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 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.     

Coherent phenomena of charge separation in reaction centers of LL131H and LL131H/LM160H/FM197H mutants of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Primary stage of charge separation and transfer of charges was studied in reaction centers (RCs) of point mutants LL131H and LL131H/LM160H/FM197H of the purple bacterium Rhodobacter sphaeroides by differential absorption spectroscopy with temporal resolution of 18 fsec at 90 K. Difference absorption spectra measured at 0–4 psec delays after excitation of dimer P at 870 nm with 30 fsec step were obtained in the spectral range of 935–1060 nm. It was found that a decay of P* due to charge separation is considerably slower in the mutant RCs in comparison with native RCs of Rba. sphaeroides. Coherent oscillations were found in the kinetics of stimulated emission of the P* state at 940 nm. Fourier analysis of the oscillations revealed a set of characteristic bands in the frequency range of 20–500 cm⁻¹. The most intense band has the frequency of −30 cm⁻¹ in RCs of mutant LL131H and in native RCs and the frequency of ∼100 cm⁻¹ in RCs of the triple mutant. It was found that an absorption band of bacteriochlorophyll anion B_A⁻ which is registered in the difference absorption spectra of native RCs at 1020 nm is absent in the analogous spectra of the mutants. The results are analyzed in terms of the participation of the B_A molecule in the primary electron transfer in the presence of a nuclear wave packet moving along the inharmonic surface of P* potential energy.     

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.     

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*.     

Femtosecond charge separation in dry films of reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> and <Emphasis Type="Italic">Chloroflexus aurantiacus</Emphasis>

In this work, the influence of the crystallographic water on electron transfer between primary donor P and acceptor B_A was studied in reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides and the green bacterium Chloroflexus aurantiacus. For this purpose, time constants and oscillations of charge separation kinetics are compared between dry film RCs and RCs in glycerol-water buffer at 90 K. A common result of the drying of Rba. sphaeroides and Cfx. aurantiacus RCs is slowing of the charge separation process, decrease in amplitude of the oscillatory components of the kinetics, and the depletion of its spectrum. Thus, the major time constant of stimulated emission decay of P* bacteriochlorophyll dimer at 940 nm is increased from 1.1 psec for water-containing Rba. sphaeroides RCs to 1.9 psec for dry films of Rba. sphaeroides RCs. An analogous increase from 3.5 to 4.2 psec takes place in Cfx. aurantiacus RCs. In dry films of Rba. sphaeroides RCs, the amplitude of coherent oscillations of the absorption band of monomeric bacteriochlorophyll B_A⁻ at 1020 nm is 1.8 times less for the 130-cm⁻¹ component and 2.3 times less for the 32-cm⁻¹ component than the analogous amplitudes for water-containing RCs. Measurements in the analogous band of Cfx. aurantiacus RCs show that strong decrease (∼5-10 times) of the B_A⁻ absorption band and strong slowing (from ∼0.8 to ∼3 psec) of B_A⁻ accumulation together with ∼3-fold decrease in oscillation amplitude occurs on drying of these RCs. The overtones of the 32-cm⁻¹ component disappeared from the oscillations of the kinetics at 940 and 1020–1028 nm after drying of the Rba. sphaeroides and Cfx. aurantiacus RCs. The results are in agreement with the results for GM203L mutant of Rba. sphaeroides, in which the HOH55 water molecule is sterically removed, and with the results for dry films of pheophytin-modified RCs of Rba. sphaeroides R-26 and for YM210W and YM210L Rba. sphaeroides mutant RCs. The data are discussed in terms of the influence (or participation) of the HOH55 water molecule on electron transfer along the chain of polar atomic groups N-Mg(P_B)-N-C-N(HisM202)-HOH55-O=(B_A) connecting P_B and B_A in Rba. sphaeroides RCs.     

Coupling of nuclear wavepacket motion and charge separation in bacterial reaction centers

The mechanism of the charge separation and stabilization of separated charges was studied using the femtosecond absorption spectroscopy. It was found that nuclear wavepacket motions on potential energy surface of the excited state of the primary electron donor P* leads to a coherent formation of the charge separated states P⁺B_A⁻, P⁺H_A⁻ and P⁺H_B⁻ (where B_A, H_B and H_A are the primary and secondary electron acceptors, respectively) in native, pheophytin-modified and mutant reaction centers (RCs) of Rhodobacter sphaeroides R-26 and in Chloroflexus aurantiacus RCs. The processes were studied by measurements of coherent oscillations in kinetics at 890 and 935 nm (the stimulated emission bands of P*), at 800 nm (the absorption band of B_A) and at 1020 nm (the absorption band of B_A⁻) as well as at 760 nm (the absorption band of H_A) and at 750 nm (the absorption band of H_B). It was found that wavepacket motion on the 130–150 cm⁻¹ potential surface of P* is accompanied by approaches to the intercrossing region between P* and P⁺B_A⁻ surfaces at 120 and 380 fs delays emitting light at 935 nm (P*) and absorbing light at 1020 nm (P⁺B_A⁻). In the presence of Tyr M210 (Rb. sphaeroides) or M195 (C. aurantiacus) the stabilization of P⁺B_A⁻ is observed within a few picosseconds in contrast to YM210W. At even earlier delay (40 fs) the emission at 895 nm and bleaching at 748 nm are observed in C. aurantiacus RCs showing the wavepacket approach to the intercrossing between the P* and P⁺H_B⁻ surfaces at that time. The 32 cm⁻¹ rotation mode of HOH was found to modulate the electron transfer rate probably due to including of this molecule in polar chain connecting P_B and B_A and participating in the charge separation. The mechanism of the charge separation and stabilization of separated charges is discussed in terms of the role of nuclear motions, of polar groups connecting P and acceptors and of proton of OH group of TyrM210.     

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.     

Nuclear wavepacket motion between P and P(+)B(A)(-) potential surfaces with subsequent electron transfer to H(A) in bacterial reaction centers. 1. Room temperature

Formation and coherent propagation of nuclear wavepackets on potential energy surfaces of the excited state of the primary electron donor P and of the charge transfer states P(+)B(A)(-) and P(+)H(A)(-) were studied in native and pheophytin-modified Rhodobacter sphaeroides R-26 reaction centers (RCs) induced by 25 fs excitation (where B(A) and H(A) are the primary and secondary electron acceptors, respectively). The processes were monitored by measuring coherent oscillations in kinetics of the time evolution of the stimulated emission band of P at 935 nm, of the absorption band of B(A)(-) at 1020 nm, and of the bleaching band of H(A) at 760 nm. It was found that the nuclear wavepacket motion on the 130-140 cm(-1) surface of P is directly induced by light absorption in P. When the wavepacket approaches the intersection between P and P(+)B(A)(-) surfaces at 120 and 380 fs delays, the formation of intermediate mixed-state emitting light at 935 nm (P) and absorbing light at 1020 nm (P(+)B(A)(-)) takes place. At the latter time, the wavepacket is transferred to the 32 cm(-1) mode which can belong to the P hypersurface effectively transferring the wavepacket to the P(+)B(A)(-) surface or can represent a diabatic surface which is formed by the states P and P(+)B(A)(-). The wavepacket motion on the P(+)B(A)(-) surface or on the P(+)B(A)(-) part of the mixing surface is accompanied by irreversible electron transfer to H(A). This process is monitored by the kinetics of 1020 nm band development and 760 nm band bleaching (delayed with respect to 1020 nm band development) which both have the enhanced 32 cm(-1) mode in Fourier transform (FT) spectra. The mechanism of wavepacket transfer from the 130-140 cm(-1) to the 32 cm(-1) mode is discussed.     

Femtosecond stage of electron transfer in reaction centers of the triple mutant SL178K/GM203D/LM214H of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Coherent processes in an initial phase of charge transfer in reaction centers (RCs) of the triple mutant S(L178)K/G(M203)D/L(M214)H of Rhodobacter sphaeroides were investigated by difference (light — dark) absorption spectroscopy with 18 fsec time resolution. Electron transfer in the B cofactor branch is activated in this mutant, while the A-branch electron transfer is slowed in comparison with native RCs of Rba. sphaeroides. A bulk of absorption difference spectra was analyzed in the 940–1060 nm range (stimulated emission of excited bacteriochlorophyll dimer P* and absorption of bacteriochlorophyll anions B_A⁻ and β⁻, where β is a bacteriochlorophyll substituting the native bacteriopheophytin H_A) and in the 735–775 nm range (bleaching of the absorption band of the bacteriopheophytin H_B in the B-branch) in the −0.1 to 4 psec range of delays with respect to the moment of photoexcitation of P at 870 nm. Spectra were measured at 293 and 90 K. The kinetics of P* stimulated emission at 940 nm shows its decay with a time constant of ∼14 psec at 90 K and ∼18 psec at 293 K, which is accompanied by oscillations with a frequency of ∼150 cm⁻¹. A weak absorption band is found at 1018 nm that is formed ∼100 fsec after excitation of P and reflects the electron transfer from P* to β and/or B_A with accumulation of the P⁺β⁻ and/or P⁺B_A⁻ states. The kinetics of ΔA at 1018 nm contains the oscillations at ∼150 cm⁻¹ and distinct low-frequency oscillations at 20–100 cm⁻¹; also, the amplitude of the oscillations at 150 cm⁻¹ is much smaller at 293 than at 90 K. The oscillations in the kinetics of the 1018 nm band do not contain a 32 cm⁻¹ mode that is characteristic for native Rba. sphaeroides RCs having water molecule HOH55 in their structure. The ΔA kinetics at 751 nm reflects the electron transfer to HB with formation of the P⁺H_B⁻ state. The oscillatory part of this kinetics has the form of a single peak with a maximum at ∼50 fsec completely decaying at ∼200 fsec, which might reflect a reversible electron transfer to the B-branch. The results are analyzed in terms of coherent nuclear wave packet motion induced in the P* excited state by femtosecond light pulses, of an influence of the incorporated mutations on the mutual position of the energy levels of charge separated states, and of the role of water HOH55 in the dynamics of the initial electron transfer.     

An alternative pathway of light-induced transmembrane electron transfer in photosynthetic reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the absorption spectrum of Rhodobacter sphaeroides reaction centers, a minor absorption band was found with a maximum at 1053 nm. The amplitude of this band is ~10,000 times less and its half-width is comparable to that of the long-wavelength absorption band of the primary electron donor P₈₇₀. When the primary electron donor is excited by femtosecond light pulses at 870 nm, the absorption band at 1053 nm is increased manifold during the earliest stages of charge separation. The growth of this absorption band in difference absorption spectra precedes the appearance of stimulated emission at 935 nm and the appearance of the absorption band of anion-radical B_A ^– at 1020 nm, reported earlier by several researchers. When reaction centers are illuminated with 1064 nm light, the absorption spectrum undergoes changes indicating reduction of the primary electron acceptor Q_A, with the primary electron donor P₈₇₀ remaining neutral. These photoinduced absorption changes reflect the formation of the long-lived radical state PB_AH_AQ_A ^–.     

Nuclear wavepacket motion producing a reversible charge separation in bacterial reaction centers

The excitation of bacterial reaction centers (RCs) at 870 nm by 30 fs pulses induces the nuclear wavepacket motions on the potential energy surface of the primary electron donor excited state P*, which lead to the fs oscillations in stimulated emission from P* [M.H. Vos, M.R. Jones, C.N. Hunter, J. Breton, J.-C. Lambry and J.-L. Martin (1994) Biochemistry 33, 6750-6757] and in Qy absorption band of the primary electron acceptor, bacteriochlorophyll monomer B(A) [A.M. Streltsov, S.I.E. Vulto, A.Y. Shkuropatov, A.J. Hoff, T.J. Aartsma and V.A. Shuvalov (1998) J. Phys. Chem. B 102, 7293-7298] with a set of fundamental frequencies in the range of 10-300 cm(-1). We have found that in pheophytin-modified RCs, the fs oscillations with frequency around 130 cm(-1) observed in the P*-stimulated emission as well as in the B(A) absorption band at 800 nm are accompanied by remarkable and reversible formation of the 1020 nm absorption band which is characteristic of the radical anion band of bacteriochlorophyll monomer B(A)-. These results are discussed in terms of a reversible electron transfer between P* and B(A) induced by a motion of the wavepacket near the intersection of potential energy surfaces of P* and P+B(A)-, when a maximal value of the Franck-Condon factor is created.     

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.     

Mutant reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> I(L177)H with strongly bound bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>: Structural properties and pigment-protein interactions

Methods of photoinduced Fourier transform infrared (FTIR) difference spectroscopy and circular dichroism were employed for studying features of pigment-protein interactions caused by replacement of isoleucine L177 by histidine in the reaction center (RC) of the site-directed mutant I(L177)H of Rhodobacter sphaeroides. A functional state of pigments in the photochemically active cofactor branch was evaluated with the method of photo-accumulation of reduced bacteriopheophytin H _A ^? . The results are compared with those obtained for wild-type RCs. It was shown that the dimeric nature of the radical cation of the primary electron donor P was preserved in the mutant RCs, with an asymmetric charge distribution between the bacteriochlorophylls P_A and P_B in the P⁺ state. However, the dimers P in the wild-type and mutant RCs are not structurally identical due probably to molecular rearrangements of the P_A and P_B macrocycles and/or alterations in their nearest amino acid environment induced by the mutation. Analysis of the electronic absorption and FTIR difference P⁺Q^?/PQ spectra suggests the 17³-ester group of the bacteriochlorophyll P_A to be involved in covalent interaction with the I(L177)H RC protein. Incorporation of histidine into the L177 position does not modify the interaction between the primary electron acceptor bacteriochlorophyll B_A and the bacteriopheophytin H_A. Structural changes are observed in the monomer bacteriochlorophyll B_B binding site in the inactive chromophore branch of the mutant RCs.     

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.     

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: Photooxidation of the primary electron donor

Photochemical oxidation of the primary electron donor P in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus (C.) aurantiacus was examined by light-induced Fourier transform infrared (FTIR) difference spectroscopy at 95 K in the spectral range of 4000–1200 cm⁻¹. The light-induced P⁺Q_A⁻/PQ_A IR spectrum of C. aurantiacus RCs is compared to the well-characterized FTIR difference spectrum of P photooxidation in the purple bacterium Rhodobacter (R.) sphaeroides R-26 RCs. The presence in the P⁺Q_A⁻/PQ_A FTIR spectrum of C. aurantiacus RCs of specific low-energy electronic transitions at ∼2650 and ∼2200 cm⁻¹, as well as of associated vibrational (phase-phonon) bands at 1567, 1481, and 1294–1285 cm⁻¹, indicates that the radical cation P⁺ in these RCs has dimeric structure, with the positive charge distributed between the two coupled bacteriochlorophyll a molecules. The intensity of the P⁺ absorbance band at ∼1250 nm (upon chemical oxidation of P at room temperature) in C. aurantiacus RCs is approximately 1.5 times lower than that in R. sphaeroides R-26 RCs. This fact, together with the decreased intensity of the absorbance band at ∼2650 cm⁻¹, is interpreted in terms of the weaker coupling of bacteriochlorophylls in the P⁺ dimer in C. aurantiacus compared to R. sphaeroides R-26. In accordance with the previous (pre)resonance Raman data, FTIR measurements in the carbonyl stretching region show that in C. aurantiacus RCs (i) the 13¹-keto C=O groups of P_A and P_B molecules constituting the P dimer are not involved in hydrogen bonding in either neutral or photooxidized state of P and (ii) the 3¹-acetyl C=O group of P_B forms a hydrogen bond (probably with tyrosine M187) absorbing at 1635 cm⁻¹. Differential signals at 1757(+)/1749(−) and 1741(+)/1733(−) cm⁻¹ in the FTIR spectrum of C. aurantiacus RCs are attributed to the 13³-ester C=O groups of P in different environments.     

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.     

Femtosecond primary charge separation in Synechocystis sp. PCC 6803 photosystem I

The ultrafast (< 100 fs) conversion of delocalized exciton into charge-separated state between the primary donor P700 (bleaching at 705 nm) and the primary acceptor A₀ (bleaching at 690 nm) in photosystem I (PS I) complexes from Synechocystis sp. PCC 6803 was observed. The data were obtained by application of pump-probe technique with 20-fs low-energy pump pulses centered at 720 nm. The earliest absorbance changes (close to zero delay) with a bleaching at 690 nm are similar to the product of the absorption spectrum of PS I complex and the laser pulse spectrum, which represents the efficiency spectrum of the light absorption by PS I upon femtosecond excitation centered at 720 nm. During the first ∼ 60 fs the energy transfer from the chlorophyll (Chl) species bleaching at 690 nm to the Chl bleaching at 705 nm occurs, resulting in almost equal bleaching of the two forms with the formation of delocalized exciton between 690-nm and 705-nm Chls. Within the next ∼ 40 fs the formation of a new broad band centered at ∼ 660 nm (attributed to the appearance of Chl anion radical) is observed. This band decays with time constant simultaneously with an electron transfer to A₁ (phylloquinone). The subtraction of kinetic difference absorption spectra of the closed (state P700⁺A₀A₁) PS I reaction center (RC) from that of the open (state P700A₀A₁) RC reveals the pure spectrum of the P700⁺A₀⁻ ion-radical pair. The experimental data were analyzed using a simple kinetic scheme: An* [(PA₀)*A₁ P⁺A₀⁻A₁] P⁺A₀A₁⁻, and a global fitting procedure based on the singular value decomposition analysis. The calculated kinetics of transitions between intermediate states and their spectra were similar to the kinetics recorded at 694 and 705 nm and the experimental spectra obtained by subtraction of the spectra of closed RCs from the spectra of open RCs. As a result, we found that the main events in RCs of PS I under our experimental conditions include very fast (< 100 fs) charge separation with the formation of the P700⁺A₀⁻A₁ state in approximately one half of the RCs, the ∼ 5-ps energy transfer from antenna Chl* to P700A₀A₁ in the remaining RCs, and ∼ 25-ps formation of the secondary radical pair P700⁺A₀A₁⁻.     

Structural and kinetic properties of Rhodobacter sphaeroides photosynthetic reaction centers containing exclusively Zn-coordinated bacteriochlorophyll as bacteriochlorin cofactors

The Zn-BChl-containing reaction center (RC) produced in a bchD (magnesium chelatase) mutant of Rhodobacter sphaeroides assembles with six Zn-bacteriochlorophylls (Zn-BChls) in place of four Mg-containing bacteriochlorophylls (BChls) and two bacteriopheophytins (BPhes). This protein presents unique opportunities for studying biological electron transfer, as Zn-containing chlorins can exist in 4-, 5-, and (theoretically) 6-coordinate states within the RC. In this paper, the electron transfer perturbations attributed exclusively to coordination state effects are separated from those attributed to the presence, absence, or type of metal in the bacteriochlorin at the H_A pocket of the RC. The presence of a 4-coordinate Zn^2 + ion in the H_A bacteriochlorin instead of BPhe results in a small decrease in the rates of the P* → P⁺H_A⁻ → P⁺Q_A⁻ electron transfer, and the charge separation yield is not greatly perturbed; however coordination of the Zn^2 + by a fifth ligand provided by a histidine residue results in a larger rate decrease and yield loss. We also report the first crystal structure of a Zn-BChl-containing RC, confirming that the H_A Zn-BChl was either 4- or 5-coordinate in the two types of Zn-BChl-containing RCs studied here. Interestingly, a large degree of disorder, in combination with a relatively weak anomalous difference electron density was found in the H_B pocket. These data, in combination with spectroscopic results, indicate partial occupancy of this binding pocket. These findings provide insights into the use of BPhe as the bacteriochlorin pigment of choice at H_A in both BChl- and Zn-BChl-containing RCs found in nature.