT at room temperature. Besides, monomeric bacteriochlorophyll B_A is found to be an immediate electron acceptor in the single mutant RCs, where electron transfer is hampered due to increased energy of the P⁺B _A ^? state with respect to P*.     

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to QB in Rhodobacter sphaeroides reaction centers

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q_A⁻Q_B→Q_AQ_B⁻ (k_AB⁽¹⁾) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k_AB⁽¹⁾, attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q_B [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (−), 1601 (−) and 1467 (+) cm⁻¹, characteristic of Q_A reduction. The time evolution of the spectra shows reoxidation of Q_A⁻ and concomitant reduction of Q_B with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q_B⁻ occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm⁻¹ [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q_B in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q_A⁻ to Q_B and the identification of Glu-L212 as the main proton acceptor in the state Q_AQ_B⁻.     

Properties of mutant reaction centers of Rhodobacter sphaeroides with substitutions of histidine L153, the axial Mg2+ ligand of bacteriochlorophyll BA

Mutant reaction centers (RC) from Rhodobacter sphaeroides have been studied in which histidine L153, the axial ligand of the central Mg atom of bacteriochlorophyll B_A molecule, was substituted by cysteine, methionine, tyrosine, or leucine. None of the mutations resulted in conversion of the bacteriochlorophyll B_A to a bacteriopheophytin molecule. Isolated H(L153)C and H(L153)M RCs demonstrated spectral properties similar to those of the wild-type RC, indicating the ability of cysteine and methionine to serve as stable axial ligands of the Mg atom of bacteriochlorophyll B_A. Because of instability of mutant H(L153)L and H(L153)Y RCs, their properties were studied without isolation of these complexes from the photosynthetic membranes. The most prominent effect of the mutations was observed with substitution of histidine by tyrosine. According to the spectral data and the results of pigment analysis, the B_A molecule is missing in the H(L153)Y RC. Nevertheless, being associated with the photosynthetic membrane, this RC can accomplish photochemical charge separation with quantum yield of approximately 7% of that characteristic of the wild-type RC. Possible pathways of the primary electron transport in the H(L153)Y RC in absence of photochemically active chromophore are discussed.     

Primary charge separation in native and plant pheophytin a-modified reaction centers of Chloroflexus aurantiacus: Ultrafast transient absorption measurements at low temperature

Ultrafast transient absorption (TA) spectroscopy was used to study electron transfer (ET) at 100 K in native (as isolated) reaction centers (RCs) of the green filamentous photosynthetic bacterium Chloroflexus (Cfl.) aurantiacus. The rise and decay of the 1028 nm anion absorption band of the monomeric bacteriochlorophyll a molecule at the B_A binding site were monitored as indicators of the formation and decay of the P⁺B_A⁻ state, respectively (P is the primary electron donor, a dimer of bacteriochlorophyll a molecules). Global analysis of the TA data indicated the presence of at least two populations of the P^⁎ excited state, which decay by distinct means, forming the state P⁺H_A⁻ (H_A is a photochemically active bacteriopheophytin a molecule). In one population (~65 %), P^⁎ decays in ~2 ps with the formation of P⁺H_A⁻ via a short-lived P⁺B_A⁻ intermediate in a two-step ET process P^⁎ → P⁺B_A⁻→ P⁺H_A⁻. In another population (~35 %), P^⁎ decays in ~20 ps to form P⁺H_A⁻ via a superexchange mechanism without producing measurable amounts of P⁺B_A⁻. Similar TA measurements performed on chemically modified RCs of Cfl. aurantiacus containing plant pheophytin a at the H_A binding site also showed the presence of two P^⁎ populations (~2 and ~20 ps), with P^⁎ decaying through P⁺B_A⁻ only in the ~2 ps population. At 100 K, the quantum yield of primary charge separation in native RCs is determined to be close to unity. The results are discussed in terms of involving a one-step P^⁎ → P⁺H_A⁻ superexchange process as an alternative highly efficient ET pathway in Cfl. aurantiacus RCs.     

Effect of the P700 pre-oxidation and point mutations near A0 on the reversibility of the primary charge separation in Photosystem I from Chlamydomonas reinhardtii

Time-resolved fluorescence studies with a 3-ps temporal resolution were performed in order to: (1) test the recent model of the reversible primary charge separation in Photosystem I (Müller et al., 2003; Holwzwarth et al., 2005, 2006), and (2) to reconcile this model with a mechanism of excitation energy quenching by closed Photosystem I (with P700 pre-oxidized to P700⁺). For these purposes, we performed experiments using Photosystem I core samples isolated from Chlamydomonas reinhardtii wild type, and two mutants in which the methionine axial ligand to primary electron acceptor, A₀, has been change to either histidine or serine. The temporal evolution of fluorescence spectra was recorded for each preparation under conditions where the “primary electron donor,” P700, was either neutral or chemically pre-oxidized to P700⁺. For all the preparations under study, and under neutral and oxidizing conditions, we observed multiexponential fluorescence decay with the major phases of ∼ 7 ps and ∼ 25 ps. The relative amplitudes and, to a minor extent the lifetimes, of these two phases were modulated by the redox state of P700 and by the mutations near A₀: both pre-oxidation of P700 and mutations caused slight deceleration of the excited state decay. These results are consistent with a model in which P700 is not the primary electron donor, but rather a secondary electron donor, with the primary charge separation event occurring between the accessory chlorophyll, A, and A₀. We assign the faster phase to the equilibration process between the excited state of the antenna/reaction center ensemble and the primary radical pair, and the slower phase to the secondary electron transfer reaction. The pre-oxidation of P700 shifts the equilibrium between the excited state and the primary radical pair towards the excited state. This shift is proposed to be induced by the presence of the positive charge on P700⁺. The same charge is proposed to be responsible for the fast A⁺A₀⁻ → AA₀ charge recombination to the ground state and, in consequence, excitation quenching in closed reaction centers. Mutations of the A₀ axial ligand shift the equilibrium in the same direction as pre-oxidation of P700 due to the up-shift of the free energy level of the state A⁺A₀⁻.     

Femtosecond nuclear oscillations under charge separation in reaction centers of photosynthesis

Results are presented of a study of primary processes of formation of the charge separated states P⁺B_A ^- and P⁺H_A ^- (where P is the primary electron donor, B_A and H_A the primary and secondary electron acceptors) in native and pheophytin-modified reaction centers (RCs) of Rhodobacter sphaeroides R-26 by methods of femtosecond spectroscopy of absorption changes at low temperature. Coherent oscillations were studied in the kinetics at 935 nm (P* stimulated emission band), at 1020 nm (B_A ^- absorption band), and at 760 nm (H_A absorption band). It was found that when the wavepacket created under femtosecond light excitation approaches the intersection between P* and P⁺B_A ^- potential surfaces at 120- and 380-fsec delays, the formation of two electron states emitting light at 935 nm (P*) and absorbing light at 1020 nm (P⁺B_A ^-) takes place. At the later time the wavepacket motion has a frequency of 32 cm^-1 and is accompanied by electron transfer from P* to B_A in pheophytin-modified and native RCs and further to H_A in native RCs. It was shown that electron transfer processes monitored by the 1020-nm absorption band development as well as by bleaching of 760-nm absorption band have the enhanced 32 cm^-1 mode in the Fourier transform spectra.     

On the efficiency of energy transfer and the different pathways of electron transfer in mutant reaction centers of Rhodobacter sphaeroides

The efficiency of energy transfer from the monomeric pigments to the primary donor was determined from 77 K steady-state fluorescence excitation spectra of three mutant reaction centers, YM210L, YM210F and LM160H / FM197H. For all three reaction centers this efficiency was not 100% and ranged between 55 and 70%. For the YM210L mutant it was shown using pump-probe spectroscopy with B band excitation at 798 nm that the excitations which are not transferred to P give rise to efficient charge separation. The results can be interpreted with a model in which excitation of the B absorbance band leads to direct formation of the radical pair state B_A ⁺H _A ^– in addition to energy transfer to P. It is also possible that some P⁺B_A ^– is formed from B^*. In previous publications we have demonstrated the operation of such alternative pathways for transmembrane electron transfer in a YM210W mutant reaction center [van Brederode et al. (1996) The Reaction center of Photosynthetic Bacteria, pp 225–238; (1997a,b) Chem Phys Lett 268: 143–149; Biochemistry 36: 6855–6861]. The results presented here demonstrate that these alternative mechanisms are not peculiar to the YM210W reaction center.     

Effect of inhibitors,redox state and isoprenoid chain length on the affinity of ubiquinone for the secondary acceptor binding site in the reaction centers of photosynthetic bacteria

Quinone and inhibitor binding to Rhodopseudomonas sphaeroides (R-26 and GA) reaction centers were studied using spectroscopic methods and by direct adsorption of reaction centers onto anion exchange filters in the presence of ¹⁴C-labelled quinone or inhibitor. These measurements show that as secondary acceptor, Q_B, ubiquinone (UQ) is tightly bound in the semiquinone form and loosely bound in the quinone and quinol forms. The quinol is probably more loosely bound than the quinone. o-Phenanthroline and terbutryn, a triazine inhibitor, compete with UQ and with each other for binding to the reaction center. Inhibition by o-phenanthroline of electron transfer from the primary to the secondary quinone acceptor (Q_A to Q_B) occurs via displacement of UQ from the Q_B binding site. Displacement of UQ by terbutryn is apparently accessory to the inhibition of electron transfer. Terbutryn binding is lowered by reduction of Q_B to Q^?_B but is practically unaffected by reduction of Q_A to Q^?_A in the absence of Q_B. UQ-9 and UQ-10 have a 5- to 6-fold higher binding affinity to the Q_B site than does UQ-1, indicating that the long isoprenoid chain facilitates the binding to the Q_B site.     

Mutations in algal and cyanobacterial Photosystem I that independently affect the yield of initial charge separation in the two electron transfer cofactor branches

In Photosystem I, light-induced electron transfer can occur in either of two symmetry-related branches of cofactors, each of which is composed of a pair of chlorophylls (ec2_A/ec3_A or ec2_B/ec3_B) and a phylloquinone (PhQ_A or PhQ_B). The axial ligand to the central Mg^2 + of the ec2_A and ec2_B chlorophylls is a water molecule that is also H-bonded to a nearby Asn residue. Here, we investigate the importance of this interaction for charge separation by converting each of the Asn residues to a Leu in the green alga, Chlamydomonas reinhardtii, and the cyanobacterium, Synechocystis sp. PCC6803, and studying the energy and electron transfer using time-resolved optical and EPR spectroscopy. Nanosecond transient absorbance measurements of the PhQ to F_X electron transfer show that in both species, the PsaA-N604L mutation (near ec2_B) results in a ~ 50% reduction in the amount of electron transfer in the B-branch, while the PsaB-N591L mutation (near ec2_A) results in a ~ 70% reduction in the amount of electron transfer in the A-branch. A diminished quantum yield of P₇₀₀⁺ PhQ^? is also observed in ultrafast optical experiments, but the lower yield does not appear to be a consequence of charge recombination in the nanosecond or microsecond timescales. The most significant finding is that the yield of electron transfer in the unaffected branch did not increase to compensate for the lower yield in the affected branch. Hence, each branch of the reaction center appears to operate independently of the other in carrying out light-induced charge separation.     

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, β, for H_A [Kirmaier et al. (1991) Science 251: 922–927] with two mutations, G(M203)D and Y(M210)W, near B_A, we have created a double and a triple mutant with long lifetimes of the excited state P^* of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P⁺Q_A ⁻ formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P⁺H_B ⁻ formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P^* lifetime of 15 ps [Heller et al. (1995) Science 269: 940–945]. We conclude that the lifetime of P^* is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q_B, was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q_A does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9–26]. It is shown that B-branch electron transfer leading to P⁺Q_B ⁻ formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P⁺Q_B ⁻ formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P⁺Q_B ⁻ state under continuous illumination were not successful. Charge recombination of P⁺Q_B ⁻ formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P⁺Q_B ⁻ trapped in wildtype RCs (10⁵ s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780–5786]. This difference is discussed in light of the different binding sites for Q_B and Q_B ⁻ that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812–816]. We present the first low-temperature absorption difference spectrum due to P⁺Q_B ⁻. This revised version was published online in June 2006 with corrections to the Cover Date.     

Primary electron transfer in Rhodobacter sphaeroides R-26 reaction centers under dehydration conditions

The photoinduced charge separation in Q_B-depleted reaction centers (RCs) from Rhodobacter sphaeroides R-26 in solid air-dried and vacuum-dried (~10⁻² Torr) films, obtained in the presence of detergent n-dodecyl-β-D-maltoside (DM), is characterized using ultrafast transient absorption spectroscopy. It is shown that drying of RC-DM complexes is accompanied by reversible blue shifts of the ground-state absorption bands of the pigment ensemble, which suggest that no dehydration-induced structural destruction of RCs occurs in both types of films. In air-dried films, electron transfer from the excited primary electron donor P^⁎ to the photoactive bacteriopheophytin H_A proceeds in 4.7 ps to form the P⁺H_A⁻ state with essentially 100% yield. P⁺H_A⁻ decays in 260 ps both by electron transfer to the primary quinone Q_A to give the state P⁺Q_A⁻ (87% yield) and by charge recombination to the ground state (13% yield). In vacuum-dried films, P^⁎ decay is characterized by two kinetic components with time constants of 4.1 and 46 ps in a proportion of ~55%/45%, and P⁺H_A⁻ decays about 2-fold slower (462 ps) than in air-dried films. Deactivation of both P^⁎ and P⁺H_A⁻ to the ground state effectively competes with the corresponding forward electron-transfer reactions in vacuum-dried RCs, reducing the yield of P⁺Q_A⁻ to 68%. The results are compared with the data obtained for fully hydrated RCs in solution and are discussed in terms of the presence in the RC complexes of different water molecules, the removal/displacement of which affects spectral properties of pigment cofactors and rates and yields of the electron-transfer reactions.     

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.     

Resistance of reaction centers from Rhodobacter sphaeroides against UV-B radiation. Effects on protein structure and electron transport

Inhibition of electron transport and damage to the protein subunits by ultraviolet-B (UV-B, 280–320 nm) radiation have been studied in isolated reaction centers of the non-sulfur purple bacterium Rhodobacter sphaeroides R26. UV-B irradiation results in the inhibition of charge separation as detected by the loss of the initial amplitude of absorbance change at 430 nm reflecting the formation of the P⁺(Q_AQ_B)^– state. In addition to this effect, the charge recombination accelerates and the damping of the semiquinone oscillation increases in the UV-B irradiated reaction centers. A further effect of UV-B is a 2 fold increase in the half- inhibitory concentration of o-phenanthroline. Some damage to the protein subunits of the RC is also observed as a consequence of UV-B irradiation. This effect is manifested as loss of the L, M and H subunits on Coomassie stained gels, but not accompanied with specific degradation products. The damaging effects of UV-B radiation enhanced in reaction centers where the quinone was semireduced (Q_B ^–) during UV-B irradiation, but decreased in reaction centers which lacked quinone at the Q_B binding site. In comparison with Photosystem II of green plant photosynthesis, the bacterial reaction center shows about 40 times lower sensitivity to UV-B radiation concerning the activity loss and 10 times lower sensitivity concerning the extent of reaction center protein damage. It is concluded that the main effect of UV-B radiation in the purple bacterial reaction center occurs at the Q_AQ_B quinone acceptor complex by decreasing the binding affinity of Q_B and shifting the electron equilibration from Q_AQ_B ^– to Q_A ^–Q_B. The inhibitory effect is likely to be caused by modification of the protein environment around the Q_B binding pocket and mediated by the semiquinone form of Q_B. The UV-resistance of the bacterial reaction center compared to Photosystem II indicates that either the Q_AQ_B acceptor complex, which is present in both types of reaction centers with similar structure and function, is much less susceptible to UV damage in purple bacteria, or, more likely, that Photosystem II contains UV-B targets which are more sensitive than its quinone complex.Abbreviations Bchl bacteriochlorophyll - P Bchl dimer - Q_A primary quinone electron acceptor - Q_B secondary quinone electron acceptor - RC reaction center - UV-B ultraviolet-B     

Redox potential of the non-heme iron complex in bacterial photosynthetic reaction center

In bacterial photosynthetic reaction centers (bRC), the electron is transferred from the special pair (P) via accessory bacteriochlorophyll (B_A), bacteriopheopytin (H_A), the primary quinone (Q_A) to the secondary quinone (Q_B). Although the non-heme iron complex (Fe complex) is located between Q_A and Q_B, it was generally supposed not to be redox-active. Involvement of the Fe complex in electron transfer (ET) was proposed in recent FTIR studies [A. Remy and K. Gerwert, Coupling of light-induced electron transfer to proton uptake in photosynthesis, Nat. Struct. Biol. 10 (2003) 637-644]. However, other FTIR studies resulted in opposite results [J. Breton, Steady-state FTIR spectra of the photoreduction of Q_A and Q_B in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones, Biochemistry 46 (2007) 4459-4465]. In this study, we calculated redox potentials of Q_A/B (E_m(Q_A/B)) and the Fe complex (E_m(Fe)) based on crystal structure of the wild-type bRC (WT-bRC), and we investigated the energetics of the system where the Fe complex is assumed to be involved in the ET. E_m(Fe) in WT-bRC is much less pH-dependent than that in PSII. In WT-bRC, we observed significant coupling of ET with Glu-L212 protonation upon oxidation of the Fe complex and a dramatic E_m(Fe) downshift by 230 mV upon formation of Q_A⁻ (but not Q_B⁻) due to the absence of proton uptake of Glu-L212. Changes in net charges of the His ligands of the Fe complex appear to be the nature of the redox event if we assume the involvement of the Fe complex in the ET.     

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.Abbreviations BChl bacteriochlorophyll - P a dimer of BChl molecules - BPh bacteriopheophytin - Q_A and Q_B quinone molecules - L, M and H light, medium and heavy polypeptides of the reaction center     

Primary electron transfer in reaction centers of YM210L and YM210L/HL168L mutants of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The role of tyrosine M210 in charge separation and stabilization of separated charges was studied by analyzing of the femtosecond oscillations in the kinetics of decay of stimulated emission from P* and of a population of the primary charge separated state P⁺B_A⁻ in YM210L and YM210L/HL168L mutant reaction centers (RCs) of Rhodobacter sphaeroides in comparison with those in native Rba. sphaeroides RCs. In the mutant RCs, TyrM210 was replaced by Leu. The HL168L mutation placed the redox potential of the P⁺/P pair 123 mV below that of native RCs, thus creating a theoretical possibility of P⁺B_A⁻ stabilization. Kinetics of P* decay at 940 nm of both mutants show a significant slowing of the primary charge separation reaction in comparison with native RCs. Distinct damped oscillations in these kinetics with main frequency bands in the range of 90–150 cm⁻¹ reflect mostly nuclear motions inside the dimer P. Formation of a very small absorption band of B_A⁻ at 1020 nm is registered in RCs of both mutants. The formation of the B_A⁻ band is accompanied by damped oscillations with main frequencies from ∼10 to ∼150 cm⁻¹. Only a partial stabilization of the P⁺B_A⁻ state is seen in the YM210L/HL168L mutant in the form of a small non-oscillating background of the 1020-nm kinetics. A similar charge stabilization is absent in the YM210L mutant. A model of oscillatory reorientation of the OH-group of TyrM210 in the electric fields of P⁺ and B_A⁻ is proposed to explain rapid stabilization of the P⁺B_A⁻ state in native RCs. Small oscillatory components at ∼330–380 cm⁻¹ in the 1020-nm kinetics of native RCs are assumed to reflect this reorientation. We conclude that the absence of TyrM210 probably cannot be compensated by lowering of the P⁺B_A⁻ free energy that is expected for the double YM210L/HL168L mutant. An oscillatory motion of the HOH55 water molecule under the influence of P⁺ and B_A⁻ is assumed to be another potential contributor to the mechanism of P⁺B_A⁻ stabilization.     

Primary charge separation in the photosystem II core from Synechocystis: a comparison of femtosecond visible/midinfrared pump-probe spectra of wild-type and two P680 mutants

It is now quite well accepted that charge separation in PS2 reaction centers starts predominantly from the accessory chlorophyll B_A and not from the special pair P₆₈₀. To identify spectral signatures of B_A, and to further clarify the process of primary charge separation, we compared the femtosecond-infrared pump-probe spectra of the wild-type (WT) PS2 core complex from the cyanobacterium Synechocystis sp. PCC 6803 with those of two mutants in which the histidine residue axially coordinated to P_B (D2-His¹⁹⁷) has been changed to Ala or Gln. By analogy with the structure of purple bacterial reaction centers, the mutated histidine is proposed to be indirectly H-bonded to the C₉O carbonyl of the putative primary donor B_A through a water molecule. The constructed mutations are thus expected to perturb the vibrational properties of B_A by modifying the hydrogen bond strength, possibly by displacing the H-bonded water molecule, and to modify the electronic properties and the charge localization of the oxidized donor . Analysis of steady-state light-induced Fourier transform infrared difference spectra of the WT and the D2-His¹⁹⁷Ala mutant indeed shows that a modification of the axially coordinating ligand to P_B induces a charge redistribution of In addition, a comparison of the time-resolved visible/midinfrared spectra of the WT and mutants has allowed us to investigate the changes in the kinetics of primary charge separation induced by the mutations and to propose a band assignment identifying the characteristic vibrations of B_A.