Accessory Bacteriochlorophyll Research Articles

Absence of electron-transfer-associated changes in the time-dependent X-ray free-electron laser structures of the photosynthetic reaction center.

Using the X-ray free-electron laser (XFEL) structures of the photosynthetic reaction center from Blastochloris viridis that show light-induced time-dependent structural changes (Dods et al., (2021) Nature 589, 310-314), we investigated time-dependent changes in the energetics of the electron-transfer pathway, considering the entire protein environment of the protein structures and titrating the redox-active sites in the presence of all fully equilibrated titratable residues. In the dark and charge separation intermediate structures, the calculated redox potential (Em) values for the accessory bacteriochlorophyll and bacteriopheophytin in the electron-transfer-active branch (BL and HL) are higher than those in the electron-transfer-inactive branch (BM and HM). However, the stabilization of the charge-separated [PLPM]•+HL•- state owing to protein reorganization is not clearly observed in the Em(HL) values in the charge-separated 5 ps ([PLPM]•+HL•- state) structure. Furthermore, the expected chlorin ring deformation upon formation of HL•- (saddling mode) is absent in the HL geometry of the original 5 ps structure. These findings suggest that there is no clear link between the time-dependent structural changes and the electron-transfer events in the XFEL structures.

Absence of electron-transfer-associated changes in the time-dependent X-ray free-electron laser structures of the photosynthetic reaction center

Using the X-ray free-electron laser (XFEL) structures of the photosynthetic reaction center from Blastochloris viridis that show light-induced time-dependent structural changes (Dods et al., (2021) Nature 589, 310–314), we investigated time-dependent changes in the energetics of the electron-transfer pathway, considering the entire protein environment of the protein structures and titrating the redox-active sites in the presence of all fully equilibrated titratable residues. In the dark and charge separation intermediate structures, the calculated redox potential (Em) values for the accessory bacteriochlorophyll and bacteriopheophytin in the electron-transfer-active branch (BL and HL) are higher than those in the electron-transfer-inactive branch (BM and HM). However, the stabilization of the charge-separated [PLPM]•+HL•– state owing to protein reorganization is not clearly observed in the Em(HL) values in the charge-separated 5 ps ([PLPM]•+HL•– state) structure. Furthermore, the expected chlorin ring deformation upon formation of HL•– (saddling mode) is absent in the HL geometry of the original 5 ps structure. These findings suggest that there is no clear link between the time-dependent structural changes and the electron-transfer events in the XFEL structures.

Investigating Primary Charge Separation in the Reaction Center of Heliobacterium modesticaldum.

We compute the primary charge separation step in the homodimeric reaction center (RC) of Heliobacterium modesticaldum from first principles. Using time-dependent density functional theory with the optimally tuned range-separated hybrid functional ωPBE, we calculate the excitations of a system comprising the special pair, the adjacent accessory bacteriochlorophylls, and the most relevant parts of the surrounding protein environment. The structure of the excitation spectrum can be rationalized from coupling of the individual bacteriochlorophyll pigments similar to molecular J- and H-aggregates. We find excited states corresponding to forward-charge transfer along the individual branches of the RC of H. modesticaldum. In the spectrum, these are located at an energy between the coupled Qy and Qx transitions. With ab initio Born-Oppenheimer molecular dynamics simulations, we reveal the influence of thermal vibrations on the excited states. The results show that the energy gap between the coupled Qy and the forward-charge transfer excitations is ∼0.4 eV, which we consider to conflict with the concept of a direct transfer mechanism. Our calculations, however, reveal a certain spectral overlap of the forward-charge transfer and the coupled Qx excitations. The reliability and robustness of the results are demonstrated by several numerical tests.

Dynamic band-shift signal in two-dimensional electronic spectroscopy: A case of bacterial reaction center.

Optical nonlinear spectroscopies carry a high amount of information about the systems under investigation; however, as they report polarization signals, the resulting spectra are often congested and difficult to interpret. To recover the landscape of energy states and physical processes such as energy and electron transfer, a clear interpretation of the nonlinear signals is prerequisite. Here, we focus on the interpretation of the electrochromic band-shift signal, which is generated when an internal electric field is established in the system following optical excitation. Whereas the derivative shape of the band-shift signal is well understood in transient absorption spectroscopy, its emergence in two-dimensional electronic spectroscopy (2DES) has not been discussed. In this work, we employed 2DES to follow the dynamic band-shift signal in reaction centers of purple bacteria Rhodobacter sphaeroides at 77 K. The prominent two-dimensional derivative-shape signal appears with the characteristic formation time of the charge separated state. To explain and characterize the band-shift signal, we use expanded double-sided Feynman diagram formalism. We propose to distinguish two types of Feynman diagrams that lead to signals with negative amplitude: excited state absorption and re-excitation. The presented signal decomposition and modeling analysis allows us to recover precise electrochromic shifts of accessory bacteriochlorophylls, identify additional signals in the B band range, and gain a further insight into the electron transfer mechanism. In a broader perspective, expanded Feynman diagram formalism will allow for interpretation of all 2D signals in a clearer and more intuitive way and therefore facilitate studying the underlying photophysics.

The nature of the lower excited state of the special pair of bacterial photosynthetic reaction center of Rhodobacter Sphaeroides and the dynamics of primary charge separation

Quantum-chemical calculations of the structure in the ground and lower singlet excited states and the vibrations (in the ground state) of special pair P of photosynthetic reaction center of purple bacteria (RCPb) Rhodobacter Sphaeroides, consisting of two bacteriochlorophyll molecules PA and PB, have been carried out. It is shown that excitation of the special pair is followed by fast relaxation dynamics, accompanied by the transformation of the initial P* state into the PAδ+PBδ- state (δ ~ 0.5) with charge separation. This behavior is due to the presence of several nonplanar vibrations with participation of the acetyl group of macrocycle PВ in the nuclear wave packet on the potential surface of the P* state; these vibrations facilitate destabilization of the lowest unoccupied molecular orbital (LUMO) and highest occupied molecular orbital (HOMO) of the macrocycle PA and formation of the PAδ+PBδ- state. The structural transformations in the P* state are due to its linking character in the contact region of the acetyl group-containing pyrrole rings of PA and PB. The transition from the P* state to specifically the PAδ+PBδ- state is related to the fact that the acetyl group PA is involved in the intermolecular hydrogen bond with amino acid residue HisL168; for this reason, this group and the pyrrole ring linked with it can hardly participate in structural transformations. The electronic matrix element Н12 of the electron transfer from the special pair in the PAδ+PBδ- state to a molecule of accessory bacteriochlorophyll ВА greatly exceeds that for the transfer to ВB. This circumstance and the fact that the PAδ+PBδ- state is energetically more favorable than the P* state facilitate the preferred directionality of the electron transfer in RCPb Rhodobacter Sphaeroides with participation of the cofactors located in its subunit L.

Induction and anisotropy of fluorescence of reaction center from photosynthetic bacterium Rhodobacter sphaeroides.

Submillisecond dark-light changes of the yield (induction) and anisotropy of fluorescence under laser diode excitation were measured in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides. Narrow band (1-2 nm) laser diodes emitting at 808 and 865 nm were used to selectively excite the accessory bacteriochlorophyll (B, 800 nm) or the upper excitonic state of the bacteriochlorophyll dimer (P-, 810 nm) and the lower excitonic state of the dimer (P+, 865 nm), respectively. The fluorescence spectrum of the wild type showed two bands centered at 850 nm (B) and 910 nm (P-). While the monotonous decay of the fluorescence yield at 910 nm tracked the light-induced oxidation of the dimer, the kinetics of the fluorescence yield at 850 nm showed an initial rise before a decrease. The anisotropy of the fluorescence excited at 865 nm (P-) was very close to the limiting value (0.4) across the whole spectral range. The excitation of both B and P- at 808 nm resulted in wavelength-dependent depolarization of the fluorescence from 0.35 to 0.24 in the wild type and from 0.30 to 0.24 in the reaction center of triple mutant (L131LH-M160LH-M197FH). The additivity law of the anisotropies of the fluorescence species accounts for the wavelength dependence of the anisotropy. The measured fluorescence yields and anisotropies are interpreted in terms of very fast energy transfer from (1)B* to (1)P- (either directly or indirectly by internal conversion from (1)P+) and to the oxidized dimer.

Quantum dynamics of radiationless electronic transitions including normal modes displacements and Duschinsky rotations: a second-order cumulant approach.

An analytical expression for the population dynamics of electronic radiationless transitions has been derived from the second order expansion of the quantum evolution operator in the Liouville space and the cumulant theory. The expression includes the effect of both normal mode displacements and Duschinsky rotations and allows to take into account both equilibrium and nonequilibrium initial conditions. The methodology has been applied to model the electron-transfer process between the accessory bacteriochlorophyll and the bacteriopheophytine in bacterial reactions centers, providing a rate in good agreement with experimental findings.

Role of Electronic-Vibrational Mixing in Enhancing Vibrational Coherences in the Ground Electronic States of Photosynthetic Bacterial Reaction Center

We describe polarization controlled two-color coherence photon echo studies of the reaction center complex from a purple bacterium Rhodobacter sphaeroides. Long-lived oscillatory signals that persist up to 2 ps are observed in neutral, oxidized, and mutant (lacking the special pair) reaction centers, for both (0°,0°,0°,0°) and (45°,-45°,90°,0°) polarization sequences. We show that the long-lived signals arise via vibronic coupling of the bacteriopheophytin (H) and accessory bacteriochlorophyll (B) pigments that leads to vibrational wavepackets in the B ground electronic state. Fourier analysis of the data suggests that the 685 cm(-1) mode of B may play a key role in the H to B energy transfer.

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 BA and HA via the appearance of the BA and HA anion bands. We observed an unexpectedly early rise of the HA− 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 BA and HA, and the effect of protein conformation on the electron transfer rate.

Proton Displacements Coupled to Primary Electron Transfer in the Rhodobacter sphaeroides Reaction Center

Using first-principles molecular dynamics (AIMD) and constrained density functional theory (CDFT) we identify the pathway of primary electron transfer in the R. Sphaeroides reaction center from the special pair excited state (P*) to the accessory bacteriochlorophyll (BA). Previous AIMD simulations on the special pair (PLPM) predicted a charge-transfer intermediate formation through the excited-state relaxation along a reaction coordinate characterized by the rotation of an axial histidine (HisM202). To account for the full electron transfer we extend the model to include the primary acceptor BA. In this extended model, the LUMO is primarily localized on the acceptor BA and extends over an interstitial water (water A) that is known to influence the rate of electron transfer (Potter et al. Biochemistry 2005 280, 27155-27164). A vibrational analysis of the dynamical trajectories gives a frequency of 30-35 cm(-1) for a molecular motion involving the hydrogen-bond network around water A, in good agreement with experimental findings (Yakovlev et al. Biochemistry, 2003, 68, 603-610). In its binding pocket water A can act as a switch by breaking and forming hydrogen bonds. With CDFT we calculate the energy required to the formation of the charge-separated state and find it to decrease along the predicted anisotropic reaction coordinate. Furthermore, we observe an increased coupling between the ground and charge-separated state. Water A adapts its hydrogen-bonding network along this reaction coordinate and weakens the hydrogen bond with HisM202. We also present AIMD simulations on the radical cation (P(•+)) showing a weakening of the hydrogen bond between HisL168 and the 3(1)-acetyl of PL. This work demonstrates how proton displacements are crucially coupled to the primary electron transfer and characterizes the reaction coordinate of the initial photoproduct formation.

Mechanism by which a single water molecule affects primary charge separation kinetics in a bacterial photosynthetic reaction center of Rhodobacter sphaeroides

Using quantum-chemical methods, we have studied the role played by water molecules W-A and W-B that are bound by hydrogen bonds to accessory bacteriochlorophyll molecules BA and BB in the process of primary charge separation in the reaction center of Rhodobacter Sphaeroides. We have found that the occurrence of a rotational mode of the W-A molecule at 32 cm−1 and/or its harmonics in stimulated emission of an electron donor P* and the dynamics of population of the states P+BA− and P+HA− may be related to the structural heterogeneity of the reaction center and the existence of a conformation in which the W-A molecule is predominantly involved in one hydrogen bond (with BA). Based on the calculated redox potentials BA and P, it has been shown that the appearance of the W-A molecule in the reaction center reduces the energy of the P+BA− state by ∼600 cm−1. This is somewhat smaller than the influence of the amino-acid residue TyrM210 (∼870 cm−1) and correlates well with a substantial decrease in the electron transfer rate in mutant forms of reaction centers GM203L (which do not contain W-A molecules) and YM210F (in which TyrM210 is replaced with Phe). The data obtained allow us to suggest that rotation of the water molecule with a fixed position of its H atom that is involved in a hydrogen bond with the keto carbonyl group of BA is initiated due to the charge separation between the halves of special pair P and the formation of the state PA+PB−. The large effect of this rotation on the kinetics of population of the states P+BA− and P+HA− after the excitation of P is quite consistent with its influence on the energy of the state P+BA−.

Resolving the Electron Transfer Kinetics in the Bacterial Reaction Center by Pulse Polarized 2-D Photon Echo Spectroscopy

At the heart of photosynthesis is excitation energy transfer toward and charge separation within highly conserved reaction centers (RCs). The function principles of RCs in purple bacteria offer a blueprint for an optoelectronic device, which efficiently utilizes the near-IR region of the solar spectrum. We present theoretical modeling of the nonlinear optical response of the bacterial RC B. viridis incorporating electron and energy transfer on equal footing. The splitting of special pair excitons P is the origin of distinct cross peaks, which allow monitoring of the kinetics of charge separation. The xxyy - xyxy signal, obtained from sequences of orthogonal polarized laser pulses, highlights the kinetics of the secondary, subpicosecond electron transfer from the accessory bacteriochlorophyll BClL to the bacteriopheophytine BPL. The increased selectivity is explained by the relative orientation of exciton transitions. The technique can resolve complex kinetics in congested signals of photosynthetic complexes that are otherwise hardly accessible.

Influence of environment induced correlated fluctuations in electronic coupling on coherent excitation energy transfer dynamics in model photosynthetic systems

Two-dimensional photon-echo experiments indicate that excitation energy transfer between chromophores near the reaction center of the photosynthetic purple bacterium Rhodobacter sphaeroides occurs coherently with decoherence times of hundreds of femtoseconds, comparable to the energy transfer time scale in these systems. The original explanation of this observation suggested that correlated fluctuations in chromophore excitation energies, driven by large scale protein motions could result in long lived coherent energy transfer dynamics. However, no significant site energy correlation has been found in recent molecular dynamics simulations of several model light harvesting systems. Instead, there is evidence of correlated fluctuations in site energy-electronic coupling and electronic coupling-electronic coupling. The roles of these different types of correlations in excitation energy transfer dynamics are not yet thoroughly understood, though the effects of site energy correlations have been well studied. In this paper, we introduce several general models that can realistically describe the effects of various types of correlated fluctuations in chromophore properties and systematically study the behavior of these models using general methods for treating dissipative quantum dynamics in complex multi-chromophore systems. The effects of correlation between site energy and inter-site electronic couplings are explored in a two state model of excitation energy transfer between the accessory bacteriochlorophyll and bacteriopheophytin in a reaction center system and we find that these types of correlated fluctuations can enhance or suppress coherence and transfer rate simultaneously. In contrast, models for correlated fluctuations in chromophore excitation energies show enhanced coherent dynamics but necessarily show decrease in excitation energy transfer rate accompanying such coherence enhancement. Finally, for a three state model of the Fenna-Matthews-Olsen light harvesting complex, we explore the influence of including correlations in inter-chromophore couplings between different chromophore dimers that share a common chromophore. We find that the relative sign of the different correlations can have profound influence on decoherence time and energy transfer rate and can provide sensitive control of relaxation in these complex quantum dynamical open systems.

Maximal Coherence at Room Temperature in the Bacterial Photosynthetic Reaction Center

The earliest steps in bacterial photosynthesis require that an antenna system efficiently capture incident photons and shuttle the excitation energy to the “special pair” bacteriochlorophylls within the membrane-bound reaction center where charge separation occurs. Previous work has shown coherent energy transfer - a wavelike transfer process - among peripheral chromophores, bacteriopheophytins and accessory bacteriochlorophylls, at cryogenic temperatures. Whether or not this coherent transfer extends to the special pair, however, has remained elusive at any temperature. Here we report direct evidence that the special pair is coherently coupled to the accessory bacteriochlophylls and that this coherence dephases only upon transfer to the special pair - the maximal amount of coherence physically possible. We employ Gradient Assisted Photon Echo Spectroscopy to simultaneously excite the bacteriopheophytins, accessory bacteriochlorophylls and the special pair in the reaction center from Rhodobacter sphaeroides. These results suggest the bacteria exploits coherent energy transfer at room temperature.

Quantum Chemical Simulations of Excited-State Absorption Spectra of Photosynthetic Bacterial Reaction Center and Antenna Complexes

The semiempirical ZINDO/S CIS configuration interaction method has been used to study the ground- and excited-state absorption spectra of wild type and heterodimer M202HL reaction centers from purple bacterium Rhodobacter sphaeroides as well as of peripheral LH2 and LH3 light harvesting complexes from purple bacterium Rhodopseudomonas acidophila. The calculations well reproduce the experimentally observed excited-state absorption spectra between 1000 and 17,000 cm(-1), despite the necessarily limited number of chromophores and protein subunits involved in the calculations. The electron density analysis reveals that the charge transfer between adjacent chromophores dominates the excited-state absorption spectra. Clear spectroscopic differences observed between the wild type and heterodimer reaction centers as well as between the LH2 and LH3 antenna complexes arise from differences in the energy level manifolds of the complexes, particularly those of the charge transfer states. The calculations also imply that the lowest excited state of the bacterial reaction centers has charge transfer character that is related to charge transfer within the special pair and between the special pair and the accessory bacteriochlorophyll of the photosynthetically active electron transfer branch.

An investigation of slow charge separation in a Tyrosine M210 to Tryptophan mutant of the Rhodobacter sphaeroides reaction center by femtosecond mid-infrared spectroscopy

Energy and electron transfer in a tyrosine M210 to tryptophan (YM210W) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated through time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy at room temperature, with the aim to further characterize the primary charge separated states in the RC. This mutant is known to display slow and multi-exponential charge separation, and was used in earlier work to prove the existence of an alternative route for charge separation starting from the accessory bacteriochlorophyll in the active branch, B(L). The mutant RCs were excited at 860 nm (direct excitation of the primary donor (P) BChls (P(L)/P(M))), 600 nm (unselective excitation), 805 nm (direct excitation of both accessory bacteriochlorophyll cofactors B(L) and B(M)) and 795 nm (direct excitation of B(L)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes of the cofactors and protein were recorded in the region between 1600 and 1775 cm(-1), and both a sequential analysis and simultaneous target analysis of the data were performed. The decay of P* in the YM210W mutant was multi-exponential with lifetimes of 29 and 63.5 ps. The decay of P(+)B(L)(-) state was approximately 10 times longer in the YM210W RC than in the R-26 RC (approximately 7 ps vs. approximately 0.7 ps), and in the mid-IR difference absorption spectrum of P(+)B(L)(-) the stretching frequency of the 9-keto C=O group of B(L) in the ground state was located around 1675-1680 cm(-1), consistent with the presence of a hydrogen bond donated by an adjacent water molecule. Excitation at 795 nm produced a small amount of B(L)*-driven charge separation, as assessed from the excitation wavelength dependence of the raw difference spectra recorded during the first few ps after excitation. This process led to the formation of P(+)B(L)(-). Only the relaxed form of the P(+)H(L)(-) radical pair was observed in the YM210W mutant, and the mid-IR difference absorption spectra of P(+)H(L)(-) and P(+)B(L)(-) showed a change in the relative amplitude of the P(L)(+) and P(M)(+) bands when compared to equivalent spectra for the R-26 RC. This indicates that the YM210W mutation causes an increased localization of the electron hole on the P(M) half of the dimer. The absorbance difference spectrum of P(+)H(L)(-) in the R-26 RC contains a feature attributable to a Stark shift of one or more amide C=O oscillators. This feature was shifted to lower frequency by approximately 5 cm(-1) in the YM210W RC, and consideration of the limited structural changes in this RC indicates that this feature arises from an amide C=O group in the immediate vicinity of the M210 residue, most probably that of the adjacent M209 amino acid.

Electron transfer in the Rhodobacter sphaeroides reaction center assembled with zinc bacteriochlorophyll

The cofactor composition and electron-transfer kinetics of the reaction center (RC) from a magnesium chelatase (bchD) mutant of Rhodobacter sphaeroides were characterized. In this RC, the special pair (P) and accessory (B) bacteriochlorophyll (BChl) -binding sites contain Zn-BChl rather than BChl a. Spectroscopic measurements reveal that Zn-BChl also occupies the H sites that are normally occupied by bacteriopheophytin in wild type, and at least 1 of these Zn-BChl molecules is involved in electron transfer in intact Zn-RCs with an efficiency of >95% of the wild-type RC. The absorption spectrum of this Zn-containing RC in the near-infrared region associated with P and B is shifted from 865 to 855 nm and from 802 to 794 nm respectively, compared with wild type. The bands of P and B in the visible region are centered at 600 nm, similar to those of wild type, whereas the H-cofactors have a band at 560 nm, which is a spectral signature of monomeric Zn-BChl in organic solvent. The Zn-BChl H-cofactor spectral differences compared with the P and B positions in the visible region are proposed to be due to a difference in the 5th ligand coordinating the Zn. We suggest that this coordination is a key feature of protein-cofactor interactions, which significantly contributes to the redox midpoint potential of H and the formation of the charge-separated state, and provides a unifying explanation for the properties of the primary acceptor in photosystems I (PS1) and II (PS2).

Multipulse spectroscopy on the wild-type and YM210W Bacterial Reaction Centre uncovers a new intermediate state in the special pair excited state

The Bacterial Reaction Centre (BRC) has a complex electronic excited state, P ∗, that evolves into subsequent charge separated product states P +H − and P +B −. Pump–dump–probe spectroscopy on the wild-type BRC and on YM210W, a mutant with a stabilized, long-lived P ∗ excited state, has uncovered a new charge-separated state in both BRC’s. When P ∗ is dumped, a fraction of its population is transferred to this state that has a strong Stark shift in the accessory bacteriochlorophyll (B M) region which serves as a signature for P + and a lifetime highly comparable to the slow phase of P ∗ decay. This lead us propose this intermediate to be P +/P −.

Kinetics and Energetics of Electron Transfer Reactions in a Photosynthetic Bacterial Reaction Center Assembled with Zinc Bacteriochlorophylls

Electron transfer processes were studied in the reaction center (RC) of a Rhodobacter sphaeroides magnesium chelatase (bchD) mutant that assembles with six chemically identical chlorin molecules. A previous study [Jaschke & Beatty, Biochemistry, 2007] and this work show the complete absence of bacteriochlorophyll (containing Mg as the metal) and bacteriopheophytin from the bchD mutant RC. Instead, bacteriochlorophylls containing a Zn atom as the metal (Zn-BChl) occupy the binding sites of the special pair (P), accessory bacteriochlorophyll (B), and primary electron acceptor (H). In spite of significant differences in cofactor composition, electron transfer from excited P through B to H proceeds with high efficiency and with rates nearly identical to the wild type RC. The rate of electron transfer from H to QA is also the same as that observed in the wild type RC. Thus, the protein-cofactor interactions, mainly through electron sharing between the metal of the BChl and the protein, play an important role in adjusting the energies of the cofactors to form an efficient electron transfer system. The study also suggests that the overall electron transfer from P to H is more sensitive to the energy change between P and B than B and H, and can tolerate a large variation in the redox energy of H.

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.