Reaction Centers Of Rhodobacter Sphaeroides Research Articles

Contribution of Protonation to the Dielectric Relaxation Arising from Bacteriopheophytin Reductions in the Photosynthetic Reaction Centers of Rhodobacter sphaeroides

The pH dependence of the free energy level of the flash-induced primary charge pair P+IA− was determined by a combination of the results from the indirect charge recombination of P+QA− and from the delayed fluorescence of the excited dimer (P*) in the reaction center of the photosynthetic bacterium Rhodobacter sphaeroides, where the native ubiquinone at the primary quinone binding site QA was replaced by low-potential anthraquinone (AQ) derivatives. The following observations were made: (1) The free energy state of P+IA− was pH independent below pH 10 (–370 ± 10 meV relative to that of the excited dimer P*) and showed a remarkable decrease (about 20 meV/pH unit) above pH 10. A part of the dielectric relaxation of the P+IA− charge pair that is not insignificant (about 120 meV) should come from protonation-related changes. (2) The single exponential decay character of the kinetics proves that the protonated/unprotonated P+IA− and P+QA− states are in equilibria and the rate constants of protonation konH +koffH are much larger than those of the charge back reaction kback ~103 s−1. (3) Highly similar pH profiles were measured to determine the free energy states of P+QA− and P+IA−, indicating that the same acidic cluster at around QB should respond to both anionic species. This was supported by model calculations based on anticooperative proton distribution in the cluster with key residues of GluL212, AspL213, AspM17, and GluH173, and the effect of the polarization of the aqueous phase on electrostatic interactions. The larger distance of IA− from the cluster (25.2 Å) compared to that of QA− (14.5 Å) is compensated by a smaller effective dielectric constant (6.5 ± 0.5 and 10.0 ± 0.5, respectively). (4) The P* → P+QA− and IA−QA → IAQA− electron transfers are enthalpy-driven reactions with the exemption of very large (>60%) or negligible entropic contributions in cases of substitution by 2,3-dimethyl-AQ or 1-chloro-AQ, respectively. The possible structural consequences are discussed.

Femtosecond Dynamics of the Excited Primary Electron Donor in Reaction Centers of the Purple Bacterium Rhodobacter sphaeroides.

Femtosecond transient absorption spectroscopy was used to study the dynamics of the excited primary electron donor in the reaction centers of the purple bacterium Rhodobacter sphaeroides. Using global analysis and the interval method, we found a correlation between the vibrational coherence damping of the excited primary electron donor and the lifetime of the charge-separated stateP+BA-, indicating the reversibility of electron transfer to the primary electron acceptor, theBA molecule. In the reaction centers, the signs of superposition of two electronic states of P were found for a delay time of less than 200fs. It is suggested that the admixture value of the charge transfer statePA+PB- with the excited primary electron donorP* is about 24%. The results obtained are discussed in terms of the two-step electron transfer mechanism.

Delocalized electronic excitations and their role in directional charge transfer in the reaction center of Rhodobacter sphaeroides.

In purple bacteria, the fundamental charge-separation step that drives the conversion of radiation energy into chemical energy proceeds along one branch-the A branch-of a heterodimeric pigment-protein complex, the reaction center. Here, we use first principles time-dependent density functional theory (TDDFT) with an optimally-tuned range-separated hybrid functional to investigate the electronic and excited-state structure of the six primary pigments in the reaction center of Rhodobacter sphaeroides. By explicitly including amino-acid residues surrounding these six pigments in our TDDFT calculations, we systematically study the effect of the protein environment on energy and charge-transfer excitations. Our calculations show that a forward charge transfer into the A branch is significantly lower in energy than the first charge transfer into the B branch, in agreement with the unidirectional charge transfer observed experimentally. We further show that the inclusion of the protein environment redshifts this excitation significantly, allowing for energy transfer from the coupled Qx excitations. Through analysis of transition and difference densities, we demonstrate that most of the Q-band excitations are strongly delocalized over several pigments and that both their spatial delocalization and charge-transfer character determine how strongly affected they are by thermally-activated molecular vibrations. Our results suggest a mechanism for charge-transfer in this bacterial reaction center and pave the way for further first-principles investigations of the interplay between delocalized excited states, vibronic coupling, and the role of the protein environment in this and other complex light-harvesting systems.

High Yield of B-Side Electron Transfer at 77 K in the Photosynthetic Reaction Center Protein from Rhodobacter sphaeroides.

The primary electron transfer (ET) processes at 295 and 77 K are compared for the Rhodobacter sphaeroides reaction center (RC) pigment-protein complex from 13 mutants including a wild-type control. The engineered RCs bear mutations in the L and M polypeptides that largely inhibit ET from the excited state P* of the primary electron donor (P, a bacteriochlorophyll dimer) to the normally photoactive A-side cofactors and enhance ET to the C2-symmetry related, and normally photoinactive, B-side cofactors. P* decay is multiexponential at both temperatures and modeled as arising from subpopulations that differ in contributions of two-step ET (e.g., P* → P+BB- → P+HB-), one-step superexchange ET (e.g., P* → P+HB-), and P* → ground state. [HB and BB are monomeric bacteriopheophytin and bacteriochlorophyll, respectively.] The relative abundances of the subpopulations and the inherent rate constants of the P* decay routes vary with temperature. Regardless, ET to produce P+HB- is generally faster at 77 K than at 295 K by about a factor of 2. A key finding is that the yield of P+HB-, which ranges from ∼5% to ∼90% among the mutant RCs, is essentially the same at 77 K as at 295 K in each case. Overall, the results show that ET from P* to the B-side cofactors in these mutants does not require thermal activation and involves combinations of ET mechanisms analogous to those operative on the A side in the native RC.

On the Mechanism of Selective Chemical Exchange of Bacteriopheophytins in the Reaction Centers of Rhodobacter sphaeroides R-26.

To elucidate the mechanism of site-selective chemical replacement of chromophores in the reaction centers (RCs) of photosynthetic bacteria by external pigments, we investigated how the efficiency of incorporation of plant pheophytin a (Pheo) into the binding sites for bacteriopheophytin a molecules (BPheo) in the isolated Rhodobacter sphaeroides R-26 RCs depended on the incubation medium temperature, Pheo aggregation state, and the presence of organic solvent (acetone). When Pheo was in a form of monomers in free detergent micelles in a water-detergent incubation medium, the degree of selective replacement of photochemically inactive BPheo HB molecules upon incubation of the RC/Pheo mixture at 5°C was ~15%. The exchange efficiency increased to 40% upon incubation at 25°C and reached 100% at the same temperature when 10% acetone was added to the incubation medium. At both 5 and 25°C, the degree of pigment exchange increased approximately twice, when a mixture of Pheo monomers and dimers in the presence of 10% acetone was used as the incubation medium. The removal of acetone from this medium with the preservation of pigment forms led to a significant decrease in the efficiency of Pheo incorporation. The effect of acetone on the pigment exchange was also observed at an elevated incubation temperature (43.5°C), when functionally active BPheo HA molecules were partially replaced. The results are discussed in terms of the mechanism according to which (i) the temperature-dependent internal movements of the RC protein facilitate the release of the BPheo molecule from the binding site with simultaneous insertion of the Pheo molecule into the same site in a coupled process, (ii) the role of temperature largely depends on the steric accessibility of binding pockets in the RC protein, (iii) the incorporation of Pheo occurs from a pool of monomeric molecules included in the RC-detergent micelles, and (iv) the presence of acetone in the incubation medium facilitates the exchange of Pheo monomers between micelles in the solution and the detergent belt of the RC complex.

Reaction Center of Rhodobacter Sphaeroides, a Photoactive Protein for pH Sensing: A Theoretical Investigation of Charge Transport Properties

In the perspective of an increasing attention to ecological aspects of science and technology, it is of interest to design devices based on architectures of modular, low cost, and low-pollutant elements, each of them able to perform simple duties. Elemental devices may be themselves green as, for example, proteins able to make simple actions, like sensing. To this aim, photosensitive proteins are often considered because of the possibility of transferring their specific reaction to visible light into electronic signals. Here, we investigate the expected electrical response of the photoactive protein Reaction Center (bRC) of Rhodobacter Sphaeroides within the proteotronics, a recent branch of molecular electronics that evaluates the electrical properties of a protein by using an impedance network protein analog based on the protein tertiary structure and the degree of electrical connectivity between neighboring amino acids. To this purpose, the linear and nonlinear regimes of the electrical response to an applied bias are studied when the protein is in its native state or in an active state. In the linear response regime, results evidence a significant difference in the electrical properties of bRC when the pH value of the solution in which the protein is embedded changes from acid to basic. In the non-linear response regime, the current-voltage characteristics experimentally reported in the recent literature are interpreted in terms of a sequential tunneling mechanism of charge transfer. The qualitative agreement of present findings with available experiments strongly suggests the use of this protein as a bio-rheostat or a pH sensor.

A pH-based bio-rheostat: A proof-of-concept

Biological matter is attracting increasing attention because it shows innovative features that have found several applications in technology, from highly sensitive sensors for medical treatments to devices for energy harvesting. Furthermore, most of its phenomenology remains unclear, thus giving hints for speculative investigations. In this Letter, we explore the possibility to use a well-known photosensitive protein, the Reaction Center of Rhodobacter sphaeroides, to build up an electrical pH sensor, i.e., a device able to change its resistance depending on the pH of the solution in which it crystalizes. By using a microscopic model previously tested on analog proteins, we investigate the electrical response of the reaction center single protein under different conditions of applied bias, showing the feasibility of the bio-rheostat hypothesis. As a matter of fact, the calculated resistance of this protein grows about 100% when going from a pH = 10 to a pH = 6.5. Moreover, calculations of the current voltage characteristics well agree with available experiments performed with current atomic force microscopy under neutral conditions. All findings are in qualitative agreement with the known role of pH in biochemical activities of reaction center and similar proteins, therefore supporting a proof-of-concept for the development of innovative electron devices based on biomaterials.

Recent Advances Towards Sustainable Materials and Processes for Energy Conversion and Storage

Recent Advances Towards Sustainable Materials and Processes for Energy Conversion and Storage

Antagonistic Effects of Point Mutations on Charge Recombination and a New View of Primary Charge Separation in Photosynthetic Proteins

Light-induced electron-transfer reactions were investigated in wild-type and three mutant Rhodobacter sphaeroides reaction centers with the secondary electron acceptor (ubiquinone QA) either removed or permanently reduced. Under such conditions, charge separation between the primary electron donor (bacteriochlorophyll dimer, P) and the electron acceptor (bacteriopheophytin, HA) was followed by P+HA– → PHA charge recombination. Two reaction centers were used that had different single amino-acid mutations that brought about either a 3-fold acceleration in charge recombination compared to that in the wild-type protein, or a 3-fold deceleration. In a third mutant in which the two single amino-acid mutations were combined, charge recombination was similar to that in the wild type. In all cases, data from transient absorption measurements were analyzed using similar models. The modeling included the energetic relaxation of the charge-separated states caused by protein dynamics and evidenced the appearance of an intermediate charge-separated state, P+BA–, with BA being the bacteriochlorophyll located between P and HA. In all cases, mixing of the states P+BA– and P+HA– was observed and explained in terms of electron delocalization over BA and HA. This delocalization, together with picosecond protein relaxation, underlies a new view of primary charge separation in photosynthesis.

Temperature dependence of nanosecond charge recombination in mutant Rhodobacter sphaeroides reaction centers: modelling of the protein dynamics

We investigated the influence of a range of factors—temperature, redox midpoint potential of an electron carrier, and protein dynamics—on nanosecond electron transfer within a protein. The model reaction was back electron transfer from a bacteriopheophytin anion, HA−, to an oxidized primary electron donor, P+, in a wild type Rhodobacter sphaeroides reaction center (RC) with a permanently reduced secondary electron acceptor (quinone, QA−). Also used were two modified RCs with single amino acid mutations near the monomeric bacteriochlorophyll, BA, located between P and HA. Both mutant RCs showed significant slowing down of this back electron transfer reaction with decreasing temperature, similar to that observed with the wild type RC, but contrasting with a number of single point mutant RCs studied previously. The observed similarities and differences are explained in the framework of a (P+BA− ↔ P+HA−) equilibrium model with an important role played by protein relaxation. The major cause of the observed temperature dependence, both in the wild type RC and in the mutant proteins, is a limitation in access to the thermally activated pathway of charge recombination via the state P+BA− at low temperatures. The data indicate that in all RCs both charge recombination pathways, the thermally activated one and a direct one without involvement of the P+BA− state, are controlled by the protein dynamics. It is concluded that the modifications of the protein environment affect the overall back electron transfer kinetics primarily by changing the redox potential of BA and not by changing the protein relaxation dynamics.

Effect of Detergents and Osmolytes on Thermal Stability of Native and Mutant Rhodobacter sphaeroides Reaction Centers.

Photosynthetic reaction center (RC) of the purple bacterium Rhodobacter sphaeroides is one of the most well-studied transmembrane pigment-protein complexes. It is a relatively stable protein with established conditions for its isolation from membranes, purification, and storage. However, it has been shown that some amino acid substitutions can affect stability of the RC, which results in a decrease of the RCs yield during its isolation and purification, disturbs spectral properties of the RCs during storage, and can lead to sample heterogeneity. To optimize conditions for studying mutant RCs, the effect of various detergents and osmolytes on thermal stability of the complex was examined. It was shown that trehalose and, to a lesser extent, sucrose, maltose, and hydroxyectoin at 1 M concentration slow down thermal denaturation of RCs. Sodium cholate was found to have significant stabilizing effect on the structure of native and genetically modified RCs. The use of sodium cholate as a detergent has several advantages and can be recommended for the storage and investigation of the unstable mutant membrane complexes of purple bacteria in long-term experiments.

Dynamic band-shift signal in two-dimensional electronic spectroscopy: A case of bacterial reaction center.

Optical nonlinear spectroscopies carry a high amount of information about the systems under investigation; however, as they report polarization signals, the resulting spectra are often congested and difficult to interpret. To recover the landscape of energy states and physical processes such as energy and electron transfer, a clear interpretation of the nonlinear signals is prerequisite. Here, we focus on the interpretation of the electrochromic band-shift signal, which is generated when an internal electric field is established in the system following optical excitation. Whereas the derivative shape of the band-shift signal is well understood in transient absorption spectroscopy, its emergence in two-dimensional electronic spectroscopy (2DES) has not been discussed. In this work, we employed 2DES to follow the dynamic band-shift signal in reaction centers of purple bacteria Rhodobacter sphaeroides at 77 K. The prominent two-dimensional derivative-shape signal appears with the characteristic formation time of the charge separated state. To explain and characterize the band-shift signal, we use expanded double-sided Feynman diagram formalism. We propose to distinguish two types of Feynman diagrams that lead to signals with negative amplitude: excited state absorption and re-excitation. The presented signal decomposition and modeling analysis allows us to recover precise electrochromic shifts of accessory bacteriochlorophylls, identify additional signals in the B band range, and gain a further insight into the electron transfer mechanism. In a broader perspective, expanded Feynman diagram formalism will allow for interpretation of all 2D signals in a clearer and more intuitive way and therefore facilitate studying the underlying photophysics.

Polydopamine/Ethylenediamine Nanoparticles Embedding a Photosynthetic Bacterial Reaction Center for Efficient Photocurrent Generation

AbstractPhotoactive biohybrid soft nanoparticles are obtained by embedding the Rhodobacter sphaeroides Reaction Center (RC) in polydopamine (PDA) suspended aggregates and treating them with ethylenediamine (EDA). Such PDA:EDA@RC nanoparticles are investigated for photocurrent generation in photoelectrochemical cells, which are able to convert sunlight into electrical energy. The photosynthetic protein retains its structural and functional integrity in the nanostructures and the PDA:EDA@RC nanoparticles exhibit better water dispersity, improved light collection ability, and higher photocurrent generation compared to the PDA@RC precursors, where the reaction center is embedded in pure PDA. The hybrid soft nanoparticles incorporating the RC bacterial photoenzyme show charge separated state generation comparable to that of the pristine enzyme in solution, overcoming the main limitation of RC encapsulation in pure PDA, which is the polymer low light transmission ability. As a consequence, photocurrents obtained by RC within the PDA:EDA environment are almost doubled with respect to the PDA@RC. The biohybrid composites described here represent an interesting example of effective functional nanostructures for sunlight photoconversion based on a biological component addressed in a tunable biocompatible polymer composite, also showing the potential of fine chemical tailoring of polydopamine biointerfaces.

Insight into Electron Transfer from a Redox Polymer to a Photoactive Protein.

Biohybrid photoelectrochemical systems in photovoltaic or biosensor applications have gained considerable attention in recent years. While the photoactive proteins engaged in such systems usually maintain an internal charge separation quantum yield of nearly 100%, the subsequent steps of electron and hole transfer beyond the protein often limit the overall system efficiency and their kinetics remain largely uncharacterized. To reveal the dynamics of one of such charge-transfer reactions, we report on the reduction of Rhodobacter sphaeroides reaction centers (RCs) by Os-complex-modified redox polymers (P-Os) characterized using transient absorption spectroscopy. RCs and P-Os were mixed in buffered solution in different molar ratios in the presence of a water-soluble quinone as an electron acceptor. Electron transfer from P-Os to the photoexcited RCs could be described by a three-exponential function, the fastest lifetime of which was on the order of a few microseconds, which is a few orders of magnitude faster than the internal charge recombination of RCs with fully separated charge. This was similar to the lifetime for the reduction of RCs by their natural electron donor, cytochrome c2. The rate of electron donation increased with increasing ratio of polymer to protein concentrations. It is proposed that P-Os and RCs engage in electrostatic interactions to form complexes, the sizes of which depend on the polymer-to-protein ratio. Our findings throw light on the processes within hydrogel-based biophotovoltaic devices and will inform the future design of materials optimally suited for this application.

Analysis of the electronic structure of the primary electron donor of photosystem I of <i>Spirodela</i> <i>oligorrhiza</i> by photochemically induced dynamic nuclear polarization (photo-CIDNP) solid-state nuclear magnetic resonance (NMR)

Abstract. The electron donor in photosystem I (PSI), the chlorophyll dimer P700, is studied by photochemically induced dynamic nuclear polarization (photo-CIDNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) on selectively 13C and uniformly 15N labeled PSI core preparations (PSI-100) obtained from the aquatic plant duckweed (Spirodela oligorrhiza). Light-induced signals originate from the isotope-labeled nuclei of the cofactors involved in the spin-correlated radical pair forming upon light excitation. Signals are assigned to the two donor cofactors (Chl a and Chl a') and the two acceptor cofactors (both Chl a). Light-induced signals originating from both donor and acceptor cofactors demonstrate that electron transfer occurs through both branches of cofactors in the pseudo-C2 symmetric reaction center (RC). The experimental results supported by quantum chemical calculations indicate that this functional symmetry occurs in PSI despite similarly sized chemical shift differences between the cofactors of PSI and the functionally asymmetric special pair donor of the bacterial RC of Rhodobacter sphaeroides. This contributes to converging evidence that local differences in time-averaged electronic ground-state properties, over the donor are of little importance for the functional symmetry breaking across photosynthetic RC species.

Mutation H(M202)L does not lead to the formation of a heterodimer of the primary electron donor in reaction centers of Rhodobacter sphaeroides when combined with mutation I(M206)H

In photosynthetic reaction centers (RCs) of purple bacteria, conserved histidine residues [His L173 and His M202 in Rhodobacter (Rba.) sphaeroides] are known to serve as fifth axial ligands to the central Mg atom of the bacteriochlorophyll (BChl) molecules (PA and PB, respectively) that constitute the homodimer (BChl/BChl) primary electron donor P. In a number of previous studies, it has been found that replacing these residues with leucine, which cannot serve as a ligand to the Mg ion of BChl, leads to the assembly of heterodimer RCs with P represented by the BChl/BPheo pair. Here, we show that a homodimer P is assembled in Rba. sphaeroides RCs if the mutation H(M202)L is combined with the mutation of isoleucine to histidine at position M206 located in the immediate vicinity of PB. The resulting mutant H(M202)L/I(M206)H RCs are characterized using pigment analysis, redox titration, and a number of spectroscopic methods. It is shown that, compared to wild-type RCs, the double mutation causes significant changes in the absorption spectrum of the P homodimer and the electronic structure of the radical cation P+, but has only minor effect on the pigment composition, the P/P+ midpoint potential, and the initial electron-transfer reaction. The results are discussed in terms of the nature of the axial ligand to the Mg of PB in mutant H(M202)L/I(M206)H RCs and the possibility of His M202 participation in the previously proposed through-bond route for electron transfer from the excited state P* to the monomeric BChl BA in wild-type RCs.

New approach for membrane protein reconstitution into peptidiscs and basis for their adaptability to different proteins.

Previously we introduced peptidiscs as an alternative to detergents to stabilize membrane proteins in solution (Carlson et al., 2018). Here, we present 'on-gradient' reconstitution, a new gentle approach for the reconstitution of labile membrane-protein complexes, and used it to reconstitute Rhodobacter sphaeroides reaction center complexes, demonstrating that peptidiscs can adapt to transmembrane domains of very different sizes and shapes. Using the conventional 'on-bead' approach, we reconstituted Escherichia coli proteins MsbA and MscS and find that peptidiscs stabilize them in their native conformation and allow for high-resolution structure determination by cryo-electron microscopy. The structures reveal that peptidisc peptides can arrange around transmembrane proteins differently, thus revealing the structural basis for why peptidiscs can stabilize such a large variety of membrane proteins. Together, our results establish the gentle and easy-to-use peptidiscs as a potentially universal alternative to detergents as a means to stabilize membrane proteins in solution for structural and functional studies.

In situ spectroelectrochemical investigation of a biophotoelectrode based on photoreaction centers embedded in a redox hydrogel

The field of biophotoelectrochemistry and its application in biophotovoltaics and biosensors has gained more and more attention in recent years. Knowledge of the redox potentials of the catalytically active protein cofactors in biophotovoltaic devices is crucial for accurate modelling and in discerning the mechanisms of their operation. Here, for the first time, we used spectroelectrochemical methods to investigate thermodynamic parameters of a biophotoelectrode in situ. We determined redox potentials of two elements of the system: the primary electron donor in photosynthetic reaction centers (RCs) of the bacterium Rhodobacter sphaeroides and osmium-complex based redox mediators that are bound to a hydrogel matrix. We observe that the midpoint potential of the primary donor is shifted towards more positive potentials in comparison to literature data for RCs solubilized in buffered water solution, likely due to interaction with the polymer matrix. We also demonstrate that the osmium-complex modified redox polymer efficiently wires the RCs to the electrode, maintaining a high Internal Quantum Efficiency with approximately one electron per two photons generated (IQE=50±12%). Overall, this biophotoelectrode may be attractive for controlling the redox state of the protein when performing other types of experiments, e.g. time resolved absorption or fluorescence measurements, in order to gain insights into kinetic limitations and thereby help in the rational design of bioelectronic devices.

Analysis are of the hidden properties of the macromolecular system as an example of the reaction centers of bacteria Rhodobacter sphaeroides

Relevant: Is the study of the response of biological macromolecules to external stimuli. Often the reaction of macromolecules has an effect of structural self-regulation. In this case, their reaction is not only external infl uence, but also the spatial-temporal motions of the macromolecule. In this situation deserves the attention of electronic-conformational interactions of macromolecules.

Use of a system of differential equations to analyze the functioning of a catalytic bio macromolecule under non equilibrium conditions

The aim of the work is to analyze the response of a biomolecule to an external influence based on the study of its hidden states by identifying differential equations with constant coefficients.The relevance of the work lies in the fact that often the main reaction of an object to an external action can be represented as a sum of various exponential functions with a common starting point and a material balance equation. In this case, the response of an object to an external action corresponds to a system of differential equations with constant coefficients. This character of the main reaction may be due to the influence of the hidden properties of the object, which play the role of regulatory parameters.The problem is that the hidden factors and the system of differential equations are not identified. As an object, isolated reaction centers (RC) of the bacteria Rhodobacter sphaeroides, which possess the above properties, has used. Their structure is well studied.As result of studying of photo excitation processes of the reaction center has shown that electron transfer kinetics (the main reaction) can be approximated by three normalized exponential functions. Program was developed to identify for four differential equations of electron transfer and the balance equation, the behavior of hidden states of the reaction center.It was concluded that time the dependence the probability density of finding an electron in different conformational states of the reaction center characterizes the space-time changes in the structure of the reaction center.