bacteriochlorophylls and bacteriopheophytin

Topics: Bacterial Physiological Phenomena; Bacteriochlorophylls; Chlorophyll; Chromatium; Pheophytins; Photosynthesis; Pigments, Biological; Rhodopseudomonas; Rhodospirillum rubrum

Bacteriochlorophyll (BChl) b has a unique π-conjugation system, in which the bacteriochlorin macrocycle is conjugated with the C8-ethylidene group. This π-system is converted easily to the chlorin macrocycle. However, the effects of the central magnesium in BChl b on this conversion are unclear. In this study, the isomerization kinetics of BChl b and its demetalated pigment, bacteriopheophytin (BPhe) b, was analyzed under weakly acidic conditions. BChl b exhibited faster acid-induced isomerization than BPhe b. These results were attributed to the stabilization of a cationic intermediate, whose C8-ethylidene group is protonated, during the isomerization of BChl b compared to BPhe b because of a difference in the electron densities of the π-conjugation systems between BChl b and BPhe b. High-performance liquid chromatography analyses indicated that BChl b was primarily isomerized to 3-acetyl Chl a, followed by demetalation. The reaction order was due to the slower demetalation kinetics of metallobacteriochlorins than metallochlorins. These results will be helpful for handling unstable BChl b and BPhe b. The reaction properties of BChl b and BPhe b demonstrated here will be helpful for understanding the in vivo formation of BPhe b, which acts as the primary electron acceptor in photosynthetic reaction center complexes in BChl b-containing purple photosynthetic bacteria.

Topics: Bacteriochlorophylls; Isomerism; Kinetics; Pheophytins

Photosynthetic reaction centres harvest the energy content of sunlight by transporting electrons across an energy-transducing biological membrane. Here we use time-resolved serial femtosecond crystallography

Topics: Bacteriochlorophylls; Binding Sites; Chlorophyll; Crystallography; Cytoplasm; Electron Transport; Electrons; Hyphomicrobiaceae; Lasers; Models, Molecular; Oxidation-Reduction; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protons; Ubiquinone; Vitamin K 2

We report 90% yield of electron transfer (ET) from the singlet excited state P* of the primary electron-donor P (a bacteriochlorophyll dimer) to the B-side bacteriopheophytin (H

Topics: Amino Acid Substitution; Bacteriochlorophylls; Electron Transport; Molecular Dynamics Simulation; Mutation; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protein Engineering; Rhodobacter sphaeroides

Although the cofactors in the bacterial reaction centre of Rhodobacter sphaeroides wild type (WT) are arranged almost symmetrically in two branches, the light-induced electron transfer occurs selectively in one branch. As origin of this functional symmetry break, a hydrogen bond between the acetyl group of P

Topics: Bacteriochlorophyll A; Electron Transport; Hydrogen Bonding; Models, Molecular; Molecular Structure; Nuclear Magnetic Resonance, Biomolecular; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides

Using high-throughput methods for mutagenesis, protein isolation and charge-separation functionality, we have assayed 40 Rhodobacter capsulatus reaction center (RC) mutants for their P(+)QB(-) yield (P is a dimer of bacteriochlorophylls and Q is a ubiquinone) as produced using the normally inactive B-side cofactors BB and HB (where B is a bacteriochlorophyll and H is a bacteriopheophytin). Two sets of mutants explore all possible residues at M131 (M polypeptide, native residue Val near HB) in tandem with either a fixed His or a fixed Asn at L181 (L polypeptide, native residue Phe near BB). A third set of mutants explores all possible residues at L181 with a fixed Glu at M131 that can form a hydrogen bond to HB. For each set of mutants, the results of a rapid millisecond screening assay that probes the yield of P(+)QB(-) are compared among that set and to the other mutants reported here or previously. For a subset of eight mutants, the rate constants and yields of the individual B-side electron transfer processes are determined via transient absorption measurements spanning 100 fs to 50 μs. The resulting ranking of mutants for their yield of P(+)QB(-) from ultrafast experiments is in good agreement with that obtained from the millisecond screening assay, further validating the efficient, high-throughput screen for B-side transmembrane charge separation. Results from mutants that individually show progress toward optimization of P(+)HB(-)→P(+)QB(-) electron transfer or initial P*→P(+)HB(-) conversion highlight unmet challenges of optimizing both processes simultaneously.

Topics: Amino Acid Motifs; Bacteriochlorophylls; Electron Transport; Gene Expression; Hydrogen Bonding; Kinetics; Light; Light-Harvesting Protein Complexes; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutation; Pheophytins; Photosynthesis; Rhodobacter capsulatus; Static Electricity; Structure-Activity Relationship; Ubiquinone

The influence of amino acid substitutions at position M214 (M-subunit, residue 214) on the rate and pathway of electron transfer involving the bacteriopheophytin cofactor, HA, in a bacterial photosynthetic reaction center has been explored in a series of Rhodobacter sphaeroides mutants. The M214 leucine (L) residue of the wild type was replaced with histidine (H), glutamine (Q), and asparagine (N), creating the mutants M214LH, M214LQ, and M214LN, respectively. As has been reported previously for M214LH, each of these mutations resulted in a bacteriochlorophyll molecule in place of a bacteriopheophytin in the HA pocket, forming so-called β-type mutants (in which the HA cofactor is called βA). In addition, these mutations changed the properties of the surrounding protein environment in terms of charge distribution and the amino acid side chain volume. Electron transfer reactions from the excited primary donor P to the acceptor QA were characterized using ultrafast transient absorption spectroscopic techniques. Similar to that of the previously characterized M214LH (β mutant), the strong energetic mixing of the P(+)BA(-) and P(+)βA(-) states (the mixed anion is denoted I(-)) increased the rate of charge recombination between P(+) and I(-) in competition with the I(-) → QA forward reaction. This reduced the overall yield of charge separation forming the P(+)QA(-) state. While the kinetics of the primary electron transfer forming P(+)I(-) were essentially identical in all three β mutants, the rates of the βA(-) (I(-)) → QA electron transfer in M214LQ and M214LH were very similar but quite different from that of the M214LN mutant. The observed yield changes and the differences in kinetics are correlated more closely with the volume of the mutated amino acid than with their charge characteristics. These results are consistent with those of previous studies of a series of M214 mutants with different sizes of amino acid side chains that did not alter the HA cofactor composition [Pan, J., et al. (2013) J. Phys. Chem. B 117, 7179-7189]. Both studies indicate that protein relaxation in this region of the reaction center plays a key role in stabilizing charge-separated states involving the HA or βA cofactor. The effect is particularly pronounced for reactions occurring on time scales of tens and hundreds of picoseconds (forward transfer to the QA and charge recombination).

Topics: Bacteriochlorophylls; Electron Transport; Kinetics; Ligands; Pheophytins; Photosynthetic Reaction Center Complex Proteins

Most purple photosynthetic bacteria contain bacteriochlorophyll(BChl)-a (a magnesium complex) and bacteriopheophytin(BPhe)-a (its free base) as their photoactive pigments. These pigments are composed of two parts: a cyclic tetrapyrrole as the chromophore and a long hydrocarbon-chain as the propionate-type esterifying group at the 17-position. The hydrocarbon-chain is usually an isoprenoid-type C20 phytyl (Phy) group in both the pigments. In the ester group of BChl-a, several variants such as geranylgeranyl (GG), dihydrogeranylgeranyl (DHGG) and tetrahydrogeranylgeranyl (THGG) groups were found in the final stage of BChl-a biosynthesis. On the other hand, the esterifying variants in BPhe-a have not been studied as much due to the lower levels of this pigment relative to BChl-a. The esterifying group does not affect the electronic absorption properties of such pigments in the monomeric state, but drastically alters the hydrophobicity. In this study, BChl-a and BPhe-a in the six phylogenetically distinct classes of purple bacteria were analyzed in terms of their esterifying groups in the 17-propionate residues, using high-performance liquid chromatography. Both BChls-a and BPhes-a carrying GG, DHGG and THGG in addition to the usual Phy were found for all the bacterial species studied at measurable levels. In some of the species, the ratio of BPhes-a esterified with GG, DHGG and THGG over the total BPhe-a drastically decreased in comparison with that of the corresponding BChls-a. Especially, the relative content of BPhe-a with GG largely decreased. This observation might indicate that BPhe-a as a cofactor of reaction centers was preferentially esterified with partially reduced and flexible chains (THGG and Phy) rather than less reduced and rigid ones (GG and DHGG).

Topics: Bacteriochlorophyll A; Chromatography, High Pressure Liquid; Esterification; Hydrophobic and Hydrophilic Interactions; Pheophytins; Propionates; Proteobacteria; Terpenes

A Rhodobacter capsulatus strain, designated XJ-1, isolated from saline soil, accumulated almost only one kind of bacteriochlorophyll a anaerobically in the light, aerobically in the light and dark, and the relative contents of the bacteriochlorophyll a were 44.61, 74.89, and 77.53% of the total pigments, respectively. A new purple pigment appeared only in aerobic-light grown cells, exhibited absorption maxima at 355, 389, 520, 621, and 755 nm, especially distinctly unusual peak at 621 nm, whereas vanished in anaerobic-light and in aerobic-dark culture. Spheroidene and OH-spheroidene predominated in anaerobic phototrophic cultures. Spheroidenone was the sole carotenoid when exposed to both light and oxygen. The second keto-carotenoids, OH-spheroidenone, presented only in aerobic-dark culture in addition to spheroidenone. Strain XJ-1 would be a good model organism for the further illustration of the regulation of bacteriochlorophyll biosynthesis gene expression in response to unique habitat.

Topics: Bacterial Proteins; Bacteriochlorophylls; Carotenoids; Light; Mass Spectrometry; Oxygen; Pheophytins; Rhodobacter capsulatus; Salinity; Sodium Chloride; Soil; Soil Microbiology

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.

Topics: Bacteriochlorophylls; Electrons; Energy Transfer; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Vibration

Time-resolved spectroscopic studies of recombination of the P(+)HA(-) radical pair in photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides give an opportunity to study protein dynamics triggered by light and occurring over the lifetime of P(+)HA(-). The state P(+)HA(-) is formed after the ultrafast light-induced electron transfer from the primary donor pair of bacteriochlorophylls (P) to the acceptor bacteriopheophytin (HA). In order to increase the lifetime of this state, and thus increase the temporal window for the examination of protein dynamics, it is possible to block forward electron transfer from HA(-) to the secondary electron acceptor QA. In this contribution, the dynamics of P(+)HA(-) recombination were compared at a range of temperatures from 77 K to room temperature, electron transfer from HA(-) to QA being blocked either by prereduction of QA or by genetic removal of QA. The observed P(+)HA(-) charge recombination was significantly slower in the QA-deficient RCs, and in both types of complexes, lowering the temperature from RT to 77 K led to a slowing of charge recombination. The effects are explained in the frame of a model in which charge recombination occurs via competing pathways, one of which is thermally activated and includes transient formation of a higher-energy state, P(+)BA(-). An internal electrostatic field supplied by the negative charge on QA increases the free energy levels of the state P(+)HA(-), thus decreasing its energetic distance to the state P(+)BA(-). In addition, the dielectric response of the protein environment to the appearance of the state P(+)HA(-) is accelerated from ∼50-100 ns in the QA-deficient mutant RCs to ∼1-16 ns in WT RCs with a negatively charged QA(-). In both cases, the temperature dependence of the protein dynamics is weak.

Topics: Bacterial Proteins; Bacteriochlorophylls; Electron Transport; Mutation; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Recombinant Proteins; Rhodobacter sphaeroides; Spectrometry, Fluorescence; Static Electricity; Temperature; Time Factors; Ubiquinone

In the native reaction center (RC) of Rhodobacter sphaeroides, the side chain of (M)L214 projects orthogonally toward the plane and into the center of the A branch bacteriopheophytin (BPhe) macrocycle. The possibility that this side chain is responsible for the dechelation of the central Mg(2+) of bacteriochlorophyll (BChl) was investigated by replacement of (M)214 with residues possessing small, nonpolar side chains that can neither coordinate nor block access to the central metal ion. The (M)L214 side chain was also replaced with Cys, Gln, and Asn to evaluate further the requirements for assembly of the RC with BChl in the HA pocket. Photoheterotrophic growth studies showed no difference in growth rates of the (M)214 nonpolar mutants at a low light intensity, but the growth of the amide-containing mutants was impaired. The absorbance spectra of purified RCs indicated that although absorbance changes are associated with the nonpolar mutations, the nonpolar mutant RC pigment compositions are the same as in the wild-type protein. Crystal structures of the (M)L214G, (M)L214A, and (M)L214N mutants were determined (determined to 2.2-2.85 Å resolution), confirming the presence of BPhe in the HA pocket and revealing alternative conformations of the phytyl tail of the accessory BChl in the BA site of these nonpolar mutants. Our results demonstrate that (i) BChl is converted to BPhe in a manner independent of the aliphatic side chain length of nonpolar residues replacing (M)214, (ii) BChl replaces BPhe if residue (M)214 has an amide-bearing side chain, (iii) (M)214 side chains containing sulfur are not sufficient to bind BChl in the HA pocket, and (iv) the (M)214 side chain influences the conformation of the phytyl tail of the BA BChl.

Topics: Bacterial Proteins; Bacteriochlorophylls; Crystallography, X-Ray; Models, Molecular; Mutagenesis, Site-Directed; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protein Conformation; Rhodobacter sphaeroides

Unusual temperature behavior, observed in the initial electron transfer stages in the photosynthetic reaction centers of the purple bacteria, and a strong probing pulse wavelength dependence of transfer rates, determined in transient absorption spectroscopy, can easily be explained on assuming that the transfer takes place from dynamically unrelaxed states of protein environment. The transitions from the primary special pair (P) to a single bacteriochlorophyll (B) and next to a bacteriopheophytin (H) are controlled by diffusion down the energy value of underdamped vibrational modes of frequency 200 K, probably determining distances between the succeeding cofactors. The subsequent transition to the quinone A (Q) is controlled by diffusion in the position value of an overdamped conformational mode, probably corresponding to the local polarization. From the fit of available experimental data to simple theoretical formulas, the important physical conclusion arises that the very electronic transitions are fast as compared to the relaxation processes and, in the first approximation, only the latter contribute to the overall times of the initial electron transfer stages in photosynthesis.

Topics: Bacteriochlorophylls; Electron Transport; Pheophytins; Photosynthesis; Proteobacteria; Temperature

The reaction centers (RCs) from several species of a purple photosynthetic bacterium, Rhodopseudomonas palustris, were first isolated by ammonium-sulfate fractionation of the isolated core complexes, and were successfully purified by anion-exchange and gel-filtration chromatography as well as sucrose-density gradient centrifugation. The RCs were characterized by spectroscopic and biochemical analyses, indicating that they were sufficiently pure and had conserved their redox activity. The pigment composition of the purified RCs was carefully analyzed by LCMS. Significant accumulation of both bacteriochlorophyll(BChl)-a and bacteriopheophytin(BPhe)-a esterified with various isoprenoid alcohols in the 17-propionate groups was shown in RCs for the first time. Moreover, a drastic decrease in BPhe-a with the most dehydrogenated and rigid geranylgeranyl(GG) ester was observed, indicating that BPhe-a in RC preferably took partially hydrogenated and flexible ester groups, i.e. dihydro-GG and tetrahydro-GG in addition to phytyl. Based on the reported X-ray crystal structures of purple bacterial RCs, the meaning of flexibility of the ester groups in BChl-a and BPhe-a as the cofactors of RCs is proposed.

Topics: Bacteriochlorophyll A; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodopseudomonas

Photochemical charge separation in isolated reaction center-light harvesting 1 (RC-LH1) complexes from Rhodobacter sphaeroides was examined using time-resolved mid-infrared pump-probe spectroscopy. Absorption difference spectra were recorded between 1760 and 1610 cm(-1) with subpicosecond time resolution to characterize excited-state and radical pair dynamics in these complexes, via the induced absorption changes in the keto carbonyl modes of the bacteriochlorophylls and bacteriopheophytins. Experiments on RC-LH1 complexes with and without the polypeptide PufX show that its presence is required to achieve generation of the radical pair P(+)Q(A)(-) under mildly reducing conditions. In the presence of PufX, the final radical pair formed over a ~3 ns period was P(+)Q(A)(-), but in its absence the corresponding radical pair was P(+)H(A)(-), implying that Q(A) was either absent in these PufX-deficient complexes or was prereduced. However, P(+)Q(A)(-) could be generated in PufX-deficient complexes following addition of the oxidant DMSO, showing that Q(A) was present in these complexes and allowing the conclusion that under mildly reducing conditions charge separation was blocked after P(+)H(A)(-) due to the presence of an electron on Q(A). The data provide strong support for the hypothesis that one of the functions of PufX is to regulate the stability of Q(B)(-), ensuring the oxidation of Q(A)(-) in the presence of a reduced quinone pool and so preserving efficient photochemical charge separation under anaerobic conditions.

Topics: Bacterial Proteins; Bacteriochlorophylls; Light-Harvesting Protein Complexes; Pheophytins; Photochemical Processes; Rhodobacter sphaeroides; Spectrophotometry, Infrared

The role of protein dynamics in guiding multistep electron transfer is explored in the photosynthetic reaction center of Rhodobacter sphaeroides . The energetics of the charge-separated intermediates, P(+)B(A)(-) and P(+)H(A)(-) (P is the initial electron donor bacteriochlorophyll pair and B(A) and H(A) are early bacteriochlorophyll and bacteriopheophytin acceptors, respectively), were systematically varied in a series of mutants. A fast phase of P(+)H(A)(-) recombination was resolved that is very sensitive to driving force. Either increasing or decreasing the relative free energy of P(+)H(A)(-) resulted in a more prominent fast recombination component, and thus a decreased yield forward electron transfer. The fast phase apparently represents P(+)H(A)(-) charge recombination via an activated state, probably P(+)B(A)(-) (B(A) is situated between P and H(A)). In wild type, this activated state is largely inaccessible, presumably due to dynamic stabilization of P(+)H(A)(-) within the first 100 ps. In mutants that change the energetics, the rate of decay via the activated state accelerates and that pathway becomes significant. The dynamic stabilization of the protein makes it possible to achieve a nearly optimum environment of H(A) in wild type on two different time scales and for two rather different reactions. On the picosecond time scale, the energetics is nearly, though not perfectly, optimized for transfer between the excited state of P and H(A). After dynamic stabilization of the state P(+)H(A)(-), the environment is optimized to avoid rapid recombination of the charge-separated state and instead carry out forward electron transfer to the quinone with very high yield on the hundreds of picosecond time scale. Thus, by employing protein dynamics, the reaction center is able to optimize multiple reactions, on very different time scales involving the same reaction intermediate.

Topics: Bacteriochlorophylls; Electron Transport; Kinetics; Mutation; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Recombinant Proteins; Rhodobacter sphaeroides; Thermodynamics; Time Factors

Key to efficient harvesting of sunlight in photosynthesis is the first energy conversion process in which electronic excitation establishes a trans-membrane charge gradient. This conversion is accomplished by the photosynthetic reaction center (RC) that is, in case of the purple photosynthetic bacterium Rhodobacter sphaeroides studied here, surrounded by light harvesting complex 1 (LH1). The RC employs six pigment molecules to initiate the conversion: four bacteriochlorophylls and two bacteriopheophytins. The excited states of these pigments interact very strongly and are simultaneously influenced by the surrounding thermal protein environment. Likewise, LH1 employs 32 bacteriochlorophylls influenced in their excited state dynamics by strong interaction between the pigments and by interaction with the protein environment. Modeling the excited state dynamics in the RC as well as in LH1 requires theoretical methods, which account for both pigment-pigment interaction and pigment-environment interaction. In the present study we describe the excitation dynamics within a RC and excitation transfer between light harvesting complex 1 (LH1) and RC, employing the hierarchical equation of motion method. For this purpose a set of model parameters that reproduce RC as well as LH1 spectra and observed oscillatory excitation dynamics in the RC is suggested. We find that the environment has a significant effect on LH1-RC excitation transfer and that excitation transfers incoherently between LH1 and RC.

Topics: Bacteriochlorophylls; Light; Light-Harvesting Protein Complexes; Pheophytins; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides

The replacement of tyrosine by aspartic acid at position M210 in the photosynthetic reaction center of Rhodobacter sphaeroides results in the generation of a fast charge recombination pathway that is not observed in the wild-type. Apparently, the initially formed charge-separated state (cation of the special pair, P, and anion of the A-side bacteriopheophytin, H(A)) can decay rapidly via recombination through the neighboring bacteriochlorophyll (B(A)) soon after formation. The charge-separated state then relaxes over tens of picoseconds and recombination slows to the hundreds-of-picoseconds or nanosecond timescale. This dielectric relaxation results in a time-dependent blue shift of B(A)(-) absorption, which can be monitored using transient absorbance measurements. Protein dynamics also appear to modulate the electron transfer between H(A) and the next electron carrier, Q(A) (a ubiquinone). The kinetics of this reaction are complex in the mutant, requiring two kinetic terms, and the spectra associated with the two terms are distinct; a red shift of the H(A) ground-state bleaching is observed between the shorter and longer H(A)-to-Q(A) electron-transfer phases. The kinetics appears to be pH-independent, suggesting a negligible contribution of static heterogeneity originating from protonation/deprotonation in the ground state. A dynamic model based on the energy levels of the two early charge-separated states, P(+)B(A)(-) and P(+)H(A)(-), has been developed in which the energetics of these states is modulated by fast protein dielectric relaxations and this in turn alters both the kinetic complexity of the reaction and the reaction pathway.

Topics: Amino Acid Sequence; Aspartic Acid; Bacterial Proteins; Bacteriochlorophylls; Electron Transport; Hydrogen-Ion Concentration; Kinetics; Light; Molecular Sequence Data; Mutation, Missense; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Static Electricity; Ubiquinone

Histidine M182 in the reaction center (RC) of Rhodobacter sphaeroides serves as the fifth ligand of the bacteriochlorophyll (BChl) B(B) Mg atom. When this His is substituted by an amino acid that is not able to coordinate Mg, bacteriopheophytin appears in the B(B) binding site instead of BChl (Katilius, E., et al. (1999) J. Phys. Chem. B, 103, 7386-7389). We have shown that in the presence of the additional mutation I(L177)H the coordination of the BChl B(B) Mg atom in the double mutant I(L177)H+H(M182)L RC still remains. Changes in the double mutant RC absorption spectrum attributed to BChl absorption suggest that BChl B(B) Mg atom axial ligation might be realized not from the usual α-side of the BChl macrocycle, but from the opposite, β-side. Weaker coordination of BChl B(B) Mg atom compared to the other mutant RC BChl molecules suggests that not an amino acid residue but a water molecule might be a possible ligand. The results are discussed in the light of the structural changes that occurred in the RC upon Ile/His substitution in the L177 position.

Topics: Amino Acid Substitution; Bacteriochlorophylls; Models, Molecular; Mutagenesis, Site-Directed; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protein Conformation; Rhodobacter sphaeroides; Spectrophotometry

Bacteriochlorins, which are tetrapyrrole macrocycles with two reduced pyrrole rings, are Nature's near-infrared (NIR) absorbers (700-900 nm). The strong absorption in the NIR region renders bacteriochlorins excellent candidates for a variety of applications including solar light harvesting, flow cytometry, molecular imaging, and photodynamic therapy. Natural bacteriochlorins are inherently unstable due to oxidative conversion to the chlorin (one reduced pyrrole ring) or the porphyrin. The natural pigments are also only modestly amenable to synthetic manipulation, owing to a nearly full complement of substituents on the macrocycle. Recently, a new synthetic methodology has afforded access to stable synthetic bacteriochlorins wherein a wide variety of substituents can be appended to the macrocycle at preselected locations. Herein, the spectroscopic and photophysical properties of 33 synthetic bacteriochlorins are investigated. The NIR absorption bands of the chromophores range from ∼700 to ∼820 nm; the lifetimes of the lowest excited singlet state range from ∼2 to ∼6 ns; the fluorescence quantum yields range from ∼0.05 to ∼0.25; and the yield of the lowest triplet excited state is ∼0.5. The spectroscopic/photophysical studies of the bacteriochlorins are accompanied by density functional theory (DFT) calculations that probe the characteristics of the frontier molecular orbitals. The DFT calculations indicate that the impact of substituents on the spectral properties of the molecules derives primarily from effects on the lowest unoccupied molecular orbital. Collectively, the studies show how the palette of synthetic bacteriochlorins extends the properties of the native photosynthetic pigments (bacteriochlorophylls). The studies have also elucidated design principles for tuning the spectral and photophysical characteristics as required for a wide variety of photochemical applications.

Topics: Bacteriochlorophylls; Electrons; Pheophytins; Photosynthesis; Porphyrins; Quantum Theory; Spectroscopy, Near-Infrared

The kinetics of recombination of the P(+)H(A)(-) radical pair were compared in wild-type reaction centers from Rhodobacter sphaeroides and in seven mutants in which the free energy gap, ΔG, between the charge separated states P(+)B(A)(-) and P(+)H(A)(-) was either increased or decreased. Five of the mutant RCs had been described previously, and X-ray crystal structures of two newly constructed complexes were determined by X-ray crystallography. The charge recombination reaction was accelerated in all mutants with a smaller ΔG than in the wild-type, and was slowed in a mutant having a larger ΔG. The free energy difference between the state P(+)H(A)(-) and the PH(A) ground state was unaffected by most of these mutations. These observations were consistent with a model in which the P(+)H(A)(-) → PH(A) charge recombination is thermally activated and occurs via the intermediate state P(+)B(A)(-), with a mean rate related to the size of the ΔG between the states P(+)B(A)(-) and P(+)H(A)(-) and not the ΔG between P(+)H(A)(-) and the ground state. A more detailed analysis of charge recombination in the mutants showed that the kinetics of the reaction were multiexponential, and characterized by ~0.5, ~1-3, and 7-17 ns lifetimes, similar to those measured for wild-type reaction centers. The exact lifetimes and relative amplitudes of the three components were strongly modulated by the mutations. Two models were considered in order to explain the observed multiexponentiality and modulation, involving heterogeneity or relaxation of P(+)H(A)(-) states, with the latter model giving a better fit to the experimental results.

Topics: Bacterial Proteins; Bacteriochlorophylls; Crystallography, X-Ray; Electron Transport; Kinetics; Models, Molecular; Mutation; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides

The aim of this study is to reveal the interaction relationships between lauryl dimethylamine N-oxide (LDAO) and peripheral light-harvesting complex (LH2) as well as the influence of LDAO on structure and function of LH2. In the present work, the effects of LDAO on the conformation and release processes of bacteriochlorophyll (BChl) of LH2 when incubated under different temperature and pH in the presence and absence of LDAO were investigated by spectroscopy. The results indicated that (1) the presence of LDAO resulted in alterations in the conformation, alpha-helix content, and spectra of Tyr and B850 band of LH2 at room temperature and pH 8.0. Moreover, energy transfer efficiency of LH2 was enhanced markedly in the presence of LDAO. (2) At 60 degrees C, both the B800 and B850 band of LH2 were released and transited into free BChl at pH 8.0. However, the release rates of bacteriochlorophylls of B800 and B850 band from LH2 were slowed down and the release processes were changed when incubated in the presence of LDAO. Hence, the stability of LH2 was improved in the presence of LDAO. (3) The accelerated release processes of bacteriochlorophylls of B800 and B850 band of LH2 were induced to transform into bacteriopheophytin (BPhe) and free BChl by LDAO in strong acid and strong alkalic solution at room temperature. However, the kinetic patterns of the B800 and B850 band were remarkably different. The release and self-assemble processes of B850 in LH2 were observed in strong acid solution without LDAO. Therefore, the different release behaviors of B800 and B850 of LH2 are induced by LDAO under different extreme environmental conditions.

Topics: Bacterial Proteins; Bacteriochlorophylls; Dimethylamines; Energy Transfer; Kinetics; Light-Harvesting Protein Complexes; Pheophytins; Protein Structure, Secondary; Rhodobacter

Heterodimer mutant reaction centers (RCs) of Blastochloris viridis were crystallized using microfluidic technology. In this mutant, a leucine residue replaced the histidine residue which had acted as a fifth ligand to the bacteriochlorophyll (BChl) of the primary electron donor dimer M site (HisM200). With the loss of the histidine-coordinated Mg, one bacteriochlorophyll of the special pair was converted into a bacteriopheophytin (BPhe), and the primary donor became a heterodimer supermolecule. The crystals had dimensions 400 x 100 x 100 microm, belonged to space group P4(3)2(1)2, and were isomorphous to the ones reported earlier for the wild type (WT) strain. The structure was solved to a 2.5 A resolution limit. Electron-density maps confirmed the replacement of the histidine residue and the absence of Mg. Structural changes in the heterodimer mutant RC relative to the WT included the absence of the water molecule that is typically positioned between the M side of the primary donor and the accessory BChl, a slight shift in the position of amino acids surrounding the site of the mutation, and the rotation of the M194 phenylalanine. The cytochrome subunit was anchored similarly as in the WT and had no detectable changes in its overall position. The highly conserved tyrosine L162, located between the primary donor and the highest potential heme C(380), revealed only a minor deviation of its hydroxyl group. Concomitantly to modification of the BChl molecule, the redox potential of the heterodimer primary donor increased relative to that of the WT organism (772 mV vs. 517 mV). The availability of this heterodimer mutant and its crystal structure provides opportunities for investigating changes in light-induced electron transfer that reflect differences in redox cascades.

Topics: Amino Acid Substitution; Bacteriochlorophylls; Crystallography, X-Ray; Cytochromes; Hyphomicrobiaceae; Oxidation-Reduction; Phenylalanine; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protein Multimerization; Spectrophotometry; Tyrosine

In this investigation we report a complete assignment of (13)C, (1)H and (15)N solution and solid state chemical shifts of two bacterial photosynthetic pigments, bacteriochlorophyll (BChl) a and bacteriopheophytin (BPheo) a. Uniform stable-isotope labelling strategies were developed and applied to biosynthetic preparation of photosynthetic pigments, namely uniformly (13)C, (15)N labelled BChl a and BPheo a. Uniform stable-isotope labelling with (13)C, (15)N allowed performing the assignment of the (13)C, (15)N and (1)H resonances. The photosynthetic pigments were isolated from the biomass of photosynthetic bacteria Rhodopseudomonas palustris 17001 grown in uniformly (13)C (99%) and (15)N (98%) enriched medium. Both pigments were characterised by NMR in solution (acetone-d(6)) and by MAS NMR in solid state and their NMR resonances were recorded and assigned through standard liquid 2D (13)C-(13)C COSY, (1)H-(13)C HMQC, (1)H-(15)N HMBC and solid 2D (13)C-(13)C RFDR, (1)H-(13)C FSLG HETCOR and (1)H-(15)N HETCOR correlation techniques at 600 MHz and 750 MHz. The characterisation of pigments is of interest from biochemical to pharmaceutical industries, photosynthesis and food research.

Topics: Bacteriochlorophyll A; Carbon Isotopes; Nitrogen Isotopes; Nuclear Magnetic Resonance, Biomolecular; Pheophytins; Pigments, Biological; Rhodopseudomonas

We present a comparative study on the influence of the quantum mechanical (QM) method (including basis set) on the evaluation of transition energies, transition densities and dipoles, and excitation energy transfer (EET) electronic couplings for a series of chromophores (and the corresponding pairs) typically found in organic electro-optical devices and photosynthetic systems. On these systems we have applied five different QM levels of description of increasing accuracy (ZINDO, CIS, TD-DFT, CASSCF, and SAC-CI). In addition, we have tested the effects of a surrounding environment (either mimicking a solvent or a protein matrix) on excitation energies, transition dipoles, and electronic couplings through the polarizable continuum model (PCM) description. Overall, the results obtained suggest that the choice of the QM level of theory affects the electronic couplings much less than it affects excitation energies. We conclude that reasonable estimates can be obtained using moderate basis sets and inexpensive methods such as configuration interaction of single excitations or time-dependent density functional theory when appropriately coupled to realistic solvation models such as PCM.

Topics: Bacteriochlorophylls; Electrons; Energy Transfer; Imides; Naphthalenes; Perylene; Pheophytins; Quantum Theory

A photosynthetic reaction center (RC) complex was isolated from a purple bacterium, Acidiphilium rubrum. The RC contains bacteriochlorophyll a containing Zn as a central metal (Zn-BChl a) and bacteriopheophytin a (BPhe a) but no Mg-BChl a. The absorption peaks of the Zn-BChl a dimer (P(Zn)), the accessory Zn-BChl a (B(Zn)), and BPhe a (H) at 4 K in the RC showed peaks at 875, 792, and 753 nm, respectively. These peaks were shorter than the corresponding peaks in Rhodobacter sphaeroides RC that has Mg-BChl a. The kinetics of fluorescence from P(Zn)(*), measured by fluorescence up-conversion, showed the rise and the major decay with time constants of 0.16 and 3.3 ps, respectively. The former represents the energy transfer from B(Zn)(*) to P(Zn), and the latter, the electron transfer from P(Zn) to H. The angle between the transition dipoles of B(Zn) and P(Zn) was estimated to be 36 degrees based on the fluorescence anisotropy. The time constants and the angle are almost equal to those in the Rb. sphaeroides RC. The high efficiency of A. rubrum RC seems to be enabled by the chemical property of Zn-BChl a and by the L168HE modification of the RC protein that modifies P(Zn).

Topics: Acidiphilium; Bacteriochlorophyll A; Electron Transport; Fluorescence; Kinetics; Oxidation-Reduction; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Spectrum Analysis; Temperature; Zinc

Topics: Bacteriochlorophylls; Chemical Phenomena; Chemistry, Physical; Energy Transfer; Pheophytins; Photons; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Time Factors

The role of quantum coherence in promoting the efficiency of the initial stages of photosynthesis is an open and intriguing question. We performed a two-color photon echo experiment on a bacterial reaction center that enabled direct visualization of the coherence dynamics in the reaction center. The data revealed long-lasting coherence between two electronic states that are formed by mixing of the bacteriopheophytin and accessory bacteriochlorophyll excited states. This coherence can only be explained by strong correlation between the protein-induced fluctuations in the transition energy of neighboring chromophores. Our results suggest that correlated protein environments preserve electronic coherence in photosynthetic complexes and allow the excitation to move coherently in space, enabling highly efficient energy harvesting and trapping in photosynthesis.

Topics: Bacteriochlorophylls; Chemical Phenomena; Chemistry, Physical; Energy Transfer; Lasers; Mathematics; Motion; Pheophytins; Photons; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Time Factors

13C photo-CIDNP MAS NMR studies have been performed on reaction centers (RCs) of Rhodobacter sphaeroides wild type (WT) that have been selectively labeled with an isotope using [5-13C]-delta-aminolevulinic acid.HCl in all the BChl and BPhe cofactors at positions C-4, C-5, C-9, C-10, C-14, C-15, C-16, and C-20. 13C CP/MAS NMR and 13C-13C dipolar correlation photo-CIDNP MAS NMR provide a chemical shift map of the cofactors involved in the electron transfer process in the RC at the atomic scale. The 13C-13C dipolar correlation photo-CIDNP spectra reveal three strong components, originating from two BChl cofactors, called P1 and P2 and assigned to the special pair, as well as one BPhe, PhiA. In addition, there is a weak component observed that arises from a third BChl cofactor, denoted P3, which appears to originate from the accessory BChl BA. An almost complete set of assignments of all the aromatic carbon atoms in the macrocycles of BChl and BPhe is achieved in combination with previous photo-CIDNP studies on site-directed BChl/BPhe-labeled RCs [Schulten, E. A. M., Matysik, J., Alia, Kiihne, S., Raap, J., Lugtenburg, J., Gast, P., Hoff, A. J., and de Groot, H. J. M. (2002) Biochemistry 41, 8708-8717], allowing a comprehensive map of the ground-state electronic structure of the photochemically active cofactors to be constructed for the first time. The reasons for the anomaly of P2 and the origin of the polarization on P3 are discussed.

Topics: Aminolevulinic Acid; Bacteriochlorophylls; Carbon Isotopes; Darkness; Light; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Pheophytins; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides

A time-nonlocal quantum master equation coupled with a perturbative scheme to evaluate the third-order polarization in the phase-matching direction k(s) = -k(1) + k(2) + k(3) is used to efficiently simulate three-pulse photon-echo signals. The present method is capable of describing photon-echo peak shifts including pulse overlap and bath memory effects. In addition, the method treats the non-Markovian evolution of the density matrix and the third-order polarization in a consistent manner, thus is expected to be useful in systems with rapid and complex dynamics. We apply the theoretical method to describe one- and two-color three-pulse photon-echo peak shift experiments performed on a bacterial photosynthetic reaction center and demonstrate that, by properly incorporating the pulse overlap effects, the method can be used to describe simultaneously all peak shift experiments and determine the electronic coupling between the localized Q(y) excitations on the bacteriopheophytin (BPhy) and accessory bateriochlorophyll (BChl) in the reaction center. A value of J = 250 cm(-1) is found for the coupling between BPhy and BChl.

Topics: Bacteriochlorophylls; Electrons; Models, Biological; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Quantum Theory; Rhodobacter sphaeroides

The photosynthetic reaction center (RC) from purple bacteria converts light into chemical energy. Although the RC shows two nearly structurally symmetric branches, A and B, light-induced electron transfer in the native RC occurs almost exclusively along the A-branch to a primary quinone electron acceptor Q(A). Subsequent electron and proton transfer to a mobile quinone molecule Q(B) converts it to a quinol, Q(B)H(2). We report the construction and characterization of a series of mutants in Rhodobacter sphaeroides designed to reduce Q(B) via the B-branch. The quantum efficiency to Q(B) via the B-branch Phi(B) ranged from 0.4% in an RC containing the single mutation Ala-M260 --> Trp to 5% in a quintuple mutant which includes in addition three mutations to inhibit transfer along the A-branch (Gly-M203 --> Asp, Tyr-M210 --> Phe, Leu-M214 --> His) and one to promote transfer along the B-branch (Phe-L181 --> Tyr). Comparing the value of 0.4% for Phi(B) obtained in the AW(M260) mutant, which lacks Q(A), to the 100% quantum efficiency for Phi(A) along the A-branch in the native RC, we obtain a ratio for A-branch to B-branch electron transfer of 250:1. We determined the structure of the most effective (quintuple) mutant RC at 2.25 A (R-factor = 19.6%). The Q(A) site did not contain a quinone but was occupied by the side chain of Trp-M260 and a Cl(-). In this structure a nonfunctional quinone was found to occupy a new site near M258 and M268. The implications of this work to trap intermediate states are discussed.

Topics: Bacteriochlorophylls; Benzoquinones; Binding Sites; Crystallization; Crystallography, X-Ray; Electron Transport; Kinetics; Lasers; Models, Chemical; Mutagenesis, Site-Directed; Oxidation-Reduction; Pheophytins; Photolysis; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrophotometry

The paper deals with some fast and slow processes of excitation energy deactivation in bacteriochlorophyll c and bacteriopheophytin c. The experiments were carried out in the air, and in oxygen or nitrogen atmosphere at different temperatures. The parameters such as fluorescence yield, the yield of triplet state generation and photostability were estimated. On the basis of these parameters an attempt was made to preliminary select the pigments best suited for medical applications. It seems that the photosensitive and highly fluorescent bacteriochlorophyll c could be used as a fluorescence probe for diagnosis, whereas its derivative without the magnesium ion is better suited for the photochemical reactions occurring during therapeutic treatment.

Topics: Bacterial Proteins; Bacteriochlorophylls; Fluorescence; Molecular Structure; Nitrogen; Oxygen; Pheophytins; Photochemistry; Solutions; Temperature

Subpicosecond transient absorption studies are reported for a set of Rhodobacter (R.) capsulatus bacterial photosynthetic reaction centers (RCs) designed to probe the origins of the unidirectionality of charge separation via one of two electron transport chains in the native pigment-protein complex. All of the RCs have been engineered to contain a heterodimeric primary electron donor (D) consisting of a bacteriochlorophyll (BChl) and a bacteriopheophytin (BPh). The BPh component of the M heterodimer (Mhd) or L heterodimer (Lhd) is introduced by substituting a Leu for His M200 or His L173, respectively. Previous work on primary charge separation in heterodimer mutants has not included the Lhd RC from R. capsulatus, which we report for the first time. The Lhd and Mhd RCs are used as controls against which we assess RCs that combine the heterodimer mutations with a second mutation (His substituted for Leu at M212) that results in replacement of the native L-side BPh acceptor with a BChl (beta). The transient absorption spectra reveal clear evidence for charge separation to the normally inactive M-side BPh acceptor (H(M)) in Lhd-beta RCs to form D+H(M)- with a yield of approximately 6%. This state also forms in Mhd-beta RCs but with about one-quarter the yield. In both RCs, deactivation to the ground state is the predominant pathway of D decay, as it is in the Mhd and Lhd single mutants. Analysis of the results indicates an upper limit ofV2L/V2m < or = 4 for the contribution of the electronic coupling elements to the relative rates of electron transfer to the L versus M sides of the wild-type RC. In comparison to the L/M rate ratio (kL/kM) approximately 30 for wild-type RCs, our findings indicate that electronic factors contribute approximately 35% at most to directionality with the other 65% deriving from energetic considerations, which includes differences in free energies, reorganization energies, and contributions of one- and two-step mechanisms on the two sides of the RC.

Topics: Bacteriochlorophylls; Dimerization; Electron Transport; Electrons; Molecular Structure; Mutagenesis, Site-Directed; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter capsulatus; Surface Properties

Reaction centers (RCs) of the photosynthetic bacterium Rhodobacter sphaeroides R-26 were reconstituted in liposomes after release of pigments (bacteriochlorophyll a (BChla) and bacteriopheophytin a (BPhea)) by treatment with acetone. As shown by absorption and circular dichroism spectroscopies, the reconstituted RCs had the same arrangement of pigments as the native RC and exhibited photoactivity of the special pair. The recovery yield of RCs of up to 30% was achieved by addition of 7.8-fold excess of BChla in the acetone treatment. Furthermore BChla was partially replaced with Zn-BChla by addition of the pigments during the acetone treatment. About 30% and 50% of the special pair and accessory pigments can be replaced with Zn-BChla, respectively. From this rate, an oxidation-reduction potential of 520 mV (vs. the normal hydrogen electrode NHE) was derived by the simulation of the experimental data, which is 35 mV higher than that of the native RC (484 mV vs. NHE).

Topics: Acetone; Ascorbic Acid; Bacteriochlorophyll A; Bacteriochlorophylls; Chromatography, High Pressure Liquid; Circular Dichroism; Electrodes; Hydrogen; Light; Light-Harvesting Protein Complexes; Liposomes; Oxidation-Reduction; Oxygen; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrophotometry; Time Factors; Zinc

Symmetry-related branches of electron-transfer cofactors-initiating with a primary electron donor (P) and terminating in quinone acceptors (Q)-are common features of photosynthetic reaction centers (RC). Experimental observations show activity of only one of them-the A branch-in wild-type bacterial RCs. In a mutant RC, we now demonstrate that electron transfer can occur along the entire, normally inactive B-branch pathway to reduce the terminal acceptor Q(B) on the time scale of nanoseconds. The transmembrane charge-separated state P(+)Q(B)(-) is created in this manner in a Rhodobacter capsulatus RC containing the F(L181)Y-Y(M208)F-L(M212)H-W(M250)V mutations (YFHV). The W(M250)V mutation quantitatively blocks binding of Q(A), thereby eliminating Q(B) reduction via the normal A-branch pathway. Full occupancy of the Q(B) site by the native UQ(10) is ensured (without the necessity of reconstitution by exogenous quinone) by purification of RCs with the mild detergent, Deriphat 160-C. The lifetime of P(+)Q(B)(-) in the YFHV mutant RC is >6 s (at pH 8.0, 298 K). This charge-separated state is not formed upon addition of competitive inhibitors of Q(B) binding (terbutryn or stigmatellin). Furthermore, this lifetime is much longer than the value of approximately 1-1.5 s found when P(+)Q(B)(-) is produced in the wild-type RC by A-side activity alone. Collectively, these results demonstrate that P(+)Q(B)(-) is formed solely by activity of the B-branch carriers in the YFHV RC. In comparison, P(+)Q(B)(-) can form by either the A or B branches in the YFH RC, as indicated by the biexponential lifetimes of approximately 1 and approximately 6-10 s. These findings suggest that P(+)Q(B)(-) states formed via the two branches are distinct and that P(+)Q(B)(-) formed by the B side does not decay via the normal (indirect) pathway that utilizes the A-side cofactors when present. These differences may report on structural and energetic factors that further distinguish the functional asymmetry of the two cofactor branches.

Topics: Amino Acid Substitution; Bacteriochlorophylls; Benzoquinones; Electron Transport; Energy Metabolism; Imidoesters; Kinetics; Light-Harvesting Protein Complexes; Mutagenesis, Site-Directed; Oxidation-Reduction; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Plasmids; Rhodobacter capsulatus; Spectrophotometry; Structure-Activity Relationship; Titrimetry; Triazines

We report time-resolved optical measurements of the primary electron transfer reactions in Rhodobacter capsulatus reaction centers (RCs) having four mutations: Phe(L181) --> Tyr, Tyr(M208) --> Phe, Leu(M212) --> His, and Trp(M250) --> Val (denoted YFHV). Following direct excitation of the bacteriochlorophyll dimer (P) to its lowest excited singlet state P, electron transfer to the B-side bacteriopheophytin (H(B)) gives P(+)H(B)(-) in approximately 30% yield. When the secondary quinone (Q(B)) site is fully occupied, P(+)H(B)(-) decays with a time constant estimated to be in the range of 1.5-3 ns. In the presence of excess terbutryn, a competitive inhibitor of Q(B) binding, the observed lifetime of P(+)H(B)(-) is noticeably longer and is estimated to be in the range of 4-8 ns. On the basis of these values, the rate constant for P(+)H(B)(-) --> P(+)Q(B)(-) electron transfer is calculated to be between approximately (2 ns)(-)(1) and approximately (12 ns)(-)(1), making it at least an order of magnitude smaller than the rate constant of approximately (200 ps)(-)(1) for electron transfer between the corresponding A-side cofactors (P(+)H(A)(-) --> P(+)Q(A)(-)). Structural and energetic factors associated with electron transfer to Q(B) compared to Q(A) are discussed. Comparison of the P(+)H(B)(-) lifetimes in the presence and absence of terbutryn indicates that the ultimate (i.e., quantum) yield of P(+)Q(B)(-) formation relative to P is 10-25% in the YFHV RC.

Topics: Bacteriochlorophylls; Benzoquinones; Dimerization; Electron Transport; Histidine; Imidoesters; Kinetics; Light-Harvesting Protein Complexes; Mutation; Nanotechnology; Phenylalanine; Pheophytins; Photolysis; Photosynthetic Reaction Center Complex Proteins; Rhodobacter capsulatus; Static Electricity; Triazines; Tyrosine

The core of the photosynthetic reaction center from the purple non-sulfur bacterium Rhodobacter sphaeroides is a quasi-symmetric heterodimer, providing two potential pathways for transmembrane electron transfer. Past measurements have demonstrated that only one of the two pathways (the A-side) is used to any significant extent upon excitation with red or near-infrared light. Here, it is shown that excitation with blue light into the Soret band of the reaction center gives rise to electron transfer along the alternate or B-side pathway, resulting in a charge-separated state involving the anion of the B-side bacteriopheophytin. This electron transfer is much faster than normal A-side transfer, apparently occurring within a few hundred femtoseconds. At low temperatures, the B-side charge-separated state is stable for at least 1 ns, but at room temperature, the B-side bacteriopheophytin anion is short-lived, decaying within approximately 15 ps. One possible physiological role for B-side electron transfer is photoprotection, rapidly quenching higher excited states of the reaction center.

Topics: Bacteriochlorophylls; Cations; Energy Transfer; Light; Light-Harvesting Protein Complexes; Pheophytins; Photolysis; Photons; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrophotometry; Temperature

The core light-harvesting LH1 complex of Rhodospirillum rubrum consists of an assembly of membrane-spanning alpha and beta polypeptides, each of which binds one bacteriochlorophyll (BChl) a molecule. In this work, we describe a technique that allows the replacement of the natural, Mg BChl a cofactors present in this protein by Zn-bacteriopheophytin (Zn-Bpheo). This technique makes use of the well-characterized, reversible dissociation of LH1 induced by the detergent beta-octylglucoside. Incubation of partially dissociated LH1 with exogeneous pigments induces an equilibrium between the protein-bound BChl and the exogeneous pigment. This results in the binding of chemically modified pigments to LH1, in amounts which depend on the pigment composition and concentration of the exchange buffer. This method can yield information on the relative affinities of the LH1 protein-binding sites for the different pigments BChl and Zn-Bpheo and can also be used to obtain fully reassociated LH1 proteins, with a variable content of modified pigment, which may be precisely monitored. Absorption and FT-Raman spectroscopy indicate that this exchange procedure leads to LH1 proteins containing modified pigments, but retaining a binding site structure identical to that of native LH1. Furthermore, examination of the binding curves suggests that there are two distinguishable binding sites, probably corresponding to the two polypeptides, with very different properties. One of these two binding sites shows a marked preference for Zn-Bpheo over BChl, while the other binding site appears to prefer BChl.

Topics: Bacterial Proteins; Bacteriochlorophylls; Circular Dichroism; Light-Harvesting Protein Complexes; Models, Biological; Models, Chemical; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Pigments, Biological; Rhodospirillum rubrum; Spectrophotometry; Spectrum Analysis, Raman; Structure-Activity Relationship; Zinc

The aim of this study was to investigate the spectral modifications of the LHII antenna complex from the purple bacterium Ectothiorhodospira sp. upon acid pH titration both in the presence and absence of urea. A blue shift specifically and reversibly affected the B850 band around pH 5.5-6.0 suggesting that a histidine residue most probably participated in the in vivo absorption red shifting mechanism. This transition was observed in the presence and absence of urea. Under strong chaotropic conditions, a second transition occurred around pH 2.0, affecting the B800 band irreversibly and the B850 reversibly. Under these conditions a blue shift from 856 to 842 nm occurred and a new and strong circular dichroism signal from the new 842 nm band was observed. Reverting to the original experimental conditions induced a red shift of the B850 band up to 856 nm but the circular dichroism signal remained mostly unaffected. Under the same experimental conditions, i.e. pH 2.1 in the presence of urea, part of the B800 band was irreversibly destroyed with concomitant appearance of a band around 770 nm due to monomeric bacteriochlorophyll from the disrupted B800. Furthermore, Gaussian deconvolution and second derivative of the reverted spectra at pH 8.0 after strong-acid treatment indicated that the new B850 band was actually composed of two bands centered at 843 and 858 nm. We ascribed the 858 nm band to bacteriochlorophylls that underwent reversible spectral shift and the 843 nm band to oligomeric bacteriopheophytin formed from a part of the B850 bacteriochlorophyll. This new oligomer would be responsible for the observed strong and mostly conservative circular dichroism signal. The presence of bacteriopheophytin in the reverted samples was definitively demonstrated by HPLC pigment analysis. The pheophytinization process progressed as the pH decreased below 2.1, and at a certain point (i.e. pH 1.5) all bacteriochlorophylls, including those from the B800 band, became converted to oligomeric bacteriopheophytin, as shown by the presence of a single absorption band around 843 nm and by the appearance of a single main elution peak in the HPLC chromatogram which corresponded to bacteriopheophytin.

Topics: Bacterial Proteins; Bacteriochlorophylls; Chromatography, High Pressure Liquid; Circular Dichroism; Ectothiorhodospira; Hydrogen-Ion Concentration; Light-Harvesting Protein Complexes; Pheophytins; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Pigments, Biological; Spectrophotometry; Urea

Topics: Bacteriochlorophylls; Chemical Phenomena; Chemistry, Physical; Electrons; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Quantum Theory; Quinones; Rhodobacter sphaeroides; Thermodynamics

Energy transfer and the primary charge separation process are studied as a function of excitation wavelength in membrane-bound reaction centers of Rhodobacter sphaeroides in which the excitonically coupled bacteriochlorophyll homodimer is converted to a bacteriochlorophyll-bacteriopheophytin heterodimer, denoted D [Bylina, E. J., and Youvan, D. C. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 7226]. In the HM202L heterodimer reaction center, excitation of D using 880 nm excitation light results in a 43 ps decay of the excited heterodimer, D. The decay of D results for about 30% in the formation of the charge separated state D+QA- and for about 70% in a decay directly to the ground state. Upon excitation of the monomeric bacteriochlorophylls using 798 nm excitation light, approximately 60% of the excitation energy is transferred downhill to D, forming D. Clear evidence is obtained that the other 40% of the excitations results in the formation of D+QA- via the pathway BA --> BA+HA- --> D+HA- --> D+QA-. In the membrane-bound "reversed" heterodimer reaction center HL173L, the simplest interpretation of the transient absorption spectra following B excitation is that charge separation occurs solely via the slow D-driven route. However, since a bleach at 812 nm is associated with the spectrum of D in the HL173L reaction center, it cannot be excluded that a state including BB is involved in the charge separation process in this complex.

Topics: Bacteriochlorophylls; Dimerization; Energy Transfer; Kinetics; Leucine; Light-Harvesting Protein Complexes; Methionine; Models, Chemical; Mutagenesis, Site-Directed; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrometry, Fluorescence; Temperature; Ubiquinone

We report the primary charge separation events in a series of Rhodobacter capsulatus reaction centers (RCs) that have been genetically modified to contain a lysine near the bacteriochlorophyll molecule, BChl(M), on the nonphotoactive M-side of the RC. Using wild type and previously constructed mutants as templates, we substituted Lys for the native Ser residue at position 178 on the L polypeptide to make the S(L178)K single mutant, the S(L178)K/G(M201)D and S(L178)K/L(M212)H double mutants, and the S(L178)K/G(M201)D/L(M212)H triple mutant. In the triple mutant, the decay of the photoexcited primary electron donor (P) occurs with a time constant of 15 ps and is accompanied by 15% return to the ground state, 62% electron transfer to the L-side bacteriopheophytin, BPh(L), and 23% electron transfer to the M-side analogue, BPh(M). The data supporting electron transfer to the M-side include bleaching of the Q(X) band of BPh(M) at 528 nm and a spectrally and kinetically resolved anion band with a maximum at 640 nm assigned to BPh(M)(-). The decay of these features and concomitant approximately 20% decay of bleaching of the 850 nm band of P give a P(+)BPh(M)(-) lifetime on the order of 1-2 ns that reflects deactivation to give the ground state. These data and additional findings are compared to those from parallel experiments on the G(M201)D/L(M212)H double mutant, in which 15% electron transfer to BPh(M) has been reported previously and is reproduced here. We also compare the above results with the primary electron-transfer processes in S(L178)K, S(L178)K/G(M201)D, and S(L178)K /L(M212)H RCs and with those for the L(M212)H and G(M201)D single mutants and wild-type RCs. The comparison of extensive results that track the primary events in these eight RCs helps to elucidate key factors underlying the directionality and high yield of charge separation in the bacterial photosynthetic RC.

Topics: Amino Acid Substitution; Anions; Aspartic Acid; Bacteriochlorophylls; Benzoquinones; Electron Transport; Histidine; Kinetics; Light-Harvesting Protein Complexes; Lysine; Mutagenesis, Site-Directed; Pheophytins; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Rhodobacter capsulatus; Spectrum Analysis

Qy-excitation resonance Raman (RR) spectra are reported for two mutant reaction centers (RCs) from Rhodobacter capsulatus in which the photoactive bacteriopheophytin (BPhL) is replaced by a bacteriochlorophyll (BChl) molecule, designated beta. The pigment change in both mutants is induced via introduction of a histidine residue near the photoactive cofactor. In one mutant, L(M212)H, the histidine is positioned over the core of the cofactor and serves as an axial ligand to the Mg+2 ion. In the other mutant, F(L121)H/F(L97)V, the histidine is positioned over ring V of the cofactor, which is nominally too distant to permit bonding to the Mg+2 ion. The salient observations are as follows: (1) The beta cofactor in F(L121)H/F(L97)V RCs is a five-coordinate BChl molecule. However, there is no evidence for the formation of a Mg-His bond. This bond is either much weaker than in the L(M212)H RCs or completely absent, the latter implying coordination by an alternative ligand. The different axial ligation for beta in the F(L121)H/F(L97)V versus L(M212)H RCs in turn leads to different conformations of the BChl macrocycles. (2) The C9-keto group of beta in F(L121)H/F(L97)V RCs is free of hydrogen bonding interactions, unlike the L(M212)H RCs in which the C9-keto of beta is hydrogen bonded to Glu L104. The interactions between other peripheral substituents of beta and the protein are also different in the F(L121)H/F(L97)V RCs versus L(M212)H RCs. Accordingly, the position and orientation of beta in the protein is different in the two beta-containing RCs. Nonetheless, previous studies have shown that the primary electron transfer reactions are very similar in the two mutants but differ in significant respects compared to wild-type RCs. Collectively, these observations indicate that changes in the conformation of a photoactive tetrapyrrole macrocycle or its interactions with the protein do not necessarily lead to significantly perturbed photochemistry and do not underlie the altered primary events in beta-type RCs.

Topics: Bacteriochlorophylls; Light-Harvesting Protein Complexes; Molecular Structure; Pheophytins; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Protein Conformation; Rhodobacter capsulatus; Spectrum Analysis, Raman; Structure-Activity Relationship; Vibration

We simulate Photo-Chemically Induced Dynamic Nuclear Polarization in the 15N-solid-state NMR of 15N-labeled photosynthetic reaction centers using a Radical Pair Mechanism (RPM). According to the experimental data, the directly polarized nuclei include all eight nitrogens in the ground state of the bacteriochlorophyll special pair (P), and N-II in the bacteriopheophytin acceptor (H) [M.G. Zysmilich, A.E. McDermott, J. Am. Chem. Soc., 116 (1994) 8362-8363.] [M.G. Zysmilich, A. McDermott, J. Am. Chem. Soc., 118 (1996) 5867-5873.] [M.G. Zysmilich, A. McDermott, Proc. Natl. Acad. Sci. U.S.A., 93 (1996) 6857-6860.]; other signals are polarized in nonspecifically labeled samples, but the polarization apparently results from magnetization exchange with neighboring polarized nitrogens, and these are not treated in this work. Two quantitative models for the polarization associated with the RPM are presented and are used to test the validity of the proposal that this mechanism is cooperative in the reaction centers. The kinetic models can treat the steady state polarizations as well as the approach to steady state, and in principle could be expanded to include anisotropic effects, or pulse-probe experiments. Several features of the detailed simulations of the steady-state amplitudes and the kinetics of the approach to steady-state are compared with our data, including the signs and approximate absolute magnitudes of the polarization on the nitrogen nuclei in P and H(L), and the changes in the relative amplitudes with the change in the lifetime of the molecular triplet, photoaccumulation time, nuclear relaxation rate and illumination intensity. The simulations demonstrate that the polarization intensities are in qualitative agreement with those predicted for the RPM, including the curious observation of strong polariza-tion on the pheophytin acceptor for certain experimental conditions. However, this agreement requires efficient relaxation of the nitrogens on H(L) by 3P, due to a fortuitous low nanosecond value for the spin-lattice relaxation for the electrons in the molecular triplet of the donor, T1e of 3P. Whether this fortuitous match is valid is unproven.

Topics: Anisotropy; Bacteriochlorophylls; Electron Transport; Kinetics; Light-Harvesting Protein Complexes; Magnetic Resonance Spectroscopy; Models, Chemical; Nitrogen; Pheophytins; Photochemistry; Photosynthetic Reaction Center Complex Proteins

A phenomenological analysis of the driving force effects in photosynthetic reaction centers modified by mutagenesis and also by chemical means is presented. Different parameter sets associated with different mechanisms of electron transfer are consistent with the mutagenesis experiments. However, only one parameter set--connected with a sequential mechanism of electron transfer--is consistent with all known experimental data. Arguments explaining why the sequential mechanism of electron transfer is selected by nature in the wild type reaction center are provided. Why the driving force of the wild type reaction center is about 0.25 eV is explained and new driving force effects are predicted.

Topics: Bacteriochlorophylls; Electron Transport; Light-Harvesting Protein Complexes; Models, Biological; Mutagenesis; Oxidation-Reduction; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Thermodynamics

We have reexamined the temperature dependence of resonance Raman (RR) spectra of the bacteriochlorin cofactors bound to reaction centers from Rhodobacter sphaeroides. Three types of resonant excitations were performed, namely, Soret band, bacteriopheophytin Qx-band, and near-infrared, Qy-band (pre)resonances. Sample temperature was varied from 300 to 10 K. In both Soret-resonant and Qy-preresonant Raman spectra, the ca. 1610-cm(-1) band corresponding to a bacteriochlorophyll CaCm methine bridge stretching mode is observed to increase in frequency by 4-6 cm(-1) as temperature is decreased from 300 to 15 K. This upshift is interpreted as arising from a change in conformation of the bacteriochlorophyll macrocycles. It may be nonspecific to the protein-bound cofactors, since a similar 4-cm(-1) upshift was observed in the same temperature range for BChl a in solution. Qx-resonant Raman spectra of either of the two bacteriopheophytin (BPhe) cofactors were obtained selectively using excitations at 537 and 546 nm. No significant frequency shift was observed for the CaCm stretching mode of BPheL between 200 and 15 K. We conclude, at variance with a previous report, that the macrocycle of the BPheL primary electron acceptor does not undergo any significant conformational change in the 200-15 K temperature range. Qy-preresonant excitation of RCs at 1064 nm provided selective Raman information on the primary electron donor (P primary). The stretching frequencies of the two conjugated keto and acetyl carbonyl groups of the M-branch primary donor BChl cofactor (P(M)) did not significantly change between 300 and 10 K. In contrast the keto carbonyl stretching frequency of cofactor P(L) was observed to upshift by 5 cm(-1), while its acetyl carbonyl frequency downshifted by 2 cm(-1). The latter shift indicated that the strong H-bond between the acetyl group of P(L) and His L168 may have slightly strengthened at 10 K. Excitation at 1064 nm of chemically oxidized RCs selectively provided RR spectra of the primary donor in its radical P.+ state. These spectra can be interpreted as a decrease of the localization of the positive charge on P(L) from 78% to 63% when the temperature decreased from 300 to 10 K resulting in a more electronically symmetric dimer. Possible origins of the temperature dependence of the positive charge delocalization in P.+ are discussed.

Topics: Bacteriochlorophylls; Hydrogen Bonding; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protein Conformation; Rhodobacter sphaeroides; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Thermodynamics; Vibration

Qy-excited resonance Raman spectra of the accessory bacteriochlorophylls (B), the bacteriopheophytins (H), and the primary electron donor (P) in the bacterial photosynthetic reaction center (RC) of Rhodobacter sphaeroides have been obtained at 95 and 278 K. Frequency and intensity differences are observed in the low-frequency region of the P vibrational spectrum when the sample is cooled from 278 to 95 K. The B and H spectra exhibit minimal changes of frequencies and relative intensities as a function of temperature. The mode patterns in the Raman spectra of B and H differ very little from Raman spectra of the chromophores in vitro. The Raman scattering cross sections of B and H are 6-7 times larger than those for analogous modes of P at 278 K. The cross sections of B and of H are 3-4 times larger at 95 K than at 278 K, while the cross sections of P are approximately constant with temperature. The temperature dependence of the Raman cross sections for B and H suggests that pure dephasing arising from coupling to low-frequency solvent/protein modes is important in the damping of their excited states. The weak Raman cross sections of the special pair suggest that the excited state of P is damped by very rapid (<<30 fs) electronic relaxation processes. These resonance Raman spectra provide information for developing multimode vibronic models of the excited-state structure and dynamics of the chromophores in the RC.

Topics: Bacteriochlorophylls; Electron Transport; Infrared Rays; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrophotometry; Spectrum Analysis, Raman; Temperature

Qy-excitation resonance Raman (RR) spectra are reported for two mutant reactions centers (RCs) from Rhodobacter sphaeroides in which the photoactive bacteriopheophytin (BPhL) is replaced by a bacteriochlorophyll (BChl) molecule, designated by beta L. One mutation, (M)L214H, yields the pigment change via introduction of a histidine residue at position M214. The other mutation, (M)L214H/(L)-E104V, removes the putative hydrogen bond between beta L and the native glutamic acid residue at position L104. The vibrational signatures of the beta L cofactors of the mutants are compared with one another and with those of the accessory BChls (BChlL,M) in both beta-mutant and wild-type RCs. The spectroscopic data reveal the following: (1) The beta L cofactor is a five-coordinate BChl molecule with a histidine axial ligand. The conformation of beta L and the strength of the Mg-histidine bond are very similar to that of BChlL,M. (2) The beta L cofactor is oriented in the protein pocket in a manner similar to that of BPhL of wild-type. (3) The beta L cofactor of the (M)L214H mutant forms a hydrogen bond with glutamic acid L104 via the C9-keto group of the macrocycle. The strength of this hydrogen bond is identical to that formed between this protein residue and the C9-keto group of BPhL in wild-type. (4) The hydrogen bonding interaction at the C9-keto site induces secondary cofactor-protein interactions which involve the C2a-acetyl and Cb-alkyl substituent groups. Collectively, the vibrational signatures of beta L indicate that its intrinsic physicochemical properties are very similar to those of BChlL. Consequently, the initial charge-separated intermediate in beta-type RCs is best characterized as a thermal/quantum mechanical admixture of P+ beta L- and P+ BChlL-(P is the primary electron donor), as originally proposed by Kirmaier et al. [(1995) J. Phys. Chem. 99, 8903-8909].

Topics: Bacteriochlorophylls; Histidine; Hydrogen Bonding; Infrared Rays; Light; Light-Harvesting Protein Complexes; Magnesium; Mutation; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrum Analysis, Raman

Formation of the vibronic wavepacket by 90-fs excitation of the primary electron donor P in bacteriochlorophyll(M)-modified reaction centers is shown to induce nuclear motions accompanied by (1) oscillation of the stimulated emission from excited primary electron donor P* and (2) wavepacket motions leading to electron transfer at 293 K from P* to bacteriochlorophyll (B(L)) and then to bacteriopheophytin (H(L)). The latter motions have low frequency (about 15 cm-1) and are related to protein-nuclear motions which are along the reaction coordinate. When the wavepacket approaches the intersection of the reactant (P*B(L)) and product (P+B(L)-) potential energy surfaces (approximately 1.5 ps delay), about 60% of P* is converted to the P+B(L)- state. The P+H(L)- state formation is delayed by approximately 2 ps with respect to that of P+B(L)-. It is suggested that the wavepacket is transferred to and moves also slowly on the P+B(L)- potential energy surface and approaches the intersection of the surfaces of P+B(L)- and P+H(L)- within approximately 2 ps (approximately 8 cm-1), indicating the electron transfer to H(L).

Topics: Bacteriochlorophylls; Electron Transport; Kinetics; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrophotometry, Infrared

Accurate quantum mechanical simulations of the primary charge transfer in photosynthetic reaction centers are reported. The process is modeled by three coupled electronic states corresponding to the photoexcited chlorophyll special pair (donor), the reduced bacteriopheophytin (acceptor), and the reduced accessory chlorophyll (bridge) that interact with a dissipative medium of protein and solvent degrees of freedom. The time evolution of the excited special pair is followed over 17 ps by using a fully quantum mechanical path integral scheme. We find that a free energy of the reduced accessory chlorophyll state approximately equal to 400 cm(-1) lower than that of the excited special pair state yields state populations in agreement with experimental results on wild-type and modified reaction centers. For this energetic configuration electron transfer is a two-step process.

Topics: Bacteriochlorophylls; Computer Simulation; Kinetics; Light-Harvesting Protein Complexes; Mathematics; Models, Molecular; Models, Theoretical; Pheophytins; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Quantum Theory; Rhodobacter sphaeroides; Thermodynamics; Time Factors

The properties of the primary electron donor in reaction centers from Rhodobacter sphaeroides have been investigated in mutants containing a bacteriochlorophyll (BChl)--bacteriopheophytin (BPhe) dimer with and without hydrogen bonds to the conjugated carbonyl groups. The heterodimer mutation His M202 to Leu was combined with each of the following mutations: His L168 to Phe, which should remove an existing hydrogen bond to the BChl molecule; Leu L131 to His, which should add a hydrogen bond to the BChl molecule; and Leu M160 to His and Phe M197 to His, each of which should add a hydrogen bond to the BPhe molecule [Rautter, J., Lendzian, F., Schulz, C., Fetsch, A., Kuhn M., Lin, X., Williams, J. C., Allen J. P., & Lubitz, W. (1995) Biochemistry 34, 8130-8143]. Pigment extractions and Fourier transform Raman spectra confirm that all of the mutants contain a heterodimer. The bands in the resonance Raman spectra arising from the BPhe molecule, which is selectively enhanced, exhibit the shifts expected for the addition of a hydrogen bond to the 9-keto and 2-acetyl carbonyl groups. The oxidation--reduction midpoint potential of the donor is increased by approximately 85 mV by the addition of a hydrogen bond to the BChl molecule but is only increased by approximately 15 mV by the addition of a hydrogen bond to the BPhe molecule. An increase in the rate of charge recombination from the primary quinone is correlated with an increase in the midpoint potential. The yield of electron transfer to the primary quinone is 5-fold reduced for the mutants with a hydrogen bond to the BPhe molecule. Room- and low-temperature optical absorption spectra show small differences from the features that are typical for the heterodimer, except that a large increase in absorption is observed around 860-900 nm for the donor Qy band in the mutant that adds a hydrogen bond to the BChl molecule. The changes in the optical spectra and the yield of electron transfer are consistent with a model in which the addition of a hydrogen bond to the BChl molecule increases the energy of an internal charge transfer state while the addition to the BPhe molecule stabilizes this state. The results show that the properties of the heterodimer are different depending on which side is hydrogen-bonded and suggest that the hydrogen bonds alter the energy of the internal charge transfer state in a well-defined manner.

Topics: Amino Acid Sequence; Bacteriochlorophylls; Fourier Analysis; Histidine; Hydrogen Bonding; Leucine; Light-Harvesting Protein Complexes; Macromolecular Substances; Mutagenesis, Site-Directed; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Point Mutation; Recombinant Proteins; Rhodobacter sphaeroides; Spectrophotometry; Spectrum Analysis, Raman

Site-specific pigment modifications are useful to investigate structure-function relationships in photosynthesis. In reaction centers bearing modified (bacterio)pheophytins, changed electron transfer kinetics have been related to the changed redox potentials of the pigments introduced (Huber, H. et al. (1995) Chemical Physics, Special Issue, vol 197 (Hochstrasser, R.M. and Hofacker, G.L. eds.) pp. 297-305; [1]). In order to analyze potentially interfering structural changes induced in these reaction centers by the exchange procedure, in particular mispositioning or misorientation of the pigments, low-temperature linear dichroism spectra have been measured for reaction centers from Rhodobacter sphaeroides containing modified bacteriopheophytins and bacteriochlorophylls at the sites HA,B and BA,B, respectively. They show that all modified pigments are oriented similar to the native ones, and that they do not affect significantly the linear dichroism of the monomeric bacteriocholorophylls and bacteriopheophytins or of the primary donor.

Topics: Bacteriochlorophylls; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protein Conformation; Rhodobacter sphaeroides; Spectrum Analysis; Temperature

The reaction center (RC) from the photosynthetic bacterium Rhodobacter (Rb.) capsulatus has been the subject of a considerable amount of molecular biological and spectroscopic work aimed at improving our understanding of the primary steps of photosynthesis. However, no three-dimensional structure is available for this protein. We present here a model obtained by combining information from the structure of the highly homologous RC from Rhodopseudomonas (Rps.) viridis with molecular mechanics and simulated annealing calculations. In the Rb. capsulatus model the orientations of the bacteriochlorophyll monomer and the bacteriopheophytin on the branch inactive in electron transfer differ significantly from those in the RCs of Rps. viridis and Rb. sphaeroides. The bacteriopheophytin orientational difference is in good accord with previous linear dichroism measurements. A comparison is made of interactions between the pigments and the protein environment that may be of functional significance in Rps. viridis, Rb. sphaeroides, and Rb. capsulatus.

Topics: Amino Acids; Bacteriochlorophylls; Hydrogen Bonding; Light-Harvesting Protein Complexes; Models, Molecular; Molecular Structure; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protein Conformation; Protein Structure, Tertiary; Rhodobacter; Rhodobacter capsulatus; Sequence Alignment

The photoreaction center (RC) of purple bacteria contains four bacteriochlorophyll (Bch) and two bacteriopheophytin (Bph) molecules as prosthetic groups. Their optical activity, as measured by circular dichroism (CD) spectroscopy, is largely increased in situ as compared to organic solutions. The all-exciton hypothesis posits that this enhanced optical activity is entirely due to excitonic interactions between the electronic transitions of all six bacteriochlorin molecules. Using the simple exciton theory, this model predicts that the near-infrared CD spectra should be conservative. The fact that they are not, whether the special pair of Bch (SP) that constitutes the primary electron donor is reduced or oxidized, has been explained by hyperchromic effects. The present work tests this hypothesis by successively eliminating the absorption and, therefore, the optical activity of the Bphs and of the non-special-pair (non-SP) Bchs. This was accomplished by trapping these pigments in their reduced state. RC preparations with the four non-SP bacteriochlorins trapped in their reduced state and, therefore, with an intact SP displayed conservative CD spectra. RC preparations with only the electronic transitions of SP and of one non-SP Bch also showed conservative CD spectra. These conservative CD spectra and their corresponding absorption spectra were simulated using simple exciton theory without assuming hyperchromic effects. Bleaching half of the 755-nm absorption band by phototrapping one of the two Bph molecules led to the complete disappearnce of the corresponding CD band. This cannot be explained by the all-exciton hypothesis. These results suggest that the optical activity of the SP alone, or with one non-SP Bch, is due to excitonic interactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Ascorbic Acid; Bacteriochlorophylls; Chromatiaceae; Circular Dichroism; Dithionite; Electron Transport; Light-Harvesting Protein Complexes; Optics and Photonics; Oxidation-Reduction; Pheophytins; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Spectrophotometry

An aspartic acid residue has been introduced near ring V of the L-side accessory bacteriochlorophyll (BCHlL) or the photosynthetic reaction center in a rhodobacter capsulatus mutant in which a His also replaces Leu 212 on the M-polypeptide. The initial stage of charge separation in the G(M201)D/L(M212)H double mutant yields approximately 70 percent electron transfer to the L-side cofactors, approximately 15 percent rapid deactivation to the ground state, and approximately 15 percent electron transfer to the so-called inactive M-side bacteriopheophytin (BPhM). It is suggested here that the Asp introduced at M201 modulates the reduction potential of BCHlL, thereby changing the energetics of charge separation. The results demonstrate that an individual amino acid residue can, through its influence on the free energies of the charge-separated states, effectively dictate the balance between the forward electron transfer reactions on the L-side of the RC, the charge-recombination processes, and electron transfer to the M-side chromophores.

Topics: Bacteriochlorophylls; Electron Transport; Light-Harvesting Protein Complexes; Mutation; Pheophytins; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Rhodobacter capsulatus; Spectrum Analysis; Thermodynamics

Photosynthetic reaction centres (RCs) catalyze light-driven electron, transport across photosynthetic membranes. The photosynthetic bacterium Rhodobacter, sphaeroides is often used for studies of RCs, and three groups have determined the structure of its reaction centre. There are discrepancies between these structures, however, and to resolve these we have determined the structure to higher resolution than before, using a new crystal form.. The new structure provides a more detailed description of the Rb. sphaeroides RC, and allows us to compare it with the structure of the RC from Rhodopseudomonas viridis. We find no evidence to support most of the published differences in cofactor binding between the RCs from Rps. viridis and Rb. sphaeroides. Generally, the mode of cofactor binding is conserved, particularly along the electron transfer pathway. Substantial differences are only found at ring V of one bacteriochlorophyll of the 'special pair' and for the secondary quinone, QB. A water chain with a length of about 23 A including 14 water molecules extends from the QB to the cytoplasmic side of the RC.. The cofactor arrangement and the mode of binding to the protein seem to be very similar among the non-sulphur bacterial photosynthetic RCs. The functional role of the displaced QB molecule, which might be present as quinol, rather than quinone, is not yet clear. The newly discovered water chain to the QB binding site suggests a pathway for the protonation of the secondary quinone QB.

Topics: Bacteriochlorophylls; Carotenoids; Electron Transport; Iron; Light-Harvesting Protein Complexes; Models, Molecular; Molecular Structure; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Protein Conformation; Protons; Quinones; Rhodobacter sphaeroides

The major part (> 90%) of bacteriopheophytin a in reaction centers (RCs) of Rhodobacter sphaeroides was substituted by plant pheophytin a. In modified RCs the photochemical formation of P+Qa- occurs with with a quantum efficiency of 79%. The intermediary state P+I- displayed a recombination time constant of 1.5 ns, and the electron transfer from I- to Qa was characterized by a time constant of 540 ps. On the basis of spectral properties of P+I- for native and modified RCs, it was suggested that bacteriopheophytin, as well as bacteriochlorophyll monomers located in L protein branch, have a transition at 545 nm with approx. equal extinction coefficients. Accordingly, the state P+I- in modified RCs is proposed to consist of a thermodynamic mixture of P+BL- (approximately 80%) and P+Phe- (approximately 20%).

Topics: Bacteriochlorophylls; Electron Transport; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Plant Proteins; Rhodobacter sphaeroides; Spectrophotometry; Time Factors

The native ubiquinone-10 redox cofactor has been removed from the QA site of the isolated reaction center protein from Rhodobacter sphaeroides and reconstitution attempted with 28 non-quinone molecules in order to identify factors governing cofactor function and the selectivity displayed by the site in the electron transfers that it catalyzes. Equilibrium binding, in situ electrochemistry, and the kinetics of electron transfer to and from the QA site occupant were examined. Four classes of non-quinone molecules are distinguished according to their ability to occupy the QA site and conduct intraprotein electron transfers. The minimal requirements for occupancy of the QA site are at least one ring and a heteroatom hydrogen bond acceptor. Thus, binding at the site is not highly selective. The rates of electron transfers to and from the class of non-quinone molecules (four) that satisfy the criteria for cofactor function at the QA site compare well with rates previously determined from 14 to 295 K for 14 quinone replacements with comparable values of the reaction free energy. This indicates that the rates are relatively insensitive to variations in exotic and quinone cofactor reorganization energy and the vibrational frequencies coupled to the electron transfers, and that the exotic and quinone cofactors are bound in the QA site in comparable positions. It appears that any variation in rate is determined predominantly by the value of the reaction free energy. The QA site protein-cofactor solvation contribution to the in situ electrochemical potential is roughly constant for 12 rigid quinone and 2 exotic cofactors (average value-61 +/- 2 kcal/mol). Favorable electrostatic contributions governing the reaction free energy are therefore also relatively insensitive to cofactor structure. However, flexible molecules appear to encounter in situ steric constraints that lower the electron affinity by destabilizing the reduced cofactor species. This is a strong determinant of whether a molecule, once in the QA site, will function. These findings compare well with those from studies of electron transfers in synthetic systems.

Topics: Bacteriochlorophylls; Binding Sites; Electron Transport; Kinetics; Light-Harvesting Protein Complexes; Oxidation-Reduction; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Structure-Activity Relationship; Thermodynamics; Ubiquinone

Light-induced P+QB-/PQB FTIR difference spectra of reaction centers (RCs) have been obtained from chromatophores lacking light-harvesting B800-850 antenna for Rhodobacter capsulatus wild type (WT) and for the two mutants HisM200-->Leu and HisL173-->Leu. The primary donor (P) in both mutants consists of a bacteriochlorophyll-bacteriopheophytin heterodimer. The most prominent difference between the WT and the mutant spectra is in the 1600-1200-cm-1 region. The WT spectrum displays large positive bands at approximately 1290, 1500-1430, and 1580-1530 cm-1. These three bands are either small or altogether absent in the heterodimer spectra. In addition, both heterodimer spectra compare well with the electrochemically generated BChla+/BChla spectrum [Mäntele, W.G., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472]. These observations indicate that the positive charge is localized on the monomeric BChl in the heterodimers. The overall shape of the ester and keto C = O signals in the BChla+/BChla spectrum is maintained in the in situ spectra although significant differences are observed in the frequency, width, and splitting of the bands. The shape of the signal at 1757/1744 cm-1 in HisL173-->Leu is comparable to the 1751/1737-cm-1 signal of BChla+/BChla in tetrahydrofuran, indicating a free 10a ester C = O of PM in HisL173-->Leu. The reduced amplitude of the negative 1740-cm-1 feature in both HisM200-->Leu and WT spectra suggests a hydrogen-bonded 10a ester C = O for PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Bacteriochlorophylls; Electrochemistry; Histidine; Leucine; Light-Harvesting Protein Complexes; Macromolecular Substances; Mutation; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter capsulatus; Spectrophotometry, Infrared

The effects of electric fields on the absorption spectra of the carotenoids spheroidene and spheroidenone in photosynthetic antenna and reaction center complexes (wild-type and several mutants) from purple non-sulfur bacteria are compared with those for the isolated pigments in organic glasses. In general, the field effects are substantially larger for the carotenoid in the protein complexes than for the extracted pigments and larger for spheroidenone than spheroidene. Furthermore, the electrochromic effects for carotenoids in all complexes are much larger than those for the Qx transitions of the bacteriochlorophyll and bacteriopheophytin pigments which absorb in the 450-700 nm spectral region. The underlying mechanism responsible for the Stark effect spectra in the complexes is found to be dominated by a change in permanent dipole moment of the carotenoid upon excitation. The magnitude of this dipole moment change is found to be considerably larger in the B800-850 complex compared to the reaction center for spheroidene; it is approximately equivalent in the two complexes for spheroidenone. These results are discussed in terms of the effects of differences in the carotenoid functional groups, isomers and perturbations on the electronic structure from interactions with the organized environment in the proteins. these data provide a quantitative basis for the analysis of carotenoid bandshifts which are used to measure transmembrane potential, and they highlight some of the pitfalls in making such measurements on complex membranes containing multiple populations of carotenoids. The results for spheroidenone should be useful for studies of mutant proteins, since mutant strains are often grown semi-aerobically to minimize reversion.

Topics: Bacterial Proteins; Bacteriochlorophylls; Carotenoids; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter capsulatus; Rhodobacter sphaeroides; Spectrophotometry

The absorbance and polarized absorbance spectra of single crystals of the reaction center complex isolated from Rhodobacter sphaeroides wild type strain 2.4.1 have been measured at 85 K. The crystals of the complex were obtained by the vapor diffusion technique. The spectroscopic experiments on the crystals were performed using an optical microspectrometer featuring a custom-built, liquid N2-flowing cold stage, the details of which are presented herein. These data demonstrate the feasibility of conducting optical spectroscopic experiments at cryogenic temperatures on single crystals of photosynthetic pigment-protein complexes.

Topics: Bacteriochlorophylls; Carotenoids; Crystallization; Freezing; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrophotometry; Spectrum Analysis, Raman

Subpicosecond time-resolved photodichroism measurements on Rhodobacter sphaeroides R26 reaction centers are reported in the key region between 620 and 740 nm, where the anions of both bacteriopheophytin and bacteriochlorophyll (BChl) have their most diagnostic absorption bands. These measurements fail to resolve clearly the formation of a reduced BChl species. The implications of this for elucidating the role of the accessory BChl in the initial stage of charge separation are discussed.

Topics: Bacteriochlorophylls; Electron Transport; Kinetics; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrum Analysis, Raman

Site-directed mutagenic replacement of M subunit Leu214 by His in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides results in incorporation of a bacteriochlorophyll molecule (BChl) in place of the native bacteriopheophytin (BPh) electron acceptor. Evidence supporting this conclusion includes the ground-state absorption spectrum of the (M)L214H mutant, pigment and metal analyses, and time-resolved optical experiments. The genetically modified RC supports transmembrane charge separation from the photoexcited BChl dimer to the primary quinone through the new BChl molecule, but with a reduced quantum yield of 60 percent (compared to 100 percent in wild-type RCs). These results have important implications for the mechanism of charge separation in the RC, and rationalize the choice of (bacterio)pheophytins as electron acceptors in a variety of photosynthetic systems.

Topics: Bacteriochlorophylls; Electron Transport; Histidine; Kinetics; Leucine; Light-Harvesting Protein Complexes; Mutagenesis, Site-Directed; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrophotometry

Photosynthetic reaction centers (RCs) from the photosynthetic bacteria Rhodobacter sphaeroides and Rhodopseudomonas viridis are protein complexes closely related in both structure and function. The structure of the Rps. viridis RC was used to determine the structure of the RC from Rb. sphaeroides. Small but meaningful differences between the positions of the helices and the cofactors in the two complexes were identified. The distances between helices AL and AM, between BL and BM, and between bacteriopheophytins BPL and BPM are significantly shorter in Rps. viridis than they are in Rb. sphaeroides RCs. There are a number of differences in the amino acid residues that surround the cofactors; some of these residues form hydrogen bonds with the cofactors. Differences in chemical properties and location of these residues account in some manner for the different spectral properties of the two RCs. In several instances, the hydrogen bonds, as well as the apparent distances between the histidine ligands and the Mg atoms of the bacteriochlorophylls, were found to significantly differ from the Rb. sphaeroides RC structure previously described by Yeates et al. [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7993-7997] and Allen et al. [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8487-8491].

Topics: Amino Acids; Bacteriochlorophylls; Binding Sites; Hydrogen Bonding; Iron; Light-Harvesting Protein Complexes; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Quinones; Rhodobacter sphaeroides; Rhodopseudomonas; Stereoisomerism; X-Ray Diffraction

Electron paramagnetic resonance (EPR) has been used to investigate the cation and triplet states of Rhodobacter capsulatus reaction centers (RCs) containing amino acid substitutions affecting the primary donor, monomeric bacteriochlorophylls (Bchls), and the photoactive bacteriopheophytin (Bphe). The broadened line width of the cation radical in HisM200----Leu and HisM200----Phe reaction centers, whose primary donor consists of a Bchl-Bphe heterodimer, indicates a highly asymmetric distribution of the unpaired electron over the heterodimer. A T0 polarized triplet state with reduced yield is observed in heterodimer-containing RCs. The zero field splitting parameters indicate that this triplet essentially resides on the Bchl half of the heterodimer. The cation and triplet states of reaction centers containing HisM200----Gln, HisL173----Gln, GluL104----Gln, or GluL104----Leu substitutions are similar to those observed in wild type. Oligonucleotide-mediated mutagenesis has been used to change the histidine residues that are positioned near the central Mg2+ ions of the reaction center monomeric bacteriochlorophylls. Reaction centers containing serine substitutions at M180 and L153 or a threonine substitution at L153 have unaltered pigment compositions and are photochemically active. The cation and triplet states of HisL153----Leu reaction centers are similar to those observed in wild type. Triplet energy transfer to carotenoid is not observed at 100 K in HisM180----Arg chromatophores. These results have important implications for the structural requirements of tetrapyrrole binding and for our understanding of the mechanisms of primary electron transfer in the reaction center.

Topics: Bacteriochlorophylls; Electron Spin Resonance Spectroscopy; Electron Transport; Light-Harvesting Protein Complexes; Mutation; Oxidation-Reduction; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Pyrroles; Rhodospirillaceae; Tetrapyrroles

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.

Topics: Bacterial Proteins; Bacteriochlorophylls; Electricity; Electron Transport; Energy Transfer; Light-Harvesting Protein Complexes; Mathematics; Oxidation-Reduction; Pheophytins; Photosynthesis; Photosynthetic Reaction Center Complex Proteins; Rhodopseudomonas

A comparison is made between the PQA----P+QA- and PQAQB----P+QAQB-transitions in Rps. viridis and Rb. sphaeroides reaction centers (RCs) by the use of light-induced Fourier transform infrared (FTIR) difference spectroscopy. In Rb. sphaeroides RCs, we identify a signal at 1650 cm-1 which is present in the P+QA-minus-PQA spectrum and not in the P+QAQB(-)-minus-PQAQB spectrum. In contrast, this signal is present in both P+QA(-)-minus-PQA- and P+QAQB(-)-minus-PQAQB spectra of Rps. viridis RCs. These data are interpreted in terms of a conformational change of the protein backbone near QA (possible at the peptide C = O of a conserved alanine residue in the QA pocket) and of the different bonding interactions of QB with the protein in the RC of the two species.

Topics: Bacterial Proteins; Bacteriochlorophylls; Chlorophyll; Oxidation-Reduction; Pheophytins; Photosynthesis; Protein Conformation; Quinones; Rhodopseudomonas; Spectrophotometry, Infrared

The three-dimensional structures of the cofactors and protein subunits of the reaction center (RC) from the carotenoidless mutant strain of Rhodobacter sphaeroides R-26 and the wild-type strain 2.4.1 have been determined by x-ray diffraction to resolutions of 2.8 A and 3.0 A with R values of 24% and 26%, respectively. The bacteriochlorophyll dimer (D), bacteriochlorophyll monomers (B), and bacteriopheophytin monomers (phi) form two branches, A and B, that are approximately related by a twofold symmetry axis. The cofactors are located in hydrophobic environments formed by the L and M subunits. Differences in the cofactor-protein interactions between the A and B cofactors, as well as between the corresponding cofactors of Rb, sphaeroides and Rhodopseudomonas viridis [Michel, H., Epp, O. & Deisenhofer, J. (1986) EMBO J. 3, 2445-2451], are delineated. The roles of several structural features in the preferential electron transfer along the A branch are discussed. Two bound detergent molecules of beta-octyl glucoside have been located near BA and BB. The environment of the carotenoid, C, that is present in RCs from Rb. sphaeroides 2.4.1 consists largely of aromatic residues of the M subunit. A role of BB in the triplet energy transfer from D to C and the reason for the preferential ease of removal of BB from the RC is proposed.

Topics: Bacteriochlorophylls; Carotenoids; Chlorophyll; Computer Simulation; Pheophytins; Photosynthesis; Rhodopseudomonas; Stereoisomerism; X-Ray Diffraction

The primary electron transfer processes in isolated reaction centers of Rhodopseudomonas sphaeroides have been investigated with subpicosecond and picosecond spectroscopic techniques. Spectra and kinetics of the absorbance changes following excitation with 0.7-ps 610-nm pulses, absorbed predominantly by bacteriochlorophyll (BChl), indicate that the radical pair state P+BPh-, in which an electron has been transferred from the BChl dimer (P) to a bacteriopheophytin (BPh), is formed with a time constant no greater than 4 ps. The initial absorbance changes also reveal an earlier state, which could be an excited singlet state, or a P+BChl- radical pair. The bleaching at 870 nm produced by 7 ps excitation at 530 nm (absorbed by BPh) or at 600 nm (absorbed predominantly by BChl) shows no resolvable delay with respect to standard compounds in solution, suggesting that the time for energy transfer from BPh to P is less than 7 ps. However, the bleaching in the BPh band at 545 nm following 7-ps 600-nm excitation, exhibits an 8- to 10-ps lag with respect to standard compounds. This finding is qualitatively similar to the 35-ps delay previously observed at 760 nm by Shuvalov at al. (Shuvalov, V.A., Klevanik, A.V., Sharkov, A.V., Matveetz, Y.A. and Kryukov, P.G. (1978) FEBS Lett. 91, 135-139) when 25-ps 880-nm excitation flashes were used. A delay in the bleaching approximately equal to the width of the excitation flash can be explained in terms of the opposing effects of bleaching due to the reduction of BPh, and absorbance increases due to short-lived excited states (probably of BChl) that turn over rapidly during the flash. The decay of the initial bleaching at 800 nm produced by 7-ps 530- or 600-nm excitation flashes shows a fast component with a 30-ps time constant, in addition to a slower component having the 200-ps kinetics expected for the decay of P+BPh-. the dependence on excitation intensity of the absorbance changes due to the 30-p]s component indicate that the quantum yield of the state responsible for this step is lower than that observed for the primary electron transfer reactions. This suggests that at least part of the transient bleaching at 800 nm is due to a secondary process, possibly caused by excitation with an excessive number of photons. If the 800-nm absorbing BChl (B) acts as an intermediate electron carrier in the primary photochemical reaction, electron transfer between B and the BPh must have a time constant no greater than 4 ps.

Topics: Bacteriochlorophylls; Chlorophyll; Electron Transport; Kinetics; Light; Macromolecular Substances; Oxidation-Reduction; Pheophytins; Rhodobacter sphaeroides; Spectrophotometry; Time Factors

Topics: Bacterial Proteins; Bacteriochlorophylls; Dimethylamines; Iron; Light-Harvesting Protein Complexes; Mathematics; Molecular Weight; Pheophytins; Phospholipids; Photosynthetic Reaction Center Complex Proteins; Protein Binding; Protein Conformation; Rhodobacter sphaeroides; Ubiquinone

Topics: Bacteriochlorophylls; Carotenoids; Chlorophyll; Chromatiaceae; Peptides; Pheophytins; Rhodopseudomonas; Spectrum Analysis

Linear dichroism measurements of reaction centers of Rhodopseudomonas sphaeroides in stretched gelatin films have yielded angles that various optical transition moments make with an axis of symmetry in the reaction center. Photoselection experiments have yielded angles that certain transition moments make with each other. We have combined these data so as to compute the orientations of the Qx and Qy transition moments of the two molecules of bacteriopheophytin and of the bacteriochlorophyll special pair (photochemical electron donor) in the reaction center. Orientations are expressed in spherical polar coordinates with the symmetry axis as the pole. We have also computed additional angles between pairs of transition moments. In this treatment we have assumed that the bacteriopheophytins are independent monomers with little or no exciton coupling.

Topics: Bacteriochlorophylls; Chlorophyll; Oxidation-Reduction; Pheophytins; Protein Conformation; Rhodobacter sphaeroides; Spectrophotometry

Topics: Anions; Bacteriochlorophylls; Chlorophyll; Electrochemistry; Electron Spin Resonance Spectroscopy; Pheophytins; Photochemistry

Topics: Bacterial Chromatophores; Bacteriochlorophylls; Chlorophyll; Chromatium; Kinetics; Light; Oxidation-Reduction; Pheophytins; Photosynthesis; Spectrophotometry; Spectrophotometry, Infrared