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The authors extend the technique of fluorescence depletion to measure femtosecond anisotropy decays. In cases where excited-state absorption contributes to the transient spectra, such as in photosynthetic antenna complexes, the anisotropy measured by fluorescence depletion differs from that measured by pump-probe spectroscopy. This new technique has the potential to elucidate the fate of the states created by excited-state absorption.

The photosynthetic bacterium Rhodobacter sphaeroides contains a light-harvesting antenna complex (LH2) with a ring of interacting bacteriochlorophyll molecules (B850). Excitation of membrane-bound LH2 complexes with low-intensity, femtosecond pulses causes changes in absorption and stimulated emission that initially depend on the excitation wavelength but relax to a quasiequilibrium with a time constant of 100 {+-} 20 fs. Excitation on the blue side of the B850 absorption band is followed by a shift of the signals to longer wavelengths and a decrease in amplitude, whereas the relaxations following excitation on the red side consist mainly of a decrease in amplitude. The signals have an apparent initial anisotropy of approximately 0.5 when the complex is excited with broadband pulses, and 0.35--0.4 with narrower pulses. The anisotropy decays to 0.1 with a time constant of about 30 fs. The anisotropies are similar at wavelengths on either side of the absorption band and are relatively insensitive to the excitation wavelength. Contributions of coherent pump-probe coupling and perturbed free induction decay to the measured anisotropies are considered. Pump-probe coupling could increase the initial anisotropy but cannot account for the decay kinetics. Using a density-matrix formalism, the authors show that the initial light-induced signals are consistent withmore » coherent excitation of multiple exciton levels in an inhomogeneous ensemble of LH2 complexes and that the main features of the spectral relaxations and the anisotropy can be explained by electronic dephasing and thermal equilibration within the manifold of exciton levels.« less

Oscillations in the electrostatic energy gap [ΔVelec(t)] for electron transfer from the primary electron donor (P) to the adjacent bacteriochlorophyll (B) in photosynthetic bacterial reaction centers are examined by molecular-dynamics simulations. Autocorrelation functions of ΔVelec in the reactant state (PB) include prominent oscillations with an energy of 17 cm−1. This feature is much weaker if the trajectory is propagated in the product state P+B−. The autocorrelation functions also include oscillations in the regions of 5, 80 and 390 cm−1 in both states, and near 25 and 48 cm−1 in P+B−. The strong 17-cm−1 oscillation could involve motions that modulate the distance between P and B, because a similar oscillation occurs in the direct electrostatic interactions between the electron carriers.

Absorption and CD spectra of a photosynthetic bacterial antenna complex are calculated on the basis of the crystal structure of the LH2 (B800-850) complex from Rhodopseudomonas acidophila. This complex contains a ring of 18 tightly coupled bacteriochlorophylls (B850) and a ring of 9 more weakly coupled bacteriochlorophylls (B800). Molecular orbitals for bacteriochlorophylls with the three different geometries seen in the crystal structure are obtained by semiempirical quantum mechanical calculations (QCFF/PI). Exciton and charge-transfer interactions are introduced at the level of configuration interactions. Particular attention is paid to the dependence of these interactions on the interatomic distances and on dielectric screening. Absorption band shapes are treated with the aid of vibronic parameters and homogeneous line widths that have been measured by hole burning (Reddy, N. R. S., et al., Photochem. Photobiol. 1993, 57, 35−39). Inhomogeneous broadening due to diagonal disorder in the monomeric and c...

Fushiki and Tachiya [Chem. Phys. Lett. 255 (1996) 83] recently analyzed the free energy surfaces of the initial electron-transfer processes in photosynthetic bacterial reaction centers. The authors state that when the results from simulations described by Warshel, Chu and Parson [Photochem. Photobiol. A: Chem. 82 (1994) 123] are analyzed using their formulation, the calculated energy of a key ion-pair state is inconsistent with experiment. They also state that previous analyses of the photosynthetic electron-transfer reactions had been limited to one-dimensional free energy surfaces. We show here that both these assertions are incorrect.

Spectral changes were measured with femtosecond resolution following low-intensity, broad-band excitation of the peripheral antenna complex of the purple photosynthetic bacteriumRhodobacter sphaeroides. Absorption anisotropy decays also were measured. We identified a 35-fs relaxation of the absorption and emission spectra of the excited state, as well as a 20-fs anisotropy decay. We interpret these results as interlevel relaxation and dephasing, respectively, of extensively delocalized exciton states of the circular bacteriochlorophyll aggregate.

The side chain of tyrosine (M)210 is located close to the bacteriochlorophyll dimer (P) that serves as the electron donor in the photochemical charge-separation reaction of photosynthetic bacterial reaction centers; it also is close to the monomeric bacteriochlorophyll (BL) that probably accepts an electron in this reaction. Electrostatics calculations and molecular-dynamics simulations were performed to explore the preferred orientation and the time-dependent fluctuations of the phenolic OH dipole of the tyrosine and to examine the effects of replacing the tyrosine residue by other amino acids. In resting reaction centers, the OH dipole was found to point toward BL in a way that would favor formation of the P+BL- ion pair. The molecular-dynamics simulations indicated that the most probable orientation of the OH dipole does not change significantly upon charge separation but that the potential well constraining the dipole deepens and the frequency of oscillations about the minimum increases. Replacing Tyr...

The quantum yield of tryptophan (Trp) fluorescence was measured in 30 designed miniproteins (17 β-hairpins and 13 Trp-cage peptides), each containing a single Trp residue. Measurements were made in D(2)O and H(2)O to distinguish between fluorescence quenching mechanisms involving electron and proton transfer in the hairpin peptides, and at two temperatures to check for effects of partial unfolding of the Trp-cage peptides. The extent of folding of all the peptides also was measured by NMR. The fluorescence yields ranged from 0.01 in some of the Trp-cage peptides to 0.27 in some hairpins. Fluorescence quenching was found to occur by electron transfer from the excited indole ring of the Trp to a backbone amide group or the protonated side chain of a nearby histidine, glutamate, aspartate, tyrosine, or cysteine residue. Ionized tyrosine side chains quenched strongly by resonance energy transfer or electron transfer to the excited indole ring. Hybrid classical/quantum mechanical molecular dynamics simulations were performed by a method that optimized induced electric dipoles separately for the ground and excited states in multiple π-π* and charge-transfer (CT) excitations. Twenty 0.5 ns trajectories in the tryptophan's lowest excited singlet π-π* state were run for each peptide, beginning by projections from trajectories in the ground state. Fluorescence quenching was correlated with the availability of a CT or exciton state that was strongly coupled to the π-π* state and that matched or fell below the π-π* state in energy. The fluorescence yields predicted by summing the calculated rates of charge and energy transfer are in good accord with the measured yields.

The initial electron-transfer reaction in photosynthetic bacterial reaction centers is the transfer of an electron from the excited state (P{sup *}) of a reactive bacteriochlorophyll dimer (P) to a bacteriopheophytin (H), generating a P{sup +}H{sup -} radical pair. In this investigation we have shown that models with proper dielectric boundaries lead to calculated electrostatic energies that are relatively insensitive to the assumptions made concerning the solvent and the charges assigned to ionized groups of the protein. Using such models leads to good agreement with the experimentally measured energy of the relaxed P{sup +}H{sup -} radical pair and places P{sup +}B{sup -}H close to P{sup *} in energy. By contrast, treatments that neglect the self-energies of the electron carriers and do not account for the screening of the field of the ionized residues place P{sup +}B{sup -}H substantially above P{sup *}. 35 refs., 5 figs., 9 tabs.

The energetics of the initial electron-transfer steps in photosynthetic bacterial reaction centers are evaluated by free-energy perturbation calculations using several different treatments of the ionizable amino acid residues and of solvent molecules in and around the protein. The calculation illustrate the problems with incomplete treatments of dielectric effects. Calculations that do not include mobile solvent lead to large overestimates of the effects of ionized amino acid side-chains, and can give errors of up to 20 kcal mol −1 in the free-energy difference between the ion-pair states P + B − and P + H − . When mobile solvent molecules are included, these two states are calculated to be relatively close together in free energy.

Using molecular dynamics simulations and steady-state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far-UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature-dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc.

Comparing photosynthetic and photovoltaic efficiencies is not a simple issue. Although both processes harvest the energy in sunlight, they operate in distinctly different ways and produce different types of products: biomass or chemical fuels in the case of natural photosynthesis and nonstored electrical current in the case of photovoltaics. In order to find common ground for evaluating energy-conversion efficiency, we compare natural photosynthesis with present technologies for photovoltaic-driven electrolysis of water to produce hydrogen. Photovoltaic-driven electrolysis is the more efficient process when measured on an annual basis, yet short-term yields for photosynthetic conversion under optimal conditions come within a factor of 2 or 3 of the photovoltaic benchmark. We consider opportunities in which the frontiers of synthetic biology might be used to enhance natural photosynthesis for improved solar energy conversion efficiency.

The rates of the light-driven, electron-transfer reactions in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides are examined in mutant strains in which tyrosine (M)210 is replaced by phenylalanine, isoleucine, or tryptophan. The spectra of the absorbance changes between 700 and 975 nm, following excitation by 0.6-ps pulses at 605 nm, are analyzed globally by singular value decomposition. The spectra measured at room temperature are interpreted in terms of a model in which the excited bacteriochlorophyll dimer (P*) transfers an electron to a bacteriopheophytin (HL) with time constants of 3.5 +/- 0.3, 10.5 +/- 1.0, 16 +/- 2, and 41 +/- 4 ps in wild-type RCs and the Phe, Ile, and Trp mutants, respectively, and an electron then moves from HL- to a quinone (QA) with a time constant of 0.16 ns in wild-type RCs, 0.24 ns in the Phe mutant, and 0.20 ns in the Ile and Trp mutants. The first step speeds up with decreasing temperature in wild-type RCs, remains virtually unchanged in the Phe mutant, and slows down in the Ile and Trp mutants. At 80 K, the signals in the 850-975-nm region include an apparent shift of the stimulated emission or absorption spectrum of P*, with a time constant of 5 ps in the Ile mutant and 13 pcs in the Trp mutant. Most of the electron transfer to HL occurs with time constants of 55 and 155 ps in the Ile and Trp mutants, respectively, and probably occurs from the relaxed form of P*. Electron transfer from the initial state cannot be ruled out, however. Relaxations of P* are not resolved in wild-type RCs or the Phe mutant. The midpoint potential (Em) of the P/P+ redox couple is measured by an electrochemical technique; the Em values are 500 +/- 5, 530 +/- 6, 533 +/- 3, and 552 +/- 10 mV for the wild-type and the Phe, Ile, and Trp mutant RCs, respectively. These values are corroborated by chemical titrations. The free energy change (delta G degrees) associated with formation of the P+HL-radical pair from P* also is determined by measuring the amplitude of fluorescence on the nanosecond time scale after blocking electron transfer from HL- to QA. The free energy of P+HL- is elevated by an amount comparable to that calculated from the increase in the Em of P in the Ile mutant and by about 16 meV more than this in the Phe and Trp mutants.(ABSTRACT TRUNCATED AT 400 WORDS)

This chapter discusses the pathway, kinetics and energetics of the initial electron-transfer steps in reaction centers (RCs) from purple photosynthetic bacteria. We consider the unusual dependence of the kinetics on temperature, the strong specificity of the reactions for electron acceptors on the ‘A’ side of the RC, effects of vibrational wavepackets, the multiphasic nature of the kinetics, effects of mutations and pigment substitutions, and links between the kinetics of electron transfer and vibrational equilibration. We then discuss some of the theories that have been used to rationalize the dynamics and temperature dependence of the electron-transfer reactions, including Marcus theory, coupling to quantized vibrational modes and density-matrix treatments. Our discussion illustrates the power of microscopic simulations for connecting structural information with mechanistic interpretations.

The human gene for catechol O-methyltransferase has a common single-nucleotide polymorphism that results in substitution of methionine (M) for valine (V) 108 in the soluble form of the enzyme (s-COMT). 108M s-COMT loses enzymatic activity more rapidly than 108V s-COMT at physiological temperature, and the 108M allele has been associated with increased risk of breast cancer and several neuropsychiatric disorders. We used circular dichroism (CD), dynamic light scattering, and fluorescence spectroscopy to examine how the 108V/M polymorphism affects the stability of the purified, recombinant protein to heat and guanidine hydrochloride (GuHCl). COMT contains two tryptophan residues, W143 and W38Y, which are located in loops that border the S-adenosylmethionine (SAM) and catechol binding sites. We therefore also studied the single-tryptophan mutants W38Y and W143Y in order to dissect the contributions of the individual tryptophans to the fluorescence signals. The 108V and 108M proteins differed in the stability of both the tertiary structure surrounding the active site, as probed by the fluorescence yields and emission spectra, and their global secondary structure as reflected by CD. With either probe, the midpoint of the thermal transition of 108M s-COMT was 5 to 7 degrees C lower than that of 108V s-COMT, and the free energy of unfolding at 25 degrees C was smaller by about 0.4 kcal/mol. 108M s-COMT also was more prone to aggregation or partial unfolding to a form with an increased radius of hydration at 37 degrees C. The co-substrate SAM stabilized the secondary structure of both 108V and 108M s-COMT. W143 dominates the tryptophan fluorescence of the folded protein and accounts for most of the decrease in fluorescence that accompanies unfolding by GuHCl. While replacing either tryptophan by tyrosine was mildly destabilizing, the lower stability of the 108M variant was retained in all cases.

Summary This chapter discusses computer models for evaluating electrostatic interactions in proteins, with emphasis on calculations of the free energies of electron-transfer states in photosynthetic bacterial reaction centers. We describe the microscopic Protein Dipoles Langevin Dipoles (PDLD) method, semimicroscopic approaches including the Poisson-Boltzmann, PDLD/S and Generalized Born models, a macroscopic model with a homogeneous dielectric medium, and microscopic free-energy-perturbation methods based on molecular dynamics simulations. We also describe the use of molecular dynamics simulations to obtain free energy surfaces of the reactant and product states as functions of the reaction coordinate for electron transfer.

The pH dependence of the redox midpoint potential of the QA/Q−A couple in isolated reaction centers from Rb. sphaeroides (Maroti, P. and Wraight, C.A. (1988) Biochim. Biophys. Acta 934, 329–347) is about 3-times larger than predicted from the proton uptake by DQ−A (preceding reference and McPherson, P.H., Okamura, M.Y and Feher, G. (1988) Biochim. Biophys. Acta 934, 348–368). To investigate the cause of this discrepancy, we have determined from the delayed fluorescence of reaction centers the pH dependence (7.0

Electrostatic interaction energies of the electron carriers with their surroundings in a photosynthetic bacterial reaction center are calculated. The calculations are based on the detailed crystal structure of reaction centers from Rhodopseu-domonas viridis, and use an iterative, self-consistent procedure to evaluate the effects of induced dipoles in the protein and the surrounding membrane. To obtain the free energies of radical-pair states, the calculated electrostatic interaction energies are combined with the experimentally measured midpoint redox potentials of the electron carriers and of bacteriochlorophyll (BChl) and bacteriopheophytin (BPh) in vitro. The P+HL- radical-pair, in which an electron has moved from the primary electron donor (P) to a BPh on the 'L' side of the reaction center (HL), is found to lie approx. 2.0 kcal/mol below the lowest excited singlet state (P*), when the radical-pair is formed in the static crystallographic structure. The reorganization energy for the subsequent relaxation of P+HL- is calculated to be 5.0 kcal/mol, so that the relaxed radical-pair lies about 7 kcal/mol below P*. The unrelaxed P+BL- radical-pair, in which the electron acceptor is the accessory BChl located between P and HL, appears to be essentially isoenergetic with P*.P+BM-, in which an electron moves to the BChl on the 'M' side, is calculated to lie about 5.5 kcal/mol above P*. These results have an estimated error range of +/- 2.5 kcal/mol. They are shown to be relatively insensitive to various details of the model, including the charge distribution in P+, the atomic charges used for the amino acid residues, the boundaries of the structural region that is considered microscopically and the treatments of the histidyl ligands of P and of potentially ionizable amino acids. The calculated free energies are consistent with rapid electron transfer from P* to HL by way of BL, and with a much slower electron transfer to the pigments on the M side. Tyrosine M208 appears to play a particularly important role in lowering the energy of P+BL-. Electrostatic interactions with the protein favor localization of the positive charge of P+ on PM, one of the two BChl molecules that make up the electron donor.

Photosynthetic organisms that do not evolve oxygen are found in four of the 24 phyla of bacteria: Proteobacteria (purple bacteria), Chloroflexi (green, non-sulfur bacteria), Chlorobi (green, sulfur bacteria) and Firmicutes (Heliobacteria). The reaction centers (RCs) of photosynthetic Chlorobi and Firmicutes (Type I reaction centers) are structurally and functionally similar to the RC of Photosystem I in oxygenic organisms, while those of Proteobacteria and Chloroflexi (Type II reaction centers) resemble Photosystem II. Reaction centers of Type I transfer electrons from a c-type cytochrome or plastocyanin to bound [4Fe-4S] iron-sulfur centers, which then reduce the soluble iron-sulfur protein ferredoxin. Type II RCs transfer electrons from c-type cytochromes to a quinone that picks up protons and dissociates from the RC as the fully reduced quinol. This chapter describes the patterns of electron transfer through the RCs of the four families of bacteria, with an emphasis on the relationships between function and structure. These functional patterns are reasonably clear in proteobacterial RCs, but much remains to be learned about them in some of the other families.

The discovery by Louis N. M. Duysens in the 1950s that illumination of photosynthetic purple bacteria can cause oxidation of either a bacteriochlorophyll complex (P) or a cytochrome was followed by an extended period of uncertainty as to which of these processes was the ‘primary’ photochemical reaction. Similar questions arose later about the roles of bacteriopheophytin (BPh) and quinones as the initial electron acceptor. This is a personal account of kinetic measurements that showed that electron transfer from P to BPh occurs in the initial step, and that the oxidized bacteriochlorophyll complex (P+) then oxidizes the cytochrome while the reduced BPh transfers an electron to a quinone.

Electrostatic energies provide what is perhaps the most effective tool for structure–function correlation of biological molecules. This review considers the current state of simulations of electrostatic energies in macromolecules as well as the early developments of this field. We focus on the relationship between microscopic and macroscopic models, considering the convergence problems of the microscopic models and the fact that the dielectric ‘constants’ in semimacroscopic models depend on the definition and the specific treatment. The advances and the challenges in the field are illustrated considering a wide range of functional properties including pKa's, redox potentials, ion and proton channels, enzyme catalysis, ligand binding and protein stability. We conclude by pointing out that, despite the current problems and the significant misunderstandings in the field, there is an overall progress that should lead eventually to quantitative descriptions of electrostatic effects in proteins and thus to quantitative descriptions of the function of proteins. © 2006 Elsevier B.V. All rights reserved.

Photosynthetic bacterial reaction centers provide a rich territory for exploring the nature of biological electron-transfer reactions. The crystal structures of reaction centers from two species of bacteria are known, and the structures can be modified by site-directed mutagenesis or by substituting other pigments for the natural bacteriochlorophylls, bacteriopheophytins or quinones. Femtosecond laser excitation pulses of various wavelengths can be used to prepare the initial excited state in a variety of conditions, and the kinetics of the subsequent charge separation and recombination reactions can be measured with extremely high time resolution. Measurements can be made under an extraordinarily wide range of conditions, including low temperatures and the presence of external electrical or magnetic fields. Further, there are many intriguing observations to explain. Several of the reactions occur extremely rapidly and become even faster with decreasing temperature; electron transfer along the active ‘L’ branch of the pigments is much faster than that along the ‘M’ branch; and seemingly drastic changes in the structure sometimes have little effect on the directionality or rates of the reactions. What is it, then, that determines the speed and temperature dependence of an intermolecular electron-transfer reaction in a protein?

The human gene for catechol O-methyltransferase (COMT) contains a common polymorphism that results in substitution of methionine (M) for valine (V) at residue 108 of the soluble form of the protein. While the two proteins have similar kinetic properties, 108M COMT loses activity more rapidly than 108V COMT at 37 degrees C. The cosubstrate S-adenosylmethionine (SAM) stabilizes the activity of 108M COMT at 40 degrees C. The 108M allele has been associated with increased risk for breast cancer, obsessive-compulsive disorder, and aggressive and highly antisocial manifestations of schizophrenia. In the current work, we have constructed homology models for both human COMT polymorphs and performed molecular dynamics simulations of these models at 25, 37, and 50 degrees C to explore the structural consequences of the 108V/M polymorphism. The simulations indicated that replacing valine with the larger methionine residue led to greater solvent exposure of residue 108 and heightened packing interactions between M108 and helices alpha2, alpha4 (especially with R78), and alpha5. These altered packing interactions propagated subtle changes between the polymorphic site and the active site 16 A away, leading to a loosening of the active site. At physiological temperature, 108M COMT sampled a larger distribution of conformations than 108V. 108M COMT was more prone to active-site distortion and had greater overall, and SAM binding site, solvent accessibility than 108V COMT at 37 degrees C. Similar structural perturbations were observed in the 108V protein only at 50 degrees C. Addition of SAM tightened up the cosubstrate pocket in both proteins and prevented the altered packing at the polymorphic site in 108M COMT.

The discovery by Louis N. M. Duysens in the 1950s that illumination of photosynthetic purple bacteria can cause oxidation of either a bacteriochlorophyll complex (P) or a cytochrome was followed by an extended period of uncertainty as to which of these processes was the 'primary' photochemical reaction. Similar questions arose later about the roles of bacteriopheophytin (BPh) and quinones as the initial electron acceptor. This is a personal account of kinetic measurements that showed that electron transfer from P to BPh occurs in the initial step, and that the oxidized bacteriochlorophyll complex (P(+)) then oxidizes the cytochrome while the reduced BPh transfers an electron to a quinone.

Editorial. Preface. Color Plates. I: Introduction to Light-Harvesting. 1. Photosynthetic Membranes and Their Light-Harvesting Antennas B.R. Green, J.M. Anderson, W.W. Parson. 2. The Pigments H. Scheer. 3. Optical Spectroscopy in Photosynthetic Antennas W.W. Parson, V. Nagarajan. 4. The Evolution of Light-Harvesting Antennas B.R. Green. II: Structure and Function in Light-Harvesting. 5. The Light-Harvesting System of Purple Bacteria B. Robert, R.J. Cogdell, R. van Grondelle. 6. Antenna Complexes from Green Photosynthetic Bacteria R.E. Blankenship, K. Matsuura. 7. Light-Harvesting in Photosystem II H. van Amerongen, J.P. Dekker. 8. Structure and Function of the Antenna System in Photsystem I P. Fromme, E. Schlodder, S. Jansson. 9. Antenna Systems and Energy Transfer in Cyanophyta and Rhodophyta M. Mimuro, H. Kikuchi. 10. Antenna Systems of Red Algae: Phycobilisomes with Photosystem II and Chlorophyll Complexes with Photosystem I E. Gantt, B. Grabowski, F.X. Cunningham Jr. 11. Light-Harvesting Systems in Chlorophyll c-Containing Algae A.N. Macpherson, R.G. Hiller. III: Biogenesis, Regulation and Adaptation. 12. Biogenesis of Green Plant Thylakoid Membranes K. Cline. 13. Pulse Amplitude Modulated Chlorophyll Fluorometry and its Application in Plant Science G.H. Krause, P. Jahns. 14. Photostasis in Plants, Green Algae and Cyanobacteria: The Role of Light-Harvesting Antenna Complexes N.P.A. Huner, G. Oquist, A. Melis. 15. Photoacclimation of Light-Harvesting Systems in EukaryoticAlgae P.G. Falkowski, Yi-Bu Chen. 16. Multi-level Regulation of Purple Bacterial Light-Harvesting Complexes C.S. Young, J.T. Beatty. 17. Environmental Regulation of Phycobilisome Biosynthesis A.R. Grossman, L.G. van Waasbergen, D. Kehoe. Index.

This chapter describes the main techniques of optical spectroscopy that are used to study the structure and operation of photosynthetic antenna systems. We outline the physical basis of optical absorption, fluorescence, linear and circular dichroism, exciton interactions and resonance energy transfer, and indicate the types of information that measurements of these phenomena can provide.

Light-harvesting antennas are pigment-proteins that absorb light energy and transfer it to photosynthetic reaction centers. This chapter starts with a brief non-technical explanation of how antennas harvest light. The antennas of the five divisions of photosynthetic bacteria (including cyanobacteria) are introduced; the antennas are placed in the context of their photosynthetic membranes. The evolutionary origin of chloroplasts by primary and secondary endosymbiosis is explained. A brief description of the various algal groups is followed by a more detailed discussion of the higher plant chloroplast and the roles of the LHC superfamily antennas. Throughout, readers are directed to the relevant chapters in the book where detailed information can be found.

The effects of charge-charge interactions on the midpoint reduction potential (E(m)()) of the primary electron donor (P) in the photosynthetic reaction center of Rhodobacter sphaeroides were investigated by introducing mutations of ionizable amino acids at selected sites. The mutations were designed to alter the electrostatic environment of P, a bacteriochlorophyll dimer, without greatly affecting its structure or molecular orbitals. Two arginine residues at homologous positions in the L and M subunits [residues (L135) and (M164)], Asp (L155), Tyr (L164), and Cys (L247) were changed independently. Arginine (L135) was replaced by Lys, Leu, Gln, or Glu; Arg (M164), by Leu or Glu; Asp (L155), by Asn; Tyr (L164), by Phe; and Cys (L247), by Lys or Asp. The R(L135)E/C(L247)K double mutant also was made. The shift in the E(m)() of P/P(+) was measured in each mutant and was compared with the effect predicted by electrostatics calculations using several different computational approaches. A simple distance-dependent dielectric screening factor reproduced the effects remarkably well. By contrast, microscopic methods that considered the reaction field in the protein and solvent but did not include explicit counterions overestimated the changes in the E(m)() considerably. Including counterions for the charged residues reduced the calculated effects of the mutations in molecular dynamics calculations. The results show that electrostatic interactions of P with ionizable amino acid residues are strongly screened, and suggest that counterions make major contributions to this screening. The screening also could reflect penetration of water or other relaxations not taken into account because of incomplete sampling of configurational space.

We have used molecular-dynamics simulations to examine the time-dependent energy gap between P* and P+BL- in purple bacterial reaction centers (RCs). Here P is the primary electron donor and BL(BA) is the neighboring “accessory” bacteriochlorophyll (BChl) that probably accepts an electron in the initial step of charge separation. From the fluctuations of the energy gap during molecular-dynamics trajectories in the reactant and product states one can obtain the Marcus free-energy curves that determine the activation energy of the initial charge-separation reaction. Effects of mutations on the activation energy can be explored, and protein motions that are coupled to the reaction can be characterized.

Energy transfer between the B850 (LH2) and B875 (LH1) antenna complexes of a mutant strain of Rhodobacter sphaeroides lacking reaction centers is investigated by femtosecond pump-probe spectroscopy at room temperature. Measurements are made at wavelengths between 810 and 910 nm at times extending to 200 ps after selective excitation of either B850 or B875. Assignments of the spectroscopic signals to the two types of antenna complex are made on the basis of measurements in strains that lack either LH1 or LH2 in addition to reaction centers. Energy transfer from excited B850 to B875 proceeds with two time constants, 4.6 +/- 0.3 and 26.3 +/- 1.0 ps, but a significant fraction of the excitations remain in B850 for considerably longer times. The fast step is interpreted as hopping of energy to LH1 from an associated LH2 complex; the slower steps are interpreted as migration of excitations in the LH2 pool preceding transfer to LH1. Transfer of excitations from B875 to B850 could not be detected, possibly suggesting that the average number of LH2 complexes in contact with each LH1 is small.

Two approaches for calculating the free energies of transient radical-pair states in bacterial reaction centers are discussed. Although macroscopic models that assign a homogenous dielectric constant to the protein and solvent are major oversimplifications, they help to clarify the importance of considering the self-energies of the charged species, and to put limits on the energetics of the charge-separation processes. The microscopic Protein-Dipoles-Langevin-Dipoles (PDLD) approach provides a much more realistic treatment of dielectric effects, but requires lengthy calculations that depend on numerous interrelated factors. Calculations by both approaches indicate that, in Rhodopseudomonas viridis reaction centers, the state P+B- generated by movement of an electron from the primary electron donor (P) to a neighboring bacteriochlorophyll (B) lies close to the excited state P* in energy, where it possibly could serve as an intermediate in electron transfer to the bacteriopheophytin (H). This conclusion agrees with previous free-energy-perturbation calculations and indicates that any model (macroscopic or microscopic) that includes all the relevant contributions and reproduces the energy of the relaxed P+H- should find the relaxed P+B- state near P*. In addition, the macroscopic model shows that electron transfer from P* to H is likely to be exothermic even in the absence of a strong field from the atomic charges of the protein.

The effects of multiple changes in hydrogen bond interactions between the electron donor, a bacteriochlorophyll dimer, and histidine residues in the reaction center from Rhodobacter sphaeroides have been investigated. Site-directed mutations were designed to add or remove hydrogen bonds between the 2-acetyl groups of the dimer and histidine residues at the symmetry-related sites His-L168 and Phe-M197, and between the 9-keto groups and Leu-L131 and Leu-M160. The addition of a hydrogen bond was correlated with an increase in the dimer midpoint potential. Measurements on double and triple mutants showed that changes in the midpoint potential due to alterations at the individual sites were additive. Midpoint potentials ranging from 410 to 765 mV, compared with 505 mV for wild type, were achieved by various combinations of mutations. The optical absorption spectra of the reaction centers showed relatively minor changes in the position of the donor absorption band, indicating that the addition of hydrogen bonds to histidines primarily destabilized the oxidized state of the donor and had little effect on the excited state relative to the ground state. Despite the change in energy of the charge-separated states by up to 260 meV, the mutant reaction centers were still capable of electron transfer to the primary quinone. The increase in midpoint potential was correlated with an increase in the rate of charge recombination from the primary quinone, and a fit of these data using the Marcus equation indicated that the reorganization energy for this reaction is approximately 400 meV higher than the change in free energy in wild type. The mutants were still capable of photosynthetic growth, although at reduced rates relative to the wild type. These results suggest a role for protein-cofactor interactions--in particular, histidine-donor interactions--in establishing the redox potentials needed for electron transfer in biological systems.

This chapter describes the simulations of electron transfer in bacterial reaction centers. The need for microscopic simulations is especially pressing in biological systems in understanding the mechanism of the initial charge–separation reaction. Photosynthetic reaction centers provide an ideal system to develop methods of simulating biological electron transfer reactions. The kinetics of the electron transfer steps is measured under a wide range of conditions in both wild-type and mutant-reaction centers, and the initial steps occur on a timescale that is accessible to molecular-dynamics simulations. A recurring issue is the role of the bacteriochlorophyll situated between P and H L in the crystal structure (B L ). Crystal structures are known for their high resolution. The effects of external electrical fields suggest that the initial separation of charge occurs along the axis connecting P with H L , rather than along the axis from P to B L . If P + B L – is not a kinetically resolvable state, it seems likely to connect the initial and final states—P* and P + H L – —by mixing with both of them quantum mechanically. The semiclassical trajectory approach can also deal with relaxations of the intermediate state if trajectories that cross from P* to P + B L – are monitored until they reach P + B H . This monitoring can be done in both the diabatic and adiabatic limits. However, the primary problem in elucidating the initial steps of bacterial photosynthesis is not associated with making a formal decision about whether the reaction is diabatic or adiabatic but with the uncertainties in the energy of P + B L – and the magnitude of σ 12 .

The primary electron donor in the reaction center of purple photosynthetic bacteria consists of a pair of bacteriochlorophylls (PL and PM). The oxidized dimer (P+) is expected to have an absorption band in the mid-IR, whose energy and dipole strength depend in part on the resonance interactions between the two bacteriochlorophylls. A broad absorption band with the predicted properties was found in a previously unexplored region of the spectrum, centered near 2600 cm-1 in reaction centers of Rhodobacter sphaeroides and several other species of bacteria that contain bacteriochlorophyll a, and near 2750 cm-1 in Rhodopseudomonas viridis. The band is not seen in the absorption spectrum of the monomeric bacteriochlorophyll cation in solution, and it is missing or much diminished in the reaction centers of bacterial mutants that have a bacteriopheophytin in place of either PL or PM. With the aid of a relatively simple quantum mechanical model, the measured transition energy and dipole strength of the band can be used to solve for the resonance interaction matrix element that causes an electron to move back and forth between PL and PM, and also for the energy difference between states in which a positive charge is localized on either PL or PM. (The absorption band can be viewed as representing a transition between supermolecular eigenstates that are obtained by mixing these basis states.) The values of the matrix element obtained in this way agree reasonably well with values calculated by using semiempirical atomic resonance integrals and the reaction center crystal structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Semiempirical molecular orbital treatments provide a useful framework for relating the reaction center’s structure to its unusual spectroscopic properties and to the interaction matrix elements that govern the rates of electron transfer. Such treatments encounter a number of obstacles, but perhaps the most difficult of these is to evaluate the interactions of atoms that are relatively far apart, where interatomic resonance integrals are not well calibrated. Alternative approaches to this problem have led to diverging opinions concerning the resonance interactions that mix intramolecular transitions with charge-transfer transitions of the special pair of BChls.* Many of the RC’s spectroscopic properties can be rationalized either by strong interactions with a charge-transfer state that lies considerably higher in energy than the Qy transitions of the BChls, or by weaker interactions with a state that lies closer in energy.1–3

We have measured the rate of the initial electron-transfer process as a function of temperature in reaction centers in a native strain of the photosynthetic bacterium Rhodobacter sphaeroides and two mutants generated by site-directed mutagenesis. In the mutants, a tyrosine residue in the vicinity of the primary electron donor and acceptor molecules was replaced by either phenylalanine or isoleucine. The electron-transfer reaction is slower in the mutants and has a qualitatively different dependence on temperature. In native reaction centers the rate increases as the temperature is reduced, in the phenylalanine mutant it is virtually independent of temperature, and in the isoleucine mutant it decreases with decreasing temperature. At 77 K, the electron-transfer reaction is approximately 30 times slower in the isoleucine mutant than in the native. These observations support the view that tyrosine-(M)210 plays an important role in the electron-transfer mechanism. In the isoleucine mutant at low temperatures, the stimulated emission from the excited reaction center undergoes a time-dependent shift to shorter wavelengths.

The solution of the crystal structure of reaction centers from purple photosynthetic bacteria (1–4) has raised a challenge: Can we account for the directionality, speed and efficiency of the primary photochemical electron transfer reactions on the basis of the crystal structure?

The photosynthetic reaction center (RC) of the thermophilic, green, gliding bacterium Chloroflexus aurantiacus is functionally similar to reaction centers of the well-studied purple non-sulfur photosynthetic bacteria, but it has some significant structural differences. It has 3 bacteriochlorophyll (BChl) and 3 bacteriopheophytin (BPh) molecules, rather than 4 BChl and 2 BPh,1 and it consists of 2, rather than 3 polypeptides, designated M and L.2 Sequence data indicate that the BChl on the “non-functional” M-branch of pigments in the purple bacteria is replaced by BPh in C. aurantiacus.2 As in the purple species, photochemistry in C. aurantiacus generates a charge-separated state, in which a pair of strongly interacting BChl molecules that serves as the primary donor (P) becomes oxidized, and a quinone (QA) becomes reduced. Reduction of QA in C. aurantiacus occurs with a time-constant of about 300 ps, as compared to about 200 ps in Rhodobacter sphaeroides. 3 To investigate earlier electron transfer events in C. aurantiacus, we have conducted transient absorbance measurements on RCs at 298 K and at 81 K with 1 to 2 ps time-resolution, and we report some spectral and kinetic data here.

The solution of the crystal structures of purple bacterial reaction centers has raised several puzzling questions: First, considering the symmetry of the crystal structure, why does the special pair of bacteriochlorophylls (P) transfer an electron to the bacteriopheophytin on the “L” side of the complex (HL) so much more rapidly than it does to the bacteriopheophytin on the “M” side (HM)? And what roles do the accessory bacteriochlorophylls (BL and BM) play in the electron-transfer reaction? The answers to these questions are likely to be intertwined. One possibility is that favorable electrostatic interactions with the protein lower the energy of the P+BL- radical-pair so that this state lies close to or below the excited dimer (P*), whereas the corresponding radical-pair on the M side (P+BM-) lies at a significantly higher energy. This would allow BL to act as an intermediate electron carrier betwen P* and HL as suggested by the recent work of Holzapfel et al. [1,2]. The competing route through P+BM- to P+HM- would be blocked by the need for thermal activation, particularly at low temperatures. A difference between the electrostatic interactions of the protein with the two radical-pairs might suffice to explain the directionality even if P+BL- lies above P* and facilitates the reaction only by mixing quantum-mechanically with P* and P+HL- (superexchange) [3–8]. If P+BM- is at a higher energy than P+BL-, its mixing with P* would be weaker.