Monomeric Bacteriochlorophyll Research Articles

Femtosecond charge separation in dry films of reaction centers of Rhodobacter sphaeroides and Chloroflexus aurantiacus

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.

Cofactor-specific photochemical function resolved by ultrafast spectroscopy in photosynthetic reaction center crystals

High-resolution mapping of cofactor-specific photochemistry in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides was achieved by polarization selective ultrafast spectroscopy in single crystals at cryogenic temperature. By exploiting the fixed orientation of cofactors within crystals, we isolated a single transition within the multicofactor manifold, and elucidated the site-specific photochemical functions of the cofactors associated with the symmetry-related active A and inactive B branches. Transient spectra associated with the initial excited states were found to involve a set of cofactors that differ depending upon whether the monomeric bacteriochlorophylls, BChl(A), BChl(B), or the special pair bacteriochlorophyll dimer, P, was chosen for excitation. Proceeding from these initial excited states, characteristic photochemical functions were resolved. Specifically, our measurements provide direct evidence for an alternative charge separation pathway initiated by excitation of BChl(A) that does not involve P*. Conversely, the initial excited state produced by excitation of BChl(B) was found to decay by energy transfer to P. A clear sequential kinetic resolution of BChl(A) and the A-side bacteriopheophytin, BPh(A), in the electron transfer proceeding from P* was achieved. These experiments demonstrate the opportunity to resolve photochemical function of individual cofactors within the multicofactor RC complexes using single crystal spectroscopy.

Lipid Binding to the Carotenoid Binding Site in Photosynthetic Reaction Centers

Lipid binding to the carotenoid binding site near the inactive bacteriochlorophyll monomer was probed in the reaction centers of carotenoid-less mutant, R-26 from Rhodobacter sphaeroides. Recently, a marked light-induced change of the local dielectric constant in the vicinity of the inactive bacteriochlorophyll monomer was reported in wild type that was attributed to structural changes that ultimately lengthened the lifetime of the charge-separated state by 3 orders of magnitude (Deshmukh, S. S.; Williams, J. C.; Allen, J. P.; Kalman, L. Biochemistry 2011, 50, 340). Here in the R-26 reaction centers, the combination of light-induced structural changes and lipid binding resulted in a 5 orders of magnitude increase in the lifetime of the charge-separated state involving the oxidized dimer and the reduced primary quinone in proteoliposomes. Only saturated phospholipids with fatty acid chains of 12 and 14 carbon atoms long were bound successfully at 8 °C by cooling the reaction center protein slowly from room temperature. In addition to reporting a dramatic increase of the lifetime of the charge-separated state at physiologically relevant temperatures, this study reveals a novel lipid binding site in photosynthetic reaction center. These results shed light on a new potential application of the reaction center in energy storage as a light-driven biocapacitor since the charges separated by ∼30 Å in a low-dielectric medium can be prevented from recombination for hours.

ENDOR Study of Charge Migration in Photosynthetic Arrays of Rhodobacter sphaeroides

The chemistry of bacterial photosynthesis begins in the photosynthetic reaction centre (RC), a protein complex containing a series of electron donor and acceptor molecules. Although the pigments of the RC can absorb light to operate the photochemistry, the bulk of the light is captured in special pigmented proteins, the light harvesting complexes (LHCs), that then transfer the energy to the RC. Ordinarily, the LHCs do not participate in chemical reactions during photosynthesis such that LHCs do not become oxidised upon light irradiation. However, upon chemical oxidation in the dark, cation radicals of bacteriochlorophyll (BChl) can be formed in the light harvesting complex 1 (LH1) of Rhodobacter sphaeroides. As observed by continuous-wave electron-paramagnetic resonance (EPR), the charges of the BChl(+) cations migrate rather freely about the LH1 complex as in a molecular wire. Remarkably, these LH1 molecular wires continue to function in the frozen, solid state. To investigate the nature of electron-hole transfer and to corroborate the process as revealed by EPR, electron-nuclear double resonance (ENDOR) was recorded at 80 K. ENDOR observed only monomeric bacteriochlorophyll cations. Their signal intensity decreased with increased oxidation while the EPR signal narrowed and increased in size. At the increased oxidation state, the possibility of spin-spin exchange between two BChl(+)s within LH1 versus electron-hole transfer is addressed. An energy landscape of the BChl(+)s in the LH1 is proposed to explain the EPR and ENDOR results.

Primary processes of charge separation in reaction centers of YM210L/FM197Y and YM210L mutants of Rhodobacter sphaeroides

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.

Mutant reaction centers of Rhodobacter sphaeroides I(L177)H with strongly bound bacteriochlorophyll a: 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 17(3)-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 light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: I. A review

The purpose of the review is to show that the tetrameric (bacterio)chlorophyll ((B)Chl) structures in reaction centers of photosystem II (PSII) of green plants and in bacterial reaction centers (BRCs) are similar and play a key role in the primary charge separation. The Stark effect measurements on PSII reaction centers have revealed an increased dipole moment for the transition at approximately 730 nm (Frese et al., Biochemistry 42:9205-9213, 2003). It was found (Heber and Shuvalov, Photosynth Res 84:84-91, 2005) that two fluorescent bands at 685 and 720 nm are observed in different organisms. These two forms are registered in the action spectrum of Q(A) photoreduction. Similar results were obtained in core complexes of PSII at low temperature (Hughes et al., Biochim Biophys Acta 1757: 841-851, 2006). In all cases the far-red absorption and emission can be interpreted as indication of the state with charge transfer character in which the chlorophyll monomer plays a role of an electron donor. The role of bacteriochlorophyll monomers (B(A) and B(B)) in BRCs can be revealed by different mutations of axial ligand for Mg central atoms. RCs with substitution of histidine L153 by tyrosine or leucine and of histidine M182 by leucine (double mutant) are not stable in isolated state. They were studied in antennaless membrane by different kinds of spectroscopy including one with femtosecond time resolution. It was found that the single mutation (L153HY) was accompanied by disappearance of B(A) molecule absorption near 802 nm and by 14-fold decrease of photochemical activity measured with ms time resolution. The lifetime of P(870)* increased up to approximately 200 ps in agreement with very low rate of the electron transfer to A-branch. In the double mutant L153HY + M182HL, the B(A) appears to be lost and B(B) is replaced by bacteriopheophytin Phi(B) with the absence of any absorption near 800 nm. Femtosecond measurements have revealed the electron transfer to B-branch with a time constant of approximately 2 ps. These results are discussed in terms of obligatory role of B(A) and Phi(B) molecules located near P for efficient electron transfer from P*.

WAVE PACKET MOTIONS COUPLED TO ELECTRON TRANSFER IN REACTION CENTERS OF CHLOROFLEXUS AURANTIACUS

Transient absorption difference spectroscopy with approximately 20 femtosecond (fs) resolution was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of Chloroflexus (C.) aurantiacus. In RCs, the composition of the B-branch chromophores is different with respect to that of purple bacterial RCs by occupying the B(B) binding site of accessory bacteriochlorophyll by bacteriopheophytin molecule (Phi(B)). It was found that the nuclear wave packet motion induced on the potential energy surface of the excited state of the primary electron donor P* by approximately 20 fs excitation leads to a coherent formation of the states P+Phi(B)(-) and P+B(A)(-) (B(A) is a bacteriochlorophyll monomer in the A-branch of cofactors). The processes were studied by measuring coherent oscillations in kinetics of the absorbance changes at 900 nm and 940 nm (P* stimulated emission), at 750 nm and 785 nm (Phi(B) absorption bands), and at 1,020-1028 nm (B(A)(-) absorption band). In RCs, the immediate bleaching of the P band at 880 nm and the appearance of the stimulated wave packet emission at 900 nm were accompanied (with a small delay of 10-20 fs) by electron transfer from P* to the B-branch with bleaching of the Phi(B) absorption band at 785 nm due to Phi(B)(-) formation. These data are consistent with recent measurements for the mutant HM182L Rb. sphaeroides RCs (Yakovlev et al., Biochim Biophys Acta 1757:369-379, 2006). Only at a delay of 120 fs was the electron transfer from P* to the A-branch observed with a development of the B(A)(-) absorption band at 1028 nm. This development was in phase with the appearance of the P* stimulated emission at 940 nm. The data on the A-branch electron transfer in C. aurantiacus RCs are consistent with those observed in native RCs of Rb. sphaeroides. The mechanism of charge separation in RCs with the modified B-branch pigment composition is discussed in terms of coupling between the nuclear wave packet motion and electron transfer from P* to Phi(B) and B(A) primary acceptors in the B-branch and A-branch, respectively.

Identification of the First Steps in Charge Separation in Bacterial Photosynthetic Reaction Centers of Rhodobacter sphaeroides by Ultrafast Mid-Infrared Spectroscopy: Electron Transfer and Protein Dynamics

Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm −1 was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q A, were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct excitation of the dimer of primary donor ( P) BChls ( P L/ P M)). The region between 1600 and 1800 cm −1 encompasses absorption changes associated with carbonyl (C O) stretch vibrational modes of the cofactors and protein. After photoexcitation of the RC the primary electron donor P excited singlet state ( P*) decayed on a timescale of 3.7 ps to the state P + B L − (where B L is the accessory BChl electron acceptor). This is the first report of the mid-IR absorption spectrum of P + B L − ; the difference spectrum indicates that the 9-keto C O stretch of B L is located around 1670–1680 cm −1. After subsequent electron transfer to the bacteriopheophytin H L in ∼1 ps, the state P + H L − was formed. A sequential analysis and simultaneous target analysis of the data showed a relaxation of the P + H L − radical pair on the ∼20 ps timescale, accompanied by a change in the relative ratio of the P L + and P M + bands and by a minor change in the band amplitude at 1640 cm −1 that may be tentatively ascribed to the response of an amide C O to the radical pair formation. We conclude that the drop in free energy associated with the relaxation of P + H L − , is due to an increased localization of the electron hole on the P L half of the dimer and a further consequence is a reduction in the electrical field causing the Stark shift of one or more amide C O oscillators.

REVERSIBLE OXIDATION OF ANTENNA BACTERIOCHLOROPHYLL IN TWO PHOTOREACTION CENTERLESS MUTANTS OF RHODOSPIRILLVM RUBRUM

Abstract— Reversible oxidation of bacteriochlorophyll in two independent mutants of Rhodospirillum rubrum lacking photoreaction center has been studied to obtain further information about the antenna system of that bacterium. Oxidation of antenna bacteriochlorophyll with ferricyanide is accompanied by three distinct absorbance changes in the near infrared: the appearance of a new band at 1230 nm (B1230) and the bleaching of two bands, a major one at 882 nm (B882) and a minor one at 888 nm (B888). The three absorbance changes take place at potentials higher than those required for complete oxidation of photoreaction-center bacteriochlorophyll in wild-type strains. On the other hand, the B882 bleaching and the B1230 appearance occur under similar redox conditions. The above data suggest, first, that in Rds. rubrum, contrary to the current generalized idea, antenna bacteriochlorophyll exhibits at least two absorption bands, B888 and B882; second, that B882 and B1230 correspond to the same bacteriochlorophyll component. In addition, the appearance of B1230 may indicate that antenna bacteriochlorophyll can be partially arranged in an oligomeric fashion, for bands in that spectral region are not exhibited by oxidized monomeric bacteriochlorophyll.

Electronic Transitions of the Soret Band of Reaction Centers fromRhodobacter sphaeroidesStudied by Femtosecond Transient Absorbance Spectroscopy

The Soret band of reaction centers from Rhodobacter sphaeroides has been systematically studied using femtosecond transient absorption spectroscopy. When the excitation wavelength was scanned over the entire Soret band, the approximate absorption spectra of the bacteriochlorophyll dimer, the monomer bacteriochlorophylls, and the bacteriopheophytins within the Soret band were determined by analyzing the ground state bleaching with about 100 fs resolution. The main contribution of H is on the blue end of the spectrum, peaking near 350 nm, P absorbs mostly on the red side of the spectrum, but probably has multiple bands, and the main absorbance of B likely lies between H and P, overlapping with P on the red side (particularly near 390 nm). The energy transfer from B to P in the QY band takes about 300 fs when Soret-band excitation is used and the time constant of overall energy transfer from H to B to P in the QY band when H is specifically excited near 350 nm is about 500 fs. Internal conversion after Soret-band excitation is the rate-limiting step for the energy-transfer process. The time constant of internal conversion for B and P is less than 300 fs, and for H it is about 500 fs.

Thermodynamics of Light‐induced and Thermal Degradation of Bacteriochlorins in Reaction Center Protein of Photosynthetic Bacteria

The rate constants of thermal (irreversible) damage of bacteriochlorin pigments (bacteriochlorophyll monomer [B], bacteriochlorophyll dimer [P] and bacteriopheophytine [H]) in reaction center [RC] protein from the photosynthetic bacterium Rhodobacter sphaeroides were studied in the dark and during intense (400 mW x cm(-2)) laser light excitation (wavelengths 488 and 515 nm) under deoxygenated conditions. While the kinetics of degradation of P and B were monoexponential, the decay kinetics of H were overlapped by an initial lag phase at elevated (>40 degrees C) temperature. This is explained by removal of the central metal ion from the bacteriochlorophylls as part of their degradation processes. At all temperatures, the rates of damage were very similar for all bacteriochlorin pigments and were larger in the light than in the dark. The logarithm of the rate constant of pigment degradation and loss of photochemistry as a function of reciprocal (absolute) temperature (Arrhenius/Eyring plot) showed single phase in the light and double phases in the dark. Below 20 degrees C, the rate of pigment degradation in the RC decreased so dramatically in the dark that it became limited by the natural degradation process of bacteriochlorophyll measured in solution. The function of loss of photochemistry in the dark was also biphasic and had a break point at 40 degrees C. The damage in the dark required high enthalpy change (DeltaH(++) = 64 kcal/mol for P and DeltaH(++) = 60 kcal/mol for B) and entropy increase (T x DeltaS(++) = 38 kcal/mol for P and T x DeltaS(++) = 34 kcal/mol for B at T = 300 K), whereas significantly smaller enthalpy change (DeltaH(++) = 21 kcal/mol for P and B and DeltaH(++) = 13 kcal/mol for H) and practically no (T x DeltaS(++) = -1 kcal/mol for P and B at T = 300 K) or small (T x DeltaS(++) = -9 kcal/mol for H at T = 300 K) entropy change was needed in the light. The thermodynamic parameters of activation reveal major steps common in the degradation of all bacteriochlorin pigments: ring opening reactions at C5 or C20 meso-bridges (or both) and breaking/removal of the phytyl chain. Their contribution in the degradation is probably reflected in the observed enthalpy/entropy compensation at an almost constant (DeltaG(++) = 22-26 kcal/mol at T = 300 K) free energy change of activation.

Substitution of Isoleucine L177 by Histidine Affects the Pigment Composition and Properties of the Reaction Center of the Purple Bacterium Rhodobacter sphaeroides

Using site-directed mutagenesis, we obtained the mutant of the purple bacterium Rhodobacter sphaeroides with Ile to His substitution at position 177 in the L-subunit of the photosynthetic reaction center (RC). The mutant strain forms stable and photochemically active RC complexes. Relative to the wild type RCs, the spectral and photochemical properties of the mutant RC differ significantly in the absorption regions corresponding to the primary donor P and the monomer bacteriochlorophyll (BChl) absorption. It is shown that the RC I(L177)H contains only three BChl molecules compared to four BChl molecules in the wild type RC. Considering the fact that the properties of both isolated and membrane-associated mutant RCs are similar, we conclude that the loss of a BChl molecule from the mutant RC is caused by the introduced mutation but not by the protein purification procedure. The new mutant missing one BChl molecule but still able to perform light-induced reactions forming the charge-separated state P+QA- appears to be an interesting object to study the mechanisms of the first steps of the primary electron transfer in photosynthesis.

Environmental Control of Primary Photochemistry in a Mutant Bacterial Reaction Center

The core structure of the photosynthetic reaction center is quasisymmetric with two potential pathways (called A and B) for transmembrane electron transfer. Both the pathway and products of light-induced charge separation depend on local electrostatic interactions and the nature of the excited states generated at early times in reaction centers isolated from Rhodobacter sphaeroides. Here transient absorbance measurements were recorded following specific excitation of the Q(y)() transitions of P (the special pair of bacteriochlorophylls), the monomer bacteriochlorophylls (B(A) and B(B)), or the bacteriopheophytins (H(A) and H(B)) as a function of both buffer pH and detergent in a reaction center mutant with the mutations L168 His to Glu and L170 Asn to Asp in the vicinity of P and B(B). At a low pH in any detergent, or at any pH in a nonionic detergent (Triton X-100), the photochemistry of this mutant is faster than, but similar to, wild type (i.e. electron transfer occurs along the A-side, 390 nm excitation is capable of producing short-lived B-side charge separation (B(B)(+)H(B)(-)) but no long-lived B(B)(+)H(B)(-) is observed). Certain buffering conditions result in the stabilization of the B-side charge separated state B(B)(+)H(B)(-), including high pH in the zwitterionic detergent LDAO, even following excitation with low energy photons (800 or 740 nm). The most striking result is that conditions giving rise to stable B-side charge separation result in a lack of A-side charge separation, even when P is directly excited. The mechanism that links B(B)(+)H(B)(-) stabilization to this change in the photochemistry of P in the mutant is not understood, but clearly these two processes are linked and highly sensitive to the local electrostatic environment produced by buffering conditions (pH and detergent).

Uphill Energy Trapping by Reaction Center in Bacterial Photosynthesis. 2. Unistep Charge Separation, Virtually Mediated by Special Pair, by Photoexcitation in Place of Excitation Transfer from the Antenna System

In photosynthesis of purple bacteria, excitation energy of bacteriochlorophylls in the core antenna LH1 is transferred to the excited state (P*) of the special pair (P) in the reaction center (RC). Subsequently, charge separation to the adjacent monomeric bacteriochlorophyll (B) occurs from P*, producing P+B- as initial energy fixation in the RC. Although the absorption-peak energy of P is appreciably (by 200−430 cm-1) higher than that of the lowest excited state LH1* of LH1, these sequential events occur still at low temperatures for the energy trapping in Rhodobacter sphaeroides, while in Rhodopseudomonas viridis LH1* is unistep converted to P+B- in the RC by the superexchange mechanism due to quantum-mechanical virtual mediation by P* at low temperatures, as clarified in part 1 (Sumi, H. J. Phys. Chem. B 2002, 106, 13370). Such differences arise since in the free energy LH1* is slightly (by ∼50 cm-1) higher than P* in Rb. sphaeroides, while in Rps. viridis LH1* is still considerably (∼150 cm-1) lower than P* in the free energy, after reorganization of the medium around P*. An experiment is proposed to investigate unifiedly such differences, by photoexcitation of a single RC from either Rb. sphaeroides or Rps. viridis. When the photon energy E is higher than about the free energy E0 for P*, the production rate of P+B-, measurable as the rate of absorption by the RC, will be nearly equal to that by P*, measurable as its excitation spectrum, at all temperatures by the sequential mechanism. When E is considerably lower than E0, the former will become much larger than the latter at low temperatures by the superexchange mechanism. The photoinduced production rate of P+B- mediated by P* is given as an analytic formula bridging the two limits, for analyzing such observations expected.

Electron Transfer Dynamics in Rhodobacter Sphaeroides Reaction Center Mutants with a Modified Ligand for the Monomer Bacteriochlorophyll on the Active Side

The histidine ligand of the monomer bacteriochlorophyll molecule on the active side (BA) of the photosynthetic reaction center from Rhodobacter sphaeroides was mutated to a number of other amino acids. Histidine (H) at the position L153 was replaced with aspartic acid (D), glutamic acid (E), glutamine (Q), glycine (G), leucine (L), phenylalanine (F), serine (S), valine (V) and tyrosine (Y). These mutations were created to investigate how the alteration of the ligand residue affects the properties of the BA molecule and changes the dynamics of the primary charge separation in reaction centers. In all of the mutants, the changes in the ligand result in a blue-shift of the BA absorption spectrum, in both visible and near-infrared spectral regions, with the size of the shift varying between mutants. The primary electron transfer time constants in these mutant reaction centers range from 4.5 to 18 ps, being substantially slower than the wild-type value of 3 ps. The decrease in the electron transfer rate is interpreted as being due to an increase in the free energy of the initial charge-separated state P+BA−.

Excitation Energy Transfer from Carotenoid to Bacteriochlorophyll in the Photosynthetic Purple Bacterial Reaction Center of Rhodobacter sphaeroides

The role of carotenoid in singlet excitation energy transfer in the purple bacterial reaction center of Rb. sphaeroides has been studied using femtosecond transient absorbance spectroscopy techniques at room temperature. The carotenoid S0-to-S2 transition band was directly excited at 490 nm and the transient spectral changes were recorded in the visible and near-infrared spectral regions with subpicosecond time resolution. This result was compared with transient spectral changes observed following direct excitation of the bacteriochlorophyll dimer of the reaction center (P) at 860 nm. With 490-nm excitation, a subpicosend relaxation process from the carotenoid S2-to-S1 state is observed, followed by energy transfer to the monomer bacteriochlorophylls. Excitation energy is further transferred to P and then is utilized for the formation of the charge separated state P+HA-. The majority of the excitation energy from the carotenoid is transferred to bacteriochlorophyll from its S1 state and occurs with a time constant of 2.2 ps. The overall energy transfer efficiency between the excited carotenoid and bacteriochlorophyll is estimated to be greater than or equal to 75%. The carotenoid in the purple bacterial reaction center is in a 15-cis-configuration, which differs from the all-trans configuration of carotenoids found in the vast majority of antenna systems. The efficient singlet excitation energy transfer observed in this study suggests that the configuration difference is not a key factor in determining the efficiency of carotenoid-to-bacteriochlorophyll singlet excitation energy transfer.

High Yield of Long-Lived B-Side Charge Separation at Room Temperature in Mutant Bacterial Reaction Centers

The photosynthetic reaction center of Rhodobacter (Rb.) sphaeroides is a quasi-symmetric pigment protein complex. This near symmetry results in two potential pathways for light-induced electron transfer, labeled A and B, yet under most conditions charge separation occurs almost exclusively along the A-side cofactors in wild-type reaction centers. One exception is that upon excitation with blue light (390 nm) a transient B-side charge-separated state, BB+HB- (the cation of the B-side monomer bacteriochlorophyll and the anion of the B-side bacteriopheophytin), is formed that decays in picoseconds at room temperature but is stable at cryogenic temperatures. To characterize the nature of this reaction further, a series of mutations involving the introduction of potentially negative amino acids in the vicinity of P and the monomer bacteriochlorophylls, BA and BB, were used to alter the local electrostatic environment. The most dramatic effects were observed for reaction centers with the mutations L168 His to Glu and L170 Asn to Asp. These mutant reaction centers display a stable (hundreds of picoseconds) formation of BB+HB- not only using 390-nm excitation but also upon direct excitation of the lowest excited singlet states of the reaction center bacteriopheophytins and bacteriochlorophylls at 740 and 800 nm, respectively. Reaction centers mutated at three sitesL168 His to Glu, L170 Asn to Asp and M199 Asn to Aspwere also found to form a stable BB+HB- state following excitation at 390 nm but with an apparently lower yield. In the other mutant reaction centers studied, long-lived BB+HB- formation was not observed using any excitation wavelength, though 390-nm excitation resulted in BB+HB- formation followed by the decay of this state on the picosecond time scale, as is observed in wild type. Combining these results with past work demonstrates that there is a rich variety of photochemistry possible in the reaction center. Both the pathway and products of light-induced charge separation depend on the interplay between local electrostatic interactions and the nature of the excited states available at early times.

Manipulations of the B-Side Charge-Separated States' Energetics in the Rhodobacter sphaeroides Reaction Center

The mutation HL(M182) in the Rb. sphaeroides reaction center (RC) results in the replacement of the monomer bacteriochlorophyll on the inactive side (B side) of the reaction center with a bacteriopheophytin (φB). In φB containing reaction centers, excitation of the initial electron donor, the special pair P, results in about 35% electron transfer along the normally inactive B side. However, the electron is transferred only to the exchanged cofactor φB. Several additional mutations in close proximity to bacteriopheophytins φB or HB have been created with the goal of altering the energetics of charge-separated states P+φB- and P+HB-. Aspartic acid residues were introduced to replace methionine L174 or valine M175 in the vicinity of the φB cofactor in order to raise the free energy of state P+φB-. Threonine M133 was mutated to the aspartic acid to add a hydrogen bond to the HB cofactor and lower the free energy of state P+HB-. The mutations in the environment surrounding the φB pigment resulted in a decrease in the quantum yield of P+φB- as well as a decrease in the recombination lifetime of this state. The mutation of valine M175 to aspartic acid showed the largest effect. The yield of state P+φB- decreased to about 25% and its recombination lifetime shortened from 200 to 125 ps. This additional mutation also resulted in the loss of the carotenoid molecule from the reaction centers. None of the three additional mutations altered the free energies sufficiently to result in observable electron transfer to HB. However, these measurements have allowed a more accurate assignment of the B-side charge-separated states' energetics than was previously possible.

Excited-state energy transfer pathways in photosynthetic reaction centers: 5. Oxidized and triplet excited special pairs as energy acceptors

In bacterial photosynthetic reaction centers, ultrafast singlet excited-state energy transfer occurs from the monomeric bacteriochlorophylls, B, and bacteriopheophytins, H, to the homodimer special pair, P, a pair of strongly interacting bacteriochlorophylls. Using fluorescence upconversion spectroscopy, energy transfer to the special pair can be monitored by observing the decay of 1 B emission and/or the rise of 1 P. We report 1 B decay kinetics following excitation in the H band in reaction centers where the homodimer and heterodimer (M202HL) special pairs are oxidized, P + and D + , respectively, and when the homodimer special pair is in the triplet state, 3 P. In wild type and the M71GL mutant (a carotenoid-less reaction center), the rates of 1 B decay when P + and 3 P are present, (∼260 fs) −1 and (∼235 fs) −1 , respectively, are similar to that for energy transfer to 1 P (∼190 fs) −1 in wild type measured by either the fluorescence decay of 1 B or the rise of 1 P. In contrast to the homodimer special pair in wild type where the energy transfer rates along the two branches are very similar, singlet energy transfer from the monomeric chromophores along the L and M branches to the heterodimer special pair is asymmetric and is slower along the L side. The 1 B decay in wild type is well described by a single rate constant of (∼190 fs) −1 and in M202HL exhibits two components with rate constants (∼780 fs) −1 and (∼250 fs) −1 . In M202HL reaction centers containing D + , 1 B decays with a single rate constant of (∼343 fs) −1 ; hence, the energy transfer rates along the two branches become similar. Thus, while conversion of the special pair homodimer to a heterodimer breaks the symmetry of ultrafast energy transfer along the two branches of chromophores, symmetry can be restored by oxidizing the heterodimer special pair. To our knowledge, this is the first report of such dramatic alteration of energy transfer within a single reaction center protein. These findings bear on the mechanism of energy transfer in the reaction center and may provide insight into the differences in the electronic interactions on the L vs. M sides of the RC that are relevant to unidirectional electron transfer.