Bacterial Photosynthetic Reaction Centers Research Articles

Lipidic Cubic Phase Crystal Structure of the Photosynthetic Reaction Centre from Rhodobacter sphaeroides at 2.35 Å Resolution

Well-ordered crystals of the bacterial photosynthetic reaction centre from Rhodobacter sphaeroides were grown from a lipidic cubic phase. Here, we report the type I crystal packing that results from this crystallisation medium, for which 3D crystals grow as stacked 2D crystals, and the reaction centre X-ray structure is refined to 2.35 Å resolution. In this crystal form, the location of the membrane bilayer could be assigned with confidence. A cardiolipin-binding site is found at the protein–protein interface within the membrane-spanning region, shedding light on the formation of crystal contacts within the membrane. A chloride-binding site was identified in the membrane-spanning region, which suggests a putative site for interaction with the light-harvesting complex I, the cytochrome bc 1 complex or PufX. Comparisons with the X-ray structures of this reaction centre deriving from detergent-based crystals are drawn, indicating that a slight compression occurs in this lipid-rich environment.

Modelling the bacterial photosynthetic reaction center. VI. Use of density-functional theory to determine the nature of the vibronic coupling between the four lowest-energy electronic states of the special-pair radical cation

It is now over ten years since the first FTIR spectra were recorded of the radical cation of the special-pair, a dimer of bacteriochlorophyll molecules that forms the primary electron donor responsible for primary charge separation in bacterial photosynthesis. While spectra of this type promise to reveal much concerning the role of the special pair electron donor in photosynthesis, attempts to model and interpret them have been limited by poor knowledge of the vibrationally specific aspects of the electron–phonon coupling and have thus been restricted to crude model calculations only. We develop techniques through which density-functional theory can be employed to evaluate most of the unknown properties. This includes symmetric-mode displacements, antisymmetric-mode vibronic coupling constants, and interstate electronic couplings evaluated for interactions between the four lowest-energy states of the special-pair cation radical: the ground state, the primary hole-transfer state, and states involving these two combined with SHOMO to HOMO transitions. Geometry optimizations are performed for all four states of the dimer while vibrational analyses are obtained for the first two; vibronic coupling constants are extracted from analysis of stolen infrared transition moments using Herzberg–Teller theory. Quantitatively, these results are employed in the subsequent paper in this series to simulate the observed spectra. Qualitatively, these results indicate that: (1) vibronic coupling occurs through a large number of antisymmetric modes of the dimer rather than through a small number of strongly active modes, (2) the role of symmetric vibrational motions of the dimer is only minor, (3) that the active symmetric modes are significant in number and low in frequency, (4) that vibronic coupling between the hole-transfer state and the SHOMO to HOMO state is relatively weak and influences spectra only near resonance, and (5) that the calculated electronic couplings are qualitatively realistic and may provide an explanation for the much weaker coupling observed in chlorophyll-containing reaction centers.

Modelling the bacterial photosynthetic reaction center. V. Assignment of the electronic transition observed at 2200 cm−1 in the special-pair radical-cation as a second-highest occupied molecular orbital to highest occupied molecular orbital transition

Primary charge separation in photoexcited photosynthetic reaction centers produces the radical cation P+ of a bacteriochlorophyll dimer known as the special-pair P. P+ has an intense electronic transition in the vicinity of 1800–5000 cm−1 which is usually assigned to the interchromophore hole-transfer excitation of the dimer radical cation; in principle, this spectrum can give much insight into key steps of the solar-to-electrical energy-conversion process. The extent to which this transition is localized on one-half of the dimer or delocalized over both is of utmost importance; an authoritative deduction of this quantity from purely spectroscopic arguments requires the detailed assignment of the observed high to medium resolution spectra. For reaction centers containing bacteriochlorophylls a or b, a shoulder is observed at 2200 cm−1 on the low-energy side of the main hole-transfer absorption band, a band whose maximum is near 2700 cm−1. Before quantitative analysis of the hole-transfer absorption in these well-studied systems can be attempted, the nature of the processes leading to this shoulder must be determined. We interpret it as arising from an intrachromophore SHOMO to HOMO transition whose intensity arises wholly through vibronic coupling with the hole-transfer band. A range of ab initio and density-functional calculations are performed to estimate the energy of this transition both for monomeric cations and for P+ of Blastochloris viridis, Rhodobacter sphaeroides, Chlorobium limicola, Chlorobium tepidum, Chlamydomonas reinhardtii, Synochocystis S.6803, spinach photosystems I and II, Heliobacillus mobilis, and finally Heliobacterium modesticaldum, with the results found to qualitatively describe the available experimental data. Subsequent papers in this series provide quantitative analyses of the vibronic coupling and complete spectral simulations based on the model developed herein.

Modeling the bacterial photosynthetic reaction center. VII. Full simulation of the intervalence hole–transfer absorption spectrum of the special-pair radical cation

ENDOR data suggests that the special-pair radical cation P+ from Rhodobacter sphaeroides is 68% localized on PL while simple interpretations of FTIR difference spectra based primarily on intensity information, but to some extent also bandwidths, suggest near-complete charge localization. We provide a complete a priori spectral simulation of the spectrum of P+ in the range 0–5000 cm−1, including explicit treatment of the high-resolution vibrational transitions, the low-resolution hole–transfer absorption centered at 2700 cm−1, and the resonance with the SHOMO to HOMO transition at 2200 cm−1 that resolve the issues concerning the nature of P+. The description of the vibrational aspects of the problem were taken from results of previous density-functional calculations, and a qualitatively realistic large number of vibrational modes (50 antisymmetric and 18–20 symmetric) were included. To facilitate the calculations, a new representation of the vibronic-coupling Hamiltonian for intervalence hole–transfer or electron–transfer problems is introduced, allowing the spectrum to be simulated efficiently using only up to 4×109 vibronic basis functions and leading also to new general analytical relationships. Observed spectra are fitted using seven adjustable chemical parameters describing the interactions between the four electronic states involved. The resulting fits provide unique descriptions of the parameters that are insensitive to the source of the observed spectrum or the nature of the symmetric modes used in the model, and all fitted parameters are found to be close in value to those from independent estimates. We determine the electronic coupling, antisymmetric-mode reorganization energy, and redox asymmetry to be J=0.126±0.002 eV, λ=0.139±0.003 eV, and E0=0.069±0.002 eV, respectively. Our description forms the basis of understanding for a wide range of other properties observed for Rhodobacter sphaeroides mutants, as well as the properties of the reaction centers from photosystems I, II, etc., facilitating a deeper understanding of the role of the special pair in initiating primary charge separation during photosynthesis.

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

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

Conformation transition in the protein of a photosynthetic reaction center observed at the nanometer range of distances at cryogenic temperatures

Electron spin echo (ESE) spectroscopy is applied to study magnetic dipolar interaction between electron spins in the transient P + Q A − radical pairs (P is the primary donor, a bacteriochlorophyll dimer, and Q A is the primary quinone acceptor) and the 3 PQ A − triplet-radical pairs in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides R26. Distance separation in both pairs is about 29 Å. A well-resolved reversible conformational transition of the reaction center protein holding the P and Q A cofactors was observed between 13 and 20 K. This transition results in a narrowing of the distribution of protein conformations with decreasing temperature, with the width of distance distribution between P and Q A dropping from ca. 4 Å at 20 K to ca. 1 Å at 13 K. This transition implies the existence of low barriers in the protein energy landscape and the presence of cooperatively rearranging domains of the size of several nm both in the protein and in the surrounding glassy environment.

A re-examination of spin–orbit coupling in the triplet state of the primary donor in photosynthetic reaction centers

Abstract Recent high-field EPR studies have revealed important differences between the g -matrices of the photoexcited triplet and the cation radical states of the primary donor P in bacterial photosynthetic reaction centers. One of the more unusual differences is the temperature dependence in the g -tensor of the triplet state 3 P, which contrasts with the temperature independence that has been observed in the g -tensor of the cation state P + . This difference leads us to suggest that intersystem (singlet–triplet) spin–orbit coupling could play a significant role in determining the electronic structure of the triplet state, 3 P. Such coupling may also be important for explaining other poorly understood characteristics of 3 P, including the temperature dependence of its zero-field splitting D , and the reduction of D relative to the triplet state in monomeric bacteriochlorophyll. We present a survey of ZFS data from a wide variety of bacteriochlorophyll species that supports this idea. Implications of this model for the electronic structure of the primary donor are discussed.

Lysine substitutions near photoactive cofactors in the bacterial photosynthetic reaction center have opposite effects on the rate of triplet energy transfer

Rates of triplet energy transfer have been investigated by time-resolved pulsed electron paramagnetic resonance (EPR) spectroscopy in mutant photosynthetic reaction centers (RCs) from both Rhodobacter (R.) sphaeroides and R. capsulatus. Mutant RCs of both species harbor lysines in place of native residues of the L subunit at position 178 or 181 near monomeric bacteriochlorophyll BB. Analysis of kinetic measurements acquired throughout a range of temperatures demonstrated an increased rate of triplet transfer in the L178Ser ! Lys (‘‘L178K’’) mutant RCs and a decreased rate in the L181Phe ! Lys (‘‘L181K’’) mutant RCs from both species relative to their respective wild types. Activation energies extracted from Arrhenius plots suggest that triplet transfer in the L178K mutant RC is nearly isoenergetic with wild-type transfer while triplet transfer in the L181K mutant RC requires significantly less activation energy. This latter finding is counter to the observation of slower triplet transfer in the L181K mutant RC. Preliminary structural data offer evidence for direct ligation between the L181 lysine side chain and the central magnesium of BB, but the side chain of L178K is disordered. Our spectroscopic data suggest that these lysine substitutions elicit opposing changes in the relative geometric orientation of BB and that subtle changes in the protein environment can have dramatic and opposing effects on the rates of the photoprotective triplet energy transfer reaction. 2003 Elsevier B.V. All rights reserved.

Probing Local Dynamics of the Photosynthetic Bacterial Reaction Center with a Cysteine Specific Spin Label

A multifrequency electron paramagnetic resonance (EPR) approach was used to probe the dynamic structure of the bacterial photosynthetic reaction center (RC) protein. We have demonstrated that the cysteine specific nitroxide spin label MTSL can be covalently bound to a surface cysteine residue of Rhodobacter sphaeroides RC protein. We suggest that the MTSL nitroxide is bound to an accessible cysteine residue, H156, which is located on the surface of the protein on the H-subunit. Analysis of the multifrequency EPR spectra of the spin-labeled RC proteins suggests the restricted character of the protein dynamics. These dynamics can be described as fast libration in a cone with a correlation time faster than 10-9 s. Several dynamically nonequivalent sites were observed in the EPR spectra, which may reflect distinct conformational substates of local protein structure. This work provides a foundation for future studies with the goal of correlating protein motions with photosynthetic charge-transfer reactions.

Selective excitation in pulsed EPR of a spin-correlated triplet-radical pair

Experiments are described in which a low-amplitude microwave pulse excites only one out of three allowed transitions of the quinone radical (Q A −) in a spin-correlated triplet-radical pair 3 PQ A − of the bacterial photosynthetic reaction center. A second high-amplitude pulse produces a FID whose temporal shape is strongly modulated with frequencies determined by electron–electron dipolar interaction in the pair. The FID is detected in both the in-phase and the out-of-phase channels. The out-of-phase FID is a result of switching off the magnetic dipolar interaction between 3 P and Q A − due to decay of 3 P during the time interval between the two pulses. Refocusing of FID by an additional non-selective pulse allows a dead-time free measurement of this modulation. The influence of the dead-time problem on the distance determination is discussed.

Primary quinone (QA) binding site of bacterial photosynthetic reaction centers: mutations at residue M265 probed by FTIR spectroscopy.

In the primary quinone (Q(A)) binding site of Rb. sphaeroides reaction centers (RCs), isoleucine M265 is in extensive van der Waals contact with the ubiquinone headgroup. Substitution of threonine or serine for this residue (mutants M265IT and M265IS), but not valine (mutant M265IV), lowers the redox midpoint potential of Q(A) by about 100 mV (Takahashi et al. (2001) Biochemistry 40, 1020-1028). The unexpectedly large effect of the polar substitutions is not due to reorientation of the methoxy groups as similar redox potential changes are seen for these mutants with either ubiquinone or anthraquinone as Q(A). Using FTIR spectroscopy to compare Q(A)(-)/Q(A) IR difference spectra for wild type and the M265 mutant RCs, we found changes in the polar mutants (M265IT and M265IS) in the quinone C[double bond]O and C[double bond]C stretching region (1600-1660 cm(-1)) and in the semiquinone anion band (1440-1490 cm(-1)), as well as in protein modes. Modeling the mutations into the X-ray structure of the wild-type RC indicates that the hydroxyl group of the mutant polar residues, Thr and Ser, is hydrogen bonded to the peptide C[double bond]O of Thr(M261). It is suggested that the mutational effect is exerted through the extended backbone region that includes Ala(M260), the hydrogen bonding partner to the C1 carbonyl of the quinone headgroup. The resulting structural perturbations are likely to include lengthening of the hydrogen bond between the quinone C1[double bond]O and the peptide NH of Ala(M260). Possible origins of the IR spectroscopic and redox potential effects are discussed.

The role of the iron–histidine bridge in the early steps of photosynthesis

The role of the iron–histidine bridge in bacterial photosynthetic reaction centres has been investigated by means of ab initio computations and quantum dynamics of elementary reaction steps. Full geometry optimization and wave packet dynamics show that, upon the arrival of a photo-electron, the primary quinone takes up a proton from the H-bonded histidine in a very fast process, which occurs in a few tens of femtoseconds. The proton transfer step significantly stabilizes the charge separated state, inhibiting the backward charge recombination process. Electron transfer to the secondary quinone can then take place by switching the positions of both the H-bonded hydrogens, in a Bjerrium type mechanism involving whole hydrogen atoms rather than protons.

B-side charge separation in bacterial photosynthetic reaction centers: nanosecond time scale electron transfer from HB- to QB.

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.

Sequence analysis reveals new membrane anchor of reaction centre-bound cytochromes possibly related to PufX

Most of the bacterial photosynthetic reaction centres known to date contain a cytochrome subunit with four covalently bound haem groups. In the case of Blastochloris viridis, this reaction centre subunit is anchored in the membrane by a lipid molecule covalently attached to the cysteine which forms the N-terminus of the mature protein after processing by a signal peptidase. We show that posttranslational N-terminal cleavage of the cytochrome subunit does not occur in the aerobic photosynthetic bacterium Roseobacter denitrificans. From sequence analysis of the resulting elongated N-terminus it follows that a transmembrane helix is anchoring the reaction centre-bound cytochrome in the membrane. Comparative sequence analysis strongly suggests that all cytochrome subunits lacking the lipid coupling cysteine share this structural feature. Comparison of the N-terminal segment of the cytochrome subunit of Roseobacter denitrificans with the sequences of the PufX proteins from Rhodobacter sphaeroides and Rhodobacter capsulatus suggests a phylogenetic relation.

Multifrequency Epr and Endor on Transient Radicals and Radical Pairs in Liquid Crystals and Proteins: Structure and Dynamics of Cofactors in Natural and Artifical Photosynthesis

Hotly debated issues in the area of biological electron transfer (ET) involve questions of how long-range ET processes proceed along pathways that incorporate non-covalently linked cofactors interacting with their protein microenvironment. To contribute answers to such questions, time-resolved EPR (TREPR) experiments at various microwave frequency/magnetic field settings have been performed. In this overview, representative examples of this work from our laboratory are given which comprise transient intermediates of light-induced ET in (i) bacterial photosynthetic reaction centers and (ii) biomimetic model complexes. In these complexes the donor and acceptor components are tethered together either covalently via cyclohexenyl bridges or non-covalently via Watson-Crick base-pairing. The studies are aiming at broadening our knowledge of structure-dynamics-function relationships associated with ET processes. It is demonstrated that high-field TREPR opens new perspectives in elucidating complex photochemical ET reactions with different paramagnetic states and species involved.

Structures and Vibrational Frequencies of the Quinones in Rb. sphaeroides Derived by a Combined Density Functional/Molecular Mechanics Approach

The structures of the ubiquinones QA and QB in the bacterial photosynthetic reaction center of Rhodobacter sphaeroides are calculated by a quantum mechanical/molecular mechanics hybrid technique. T...

Energetics of Primary Charge Separation in Bacterial Photosynthetic Reaction Center Mutants: Triplet Decay in Large Magnetic Fields

The triplet state of aromatic molecules forms and decays by intersystem crossing, as originally demonstrated by Kasha and Lewis. By contrast, the triplet state of the primary electron donor, 3P, in photosynthetic reaction centers is formed exclusively by spin- and magnetic-field-dependent charge recombination of the initially formed radical ion pair. 3P decays by intersystem crossing at low temperatures; however, at higher temperatures, it can also decay by activated re-formation of the radical ion pair from which it was born, followed by a spin- and magnetic-field-dependent pathway that leads ultimately to the ground state. The discovery of this activated decay pathway leads to an approach for obtaining information on the relative energies of the radical pair and 3P state (Chidsey et al. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 6850−6854); with knowledge of the absolute energy of 3P from its phosphorescence, the energy of the initial charge separation reaction can be obtained. In this paper, we present the first data on the temperature and magnetic field dependence of the formation and decay of 3P for Rb. sphaeroides reaction center mutants in a background that contains no carotenoid. The mutations have been studied in other contexts and were designed to perturb the redox potential of the primary electron donor or acceptor. The measured trends are in the same direction as expected from chemical intuition; however, the quantitative changes are typically smaller than expected. Possible reasons for this finding are discussed. Improved values are obtained for the enthalpy and free energy change associated with primary charge separation in wild-type reaction centers.

Out-of-Phase Stimulated Electron Spin−Echo Appearing in the Evolution of Spin-Correlated Photosynthetic Triplet-Radical Pairs

A strong out-of-phase stimulated electron spin-echo is observed for the Q A - radical in the spin-correlated triplet-radical pair 3 PQ A - in photosynthetic bacterial reaction centers. The formation of this echo is shown tobe induced by spin polarization of Q A - and by decay of the triplet state. The out-of-phase and in-phase echoes show deep envelope modulation induced by electron-electron dipole interaction between the partners in the pair. The analysis of this modulation provides dipolar frequencies. The interspin distance in 3 PQ A - is shown to be the same as in the radical pair P + Q A -. This new type of experiment appears to be widely applicable for the study of chemical reactions and intermolecular distances in solids.

A cytochrome b origin of photosynthetic reaction centers: an evolutionary link between respiration and photosynthesis.

The evolutionary origin of photosynthetic reaction centers has long remained elusive. Here, we use sequence and structural analysis to demonstrate an evolutionary link between the cytochrome b subunit of the cytochrome bc(1) complex and the core polypeptides of the photosynthetic bacterial reaction center. In particular, we have identified an area of significant sequence similarity between a three contiguous membrane-spanning domain of cytochrome b, which contains binding sites for two hemes, and a three contiguous membrane-spanning domain in the photosynthetic reaction center core subunits, which contains binding sites for cofactors such as (bacterio)chlorophylls, (bacterio)pheophytin and a non-heme iron. Three of the four heme ligands in cytochrome b are found to be conserved with the cofactor ligands in the reaction center polypeptides. Since cytochrome b and reaction center polypeptides both bind tetrapyrroles and quinones for electron transfer, the observed sequence, functional and structural similarities can best be explained with the assumption of a common evolutionary origin. Statistical analysis further supports a distant but significant homologous relationship. On the basis of previous evolutionary analyses that established a scenario that respiration evolved prior to photosynthesis, we consider it likely that cytochrome b is the evolutionary precursor for type II reaction center apoproteins. With a structural analysis confirming a common evolutionary origin of both type I and type II reaction centers, we further propose a novel "reaction center apoprotein early" hypothesis to account for the development of photosynthetic reaction center holoproteins.

Molecular Dynamics of QA-• and QB-• in Photosynthetic Bacterial Reaction Centers Studied by Pulsed High-Field EPR at 95 GHz

The anisotropic transverse relaxation times T2 of the primary and secondary acceptor quinone radical anions QA-• and QB-• in Zn-substituted photosynthetic bacterial reaction centers of Rhodobacter ...