Membrane-bound Reaction Center Research Articles

Identification of a Ubiquinone-Ubiquinol Quinhydrone Complex in Bacterial Photosynthetic Membranes and Isolated Reaction Centers by Time-Resolved Infrared Spectroscopy.

Ubiquinone redox chemistry is of fundamental importance in biochemistry, notably in bioenergetics. The bi-electronic reduction of ubiquinone to ubiquinol has been widely studied, including by Fourier transform infrared (FTIR) difference spectroscopy, in several systems. In this paper, we have recorded static and time-resolved FTIR difference spectra reflecting light-induced ubiquinone reduction to ubiquinol in bacterial photosynthetic membranes and in detergent-isolated photosynthetic bacterial reaction centers. We found compelling evidence that in both systems under strong light illumination-and also in detergent-isolated reaction centers after two saturating flashes-a ubiquinone-ubiquinol charge-transfer quinhydrone complex, characterized by a characteristic band at ~1565 cm-1, can be formed. Quantum chemistry calculations confirmed that such a band is due to formation of a quinhydrone complex. We propose that the formation of such a complex takes place when Q and QH2 are forced, by spatial constraints, to share a common limited space as, for instance, in detergent micelles, or when an incoming quinone from the pool meets, in the channel for quinone/quinol exchange at the QB site, a quinol coming out. This latter situation can take place both in isolated and membrane bound reaction centers Possible consequences of the formation of this charge-transfer complex under physiological conditions are discussed.

High-pressure tuning of primary photochemistry in bacterial photosynthesis: membrane-bound versus detergent-isolated reaction centers.

While photosynthesis thrives at close to normal pressures and temperatures, it is presently well known that life is similarly commonplace in the hostile environments of the deep seas as well as around hydrothermal vents. It is thus imperative to understand how key biological processes perform under extreme conditions of high pressures and temperatures. Herein, comparative steady-state and picosecond time-resolved spectroscopic studies were performed on membrane-bound and detergent-purified forms of a YM210W mutant reaction center (RC) from Rhodobacter sphaeroides under modulating conditions of high hydrostatic pressure applied at ambient temperature. A previously established breakage of the lone hydrogen bond formed between the RC primary donor and the protein scaffold was shown to take place in the membrane-bound RC at an almost 3 kbar higher pressure than in the purified RC, confirming the stabilizing role of the lipid environment for membrane proteins. The main change in the multi-exponential decay of excited primary donor emission across the experimental 10 kbar pressure range involved an over two-fold continuous acceleration, the kinetics becoming increasingly mono-exponential. The fastest component of the emission decay, thought to be largely governed by the rate of primary charge separation, was distinctly slower in the membrane-bound RC than in the purified RC. The change in character of the emission decay with pressure was explained by the contribution of charge recombination to emission decreasing with pressure as a result of an increasing free energy gap between the charge-separated and excited primary donor states. Finally, it was demonstrated that, in contrast to a long-term experimental paradigm, adding a combination of sodium ascorbate and phenazine methosulfate to the protein solution potentially distorts natural photochemistry in bacterial RCs.

Primary Electron Transfer Reactions in Membrane-Bound Open and Closed Reaction Centers from Purple Bacterium Rhodobacter sphaeroides

Most ultrafast transient absorption studies of primary electron transfer in reaction centers from purple bacteria have been performed in complexes isolated from their natural lipid membrane environment using detergent. In this contribution we present near-UV-vis transient absorption studies of reaction centers embedded in their natural membrane environment. The evolution of absorption spectra recorded with subpicosecond resolution and re ecting primarily electron transfer reactions has been compared to data obtained previously for isolated reaction centers. We conclude that the overall spectral evolution in both types of samples is similar, and the environment of the reaction center protein has only a minor e ect on the primary electron transfer reactions. The di erences between the two samples are explained in terms of di erent energetic levels (and their di erent temporal evolution) of the two initial charge separated states PBA and P HA , with P being the primary electron donor and BA and HA the two consecutive electron acceptors. Additionally, in the electric eld generated by P HA , BA in membrane-bound reaction centers undergoes a stronger electrochromic shift than in isolated reaction centers.

Reinterpretation of the electron density at the site of the eighth bacteriochlorophyll in the FMO protein from Pelodictyon phaeum

The Fenna-Matthews-Olson antenna protein from the green bacterium Pelodictyon phaeum mediates the energy transfer from a peripheral antenna complex to the membrane-bound reaction center. The three-dimensional structure of this protein has been previously modeled using X-ray diffraction to a resolution limit of 2.0 Å, with R (work) and R (free) values of 16.6 and 19.9%, respectively (Larson et al., Photosynth Res 107:139-150, 2011). This model shows the protein as consisting of β-sheets surrounding several bacteriochlorophyll cofactors. While most of the model clearly matches the electron density maps, in this paper we re-examine the electron density for a specific feature, namely the eighth bacteriochlorophyll a cofactor. This electron density is now interpreted as arising primarily from the end of an otherwise disordered polyethylene glycol molecule. Additional electron density is present but the density is weak and cannot be unambiguously assigned. The new model has R (work) and R (free) values of 16.2 and 19.0%, respectively.

Maximal Coherence at Room Temperature in the Bacterial Photosynthetic Reaction Center

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

The three-dimensional structure of the FMO protein from Pelodictyon phaeum and the implications for energy transfer

The Fenna-Matthews-Olson (FMO) antenna protein from the green bacterium Pelodictyon phaeum mediates the transfer of energy from the peripheral chlorosome antenna complex to the membrane-bound reaction center. The three-dimensional structure of this protein has been solved using protein crystallography to a resolution limit of 2.0 Å, with R(work) and R(free) values of 16.6 and 19.9%, respectively. The structure is a trimer of three identical subunits related by a threefold symmetry axis. Each subunit has two beta sheets that surround 8 bacteriochlorophylls. The bacteriochlorophylls are all five-coordinated, with the axial ligand being a histidine, serine, backbone carbonyl, or bound water molecule. The arrangement of the bacteriochlorophylls is generally well conserved in comparison to other FMO structures, but differences are apparent in the interactions with the surrounding protein. In this structure the position and orientation of the eighth bacteriochlorophyll is well defined and shows differences in its location and the coordination of the central Mg compared to previous models. The implications of this structure on the ability of the FMO protein to perform energy transfer are discussed in terms of the experimental optical measurements.

Equilibration kinetics in isolated and membrane-bound photosynthetic reaction centers upon illumination: a method to determine the photoexcitation rate

Kinetics of electron transfer, following variation of actinic light intensity, for photosynthetic reaction centers (RCs) of purple bacteria (isolated and membrane-bound) were analyzed by measuring absorbance changes in the primary photoelectron donor absorption band at 865 nm. The bleaching of the primary photoelectron donor absorption band in RCs, following a sudden increase of illumination from the dark to an actinic light intensity of I exp, obeys a simple exponential law with the rate constant (alpha I_{exp } ; + ;k_{text{rec}} ) , in which α is a parameter relating the light intensity, measured in mW/cm2, to a corresponding theoretical rate in units of reciprocal seconds, and k rec is the effective rate constant of the charge recombination in the photosynthetic RCs. In this work, a method for determining the α parameter value is developed and experimentally verified for isolated and membrane-bound RCs, allowing for rigorous modeling of RC macromolecule dynamics under varied photoexcitation conditions. Such modeling is necessary for RCs due to alterations of the forward photoexcitation rates and relaxation rates caused by illumination history and intramolecular structural dynamics effects. It is demonstrated that the classical Bouguer–Lambert–Beer formalism can be applied for the samples with relatively low scattering, which is not necessarily the case with strongly scattering media or high light intensity excitation.

Quantum teleportation across a biological membrane by means of correlated spin pair dynamics in photosynthetic reaction centers

In the process coined quantum teleportation the complete information contained in an input quantum stateΨi is teleported to a distant location at which the original quantum state is regenerated as teleported output stateΨi. This paper presents the proof-of-feasibility concept of a quantum teleportation experiment during which an arbitrary input quantum state is teleported across a biological membrane. As particular aspect it is emphasized that all essential subprocesses of the usual quantum teleportation scheme are suggested to be realized by free running reaction processes in a biological membrane-bound reaction center complex with only one significant adaptation required at the input side. The first process of generation of a spin-correlated (Einstein-Podolsky-Rosen) pair of particles (Bell-state source) is a naturally occurring process realized in photosynthetic reaction centers by the primary processes of light-induced charge separation across the membrane. The second process is the so-called Bell-state measurement, which is able to store the complete information of the input quantum state. It is suggested to be realized by a fast spin-dependent recombination between one pair partner spin and a properly engineered input spin. Under suitable recombination conditions the remaining second pair partner spin, situated at the receiver location on the other side of the membrane, is shown to end up in the quantum state identical to that of the initial input state due to the fixed spin correlation of the Bell-state source and the particular spin selectivity of the recombination process. Thus, the input (spin) quantum state is teleported from the spin near the (electron charge) donor side to the acceptor side of the membrane-bound photosynthetic reaction center complex. A comprehensive discussion is presented for this quantum teleportation concept using photosynthetic reaction centers as the quantum channel of communication. Standard electron paramagnetic resonance techniques can be used to set up the input state and read out or hand over the output state for subsequent quantum information processing.

Scanning Electrochemical Microscopy of the Photosynthetic Reaction Center ofRhodobactersphaeroidesin Different Environmental Systems

The present work uses a scanning electrochemical microscopy technique to study systems containing the membrane-bound reaction center protein (RC) from the purple photosynthetic bacteria Rhodobacter spheroides to chromatophores (spherical reorganization of cell membrane following its mechanical rupture) and liposomes (reconstituted membrane systems at lower degree of complexity). Scanning electrochemical microscopy is a useful tool to investigate redox processes involving a RC, because the effective heterogeneous rate constants for the redox reaction with different mediators can be measured. The technique is also able to provide information on the role of the outer cell membrane permeation on the kinetics of the electron-transfer processes and to obtain more insight into the nature of the species involved.

Bidirectional Electron Transfer in Photosystem I: Direct Evidence from High-Frequency Time-Resolved EPR Spectroscopy

Efficient charge separation occurring within membrane-bound reaction center proteins is the most important step of photosynthetic solar energy conversion. All reaction centers are classified into two types, I and II. X-ray crystal structures reveal that both types bind two symmetric membrane-spanning branches of potential electron-transfer cofactors. Determination of the functional roles of these pairs of branches is of fundamental importance. While it is established that in type II reaction centers only one branch functions in electron transfer, we present the first direct spectroscopic evidence that both cofactor branches are active in the type I reaction center, photosystem I.

The structure and function of the cytochrome c2: reaction center electron transfer complex from Rhodobacter sphaeroides

In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c2 (cyt c2) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c2 (cyt c2) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c2 is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association.

Light-Induced Equilibration Kinetics in Membrane-Bound Photosynthetic Reaction Centers: Nonlinear Dynamic Effects in Multiple Scattering Media

Nonequilibrium dynamic effects in membrane-bound reaction centers (RCs) from photosynthetic bacteria are studied for the first time. We show that the accumulation of slow conformational changes triggered by charge-separation events in the RCs control system dynamics and depend on illumination conditions in a way similar to that for isolated RCs. The light-induced, transient kinetics of membrane-bound RCs are described using a model of electron-conformational transitions governed by system diffusion along the surface of a double-well, effective adiabatic potential. The light-triggered conformational transitions in the chromatophores are estimated to occur at an actinic light intensity at least 10 times lower than that needed for conformational transitions in isolated RCs. This finding is attributed to efficient, multiple-scattering effects that occur in sample with membranes. Related optical properties of the chromatophores as a disordered system with multiple light scattering are characterized for the first time.

Multiple pathways for ultrafast transduction of light energy in the photosynthetic reaction center of Rhodobacter sphaeroides.

A pathway of electron transfer is described that operates in the wild-type reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides. The pathway does not involve the excited state of the special pair dimer of bacteriochlorophylls (P*), but instead is driven by the excited state of the monomeric bacteriochlorophyll (BA*) present in the active branch of pigments along which electron transfer occurs. Pump-probe experiments were performed at 77 K on membrane-bound RCs by using different excitation wavelengths, to investigate the formation of the charge separated state P+HA-. In experiments in which P or BA was selectively excited at 880 nm or 796 nm, respectively, the formation of P+HA- was associated with similar time constants of 1.5 ps and 1. 7 ps. However, the spectral changes associated with the two time constants are very different. Global analysis of the transient spectra shows that a mixture of P+BA- and P* is formed in parallel from BA* on a subpicosecond time scale. In contrast, excitation of the inactive branch monomeric bacteriochlorophyll (BB) and the high exciton component of P (P+) resulted in electron transfer only after relaxation to P*. The multiple pathways for primary electron transfer in the bacterial RC are discussed with regard to the mechanism of charge separation in the RC of photosystem II from higher plants.

Linear dichroism of membrane-bound reaction centers from Rhodobacter sphaeroides: Alterations of the B band induced by site-specific mutations

Low temperature absorption and linear dichroism (LD) measurements were performed on oriented membranes containing wild type Rhodobacter sphaeroides reaction centers, a mutant reaction center with the change Phe M197 to Arg (FM197R), and a double mutant reaction center where, in addition, Gly M203 was replaced by Asp (FM197R/GM203D). The monomeric bacteriochlorophyll band (B), which is highly congested in the wild type reaction center, was separated into two bands in the mutant reaction centers peaking 10 nm (single mutant) or 15 nm (double mutant) apart. This separation arose principally from changes in the interaction of the protein with the L-side monomer bacteriochlorophyll BL.The ability to separate the B bands is extremely useful in spectroscopic studies. The orientations of the two monomer-type transitions contributing to the B band were similar in all three reaction centres studied, and were asymmetric with respect to the orientation axis, with the transition mostly associated with BL making a smaller angle with the C2 axis. Differences in the LD observed in wild type membrane-bound or isolated reaction centers can be ascribed either to differences in shifts of the B transitions or to differences in the orientation axis.

Primary charge separation processes in reaction centers of an antenna-free mutant of Rhodobacter capsulatus

The primary charge separation processes of membrane-bound reaction centers from an antenna-free mutant of the purple bacterium Rhodobacter capsulatus have been investigated by picosecond time-resolved fluorescence and femtosecond stimulated emission measurements. Target analysis has been applied to decompose the time-resolved fluorescence emission spectrum into its components for open and closed (quinone-reduced) reaction centers. The reaction center environment, i.e. membrane vs. detergent, exerts a remarkable influence on the primary charge separation kinetics. For fully open reaction centers the main component of the primary electron transfer process from the excited special pair to bacteriopheophytin — measured both in fluorescence and in femtosecond stimulated emission — takes 5–6 ps, which is a factor of nearly two longer than in the corresponding detergent-isolated reaction centers (2.7 ps). For quinone-reduced RCs a very long charge separation time of approx. 10 ps is observed, concomitant with a more than five-fold increase in fluorescence yield. All of these parameters differ substantially from those of detergent-isolated RCs. Possible origins for the observed kinetics are discussed.

Primary Electron Transfer in Membrane-Bound Reaction Centers with Mutations at the M210 Position

The kinetics of primary electron transfer in membrane-bound Rhodobacter sphaeroides reaction centers (RCs) were measured on both wild-type (WT) and site-directed mutant RC's bearing mutations at the tyrosine M210 position. The tyrosine was replaced by histidine (H), phenylalanine (F), leucine (L), or tryptophan (W). A mutant with histidine at both the M210 and symmetry-related L181 positions (YM210H/FL181H) was also examined. Rates of primary charge separation were determined by both single and multiple wavelength pump−probe techniques. The time constants for the decay of stimulated emission in the membrane-bound mutant RC's were approximately 27 ps (F), 36 ps (L), 72 ps (W), 5.8 ps (H), and 4.2 ps (HH), compared with 4.6 ps in WT membrane-bound RC's. For all RC's, the decay of stimulated emission was found to be multiexponential, demonstrating that this phenomenon is not a consequence of the removal of the RC from the membrane. The source of the multiexponential decay of the primary donor excited state w...

P-E1-16 Effect of background illumination on the P +Q A− recombination in membrane bound reaction centers of Rhodobacter sphaeroides: Van Brederode ME 1, Van Stokkum IHM 1, Jones MR 2, Van Grondelle R 1. 1 Dept. of Physics and Astronomy Vrije Universiteit. Amsterdam (NL). 2 Dept. of Molecular Biology and Biotechnology, University of Sheffield (UK)

P-E1-16 Effect of background illumination on the P +Q A− recombination in membrane bound reaction centers of Rhodobacter sphaeroides: Van Brederode ME 1, Van Stokkum IHM 1, Jones MR 2, Van Grondelle R 1. 1 Dept. of Physics and Astronomy Vrije Universiteit. Amsterdam (NL). 2 Dept. of Molecular Biology and Biotechnology, University of Sheffield (UK)

Time-resolved and steady-state spectroscopic analysis of membrane-bound reaction centers from Rhodobacter sphaeroides: comparisons with detergent-solubilized complexes.

The spectroscopic analysis of the antenna-deficient Rhodobacter sphaeroides strain RCO1 has been extended to an investigation of the kinetics and spectroscopy of primary charge separation. Global analysis of time-resolved difference spectra demonstrated that the rate of charge separation in membrane-bound reaction centers is slightly slower than in detergent-solubilized reaction centers from the same strain. A kinetic analysis of the decay of the primary donor excited state at single wavelengths was carried out using a high repetition rate laser system, with the reaction centers being maintained in the open state using a combination of phenazine methosulfate and horse heart cytochrome c. The kinetics of primary charge separation in both membrane-bound and solubilized reaction centers were found to be non-monoexponential, with two exponential decay components required for a satisfactory description of the decay of the primary donor excited state. The overall rate of charge separation in membrane-bound reaction centers was slowed if the primary acceptor quinone was reduced using sodium ascorbate. This slowing was caused, in part, by an increase in the relative amplitude of the slower of the two exponential components. The acceleration in the rate of charge separation observed on removal of the reaction center from the membrane did not appear to be caused by a significant change in the electrochemical properties of the primary donor. The influence of the environment of the reaction center on primary charge separation is discussed together with the origins of the non-monoexponential decay of the primary donor excited state.

Coherent dynamics during the primary electron-transfer reaction in membrane-bound reaction centers of Rhodobacter sphaeroides.

The temporal evolution of the near-infrared stimulated emission band of the special pair excited state (P*) in the reaction center of Rhodobacter sphaeroides has been studied in intracytoplasmic membranes of the antenna-deficient RCO1 mutant at 10 K with a resolution of 30 fs. On the 100-fs time scale the emission band gradually shifts to longer wavelengths. After 150 fs the band shifts back to shorter wavelengths and continues to develop on the picosecond time scale in a damped oscillatory manner (most prominent fundamental frequencies around 15 cm-1 and at 92, 122, and 153 cm-1). These phenomena are shown to be due to low-frequency vibrational motions in the P* excited state that conserve their phase on the time scale of electron transfer. These results imply that the vibrational manifold of P* is not thermalized during the electron-transfer reaction in functional reaction centers. The initial Stokes shift dynamics are largely determined by the modes in the 90-160-cm-1 frequency range, which probably involve motions of several chromophores, including the bacteriopheophytin electron acceptor HL.

Time-resolved spectroscopy of the primary photosynthetic processes of membrane-bound reaction centers from an antenna-deficient mutant of Rhodobacter capsulatus

The primary photosynthetic reactions in whole membranes of the antenna-deficient mutant strain U43 (pTXA6–10) of Rhodobacter capsulatus are studied by transient absorption and emission spectroscopy with subpicosecond time resolution. Extensive similarities between the transient absorption data on whole membranes and on isolated reaction centers support the idea that the primary processes in isolated reaction centers are not modified by the isolation procedure.