Bacterial Photosynthetic Reaction Centers Research Articles

Lel A. Drachev and the direct electrometric method

In the bioenergetics studies, the direct electrometric method played an important role. This method is based on measuring the electrical potential difference (Δψ) between two compartments of the experimental cell generated by some membrane proteins. These proteins are incorporated into closed lipid-protein membrane vesicles associated with an artificial lipid membrane that separates the compartments. The very existence of such proteins able to generate Δψ was one of the consequences of Peter Mitchell’ s chemiosmotic concept. The discovery and investigation of their functioning contributed to the recognition of this concept and, eventually the well-deserved awarding of the Nobel Prize to P. Mitchell. Lel A. Drachev (1926-2022) was one of the main authors of the direct electrometrical method. With his participation, key studies were carried out on the electrogenesis of photosynthetic and respiratory membrane proteins, including bacteriorhodopsin, visual rhodopsin, photosynthetic bacterial reaction centers, cytochrome oxidase and others.

Homogeneous Dephasing in Photosynthetic Bacterial Reaction Centers: Time Correlation Function Approach.

A new tractable linear electronic transition dipole moment time correlation function (ETDMTCF) that accurately accounts for electronic dephasing, asymmetry, and width of 1-phonon profile, which the zero-phonon line (ZPL) contributes to it, in Rhodopseudomonas viridis bacterial reaction center is derived. This time correlation function proves to be superior to other frequency-domain expressions in case of strong electron-phonon coupling (which is often the case in bacterial RCs and pigment-protein complexes), many vibrational modes involved, and high temperature, whereby more vibronic and electronic (sequence) transitions would arise. The Fourier transform of this ETDMTCF leads to asymmetric multiphonon profiles composed of Lorentzian distribution and Gaussian distribution on the high- and low-energy sides, respectively, whereby the overtone widths fold themselves with that of the one-phonon profile. This ETDMTCF also features expedient computation in large systems using asymmetric phonon profiles to account correctly for dephasing and pigment-protein interaction (electron-phonon coupling). The derived ETDMTCF allows computing all nonlinear optical signals in both time and frequency domains, through the nonlinear dipole moment time correlation functions (as guided by nonlinear optical response theory) in line with the eight Liouville space pathways. The linear transition dipole moment time correlation function is of a central value as the nonlinear transition dipole moment time correlation function is expressed in terms of the linear transition dipole moment time correlation function, derived herein. One of the great advantages of presenting this ETDMTCF is its applicability to nonlinear transition dipole moment time correlation functions in line with the eight Liouville space pathways needed in computing nonlinear signals. As such, there is more to the utility and applicability of the presented ETDMTCF besides computational expediency and efficiency. Results show good agreement with the reported literature. The intimate connection between a one-phonon profile and the corresponding bath spectral density in photosynthetic complexes is discussed.

Primary processes in bacterial reaction center revealed by femtosecond broadband fluorescence spectroscopy

To gain a deeper understanding of the highly efficient mechanisms within the photosynthetic bacterial reaction center (BRC), we have employed femtosecond broadband fluorescence spectroscopy to investigate the dynamics of initial photo-induced energy transfer and charge separation in BRC at room temperature. Benefiting from the broadband spectral coverage inherent of this technique, two distinct transient emission species associated with bacteriochlorophylls B and P are directly identified, with Stokes shifts determined to be ~197 and 450 cm−1, respectively. The ultrafast energy transfers from bacteriopheophytin H to B (98 fs) and from B to P (170 fs) are unveiled through fitting the emission dynamics. Notably, the anticipated sub-200 fs lifetime of B emission significantly extends to ~400 fs, suggesting a plausible coupling between the electronic excited state of Band the vibronic states of P, potentially influencing the acceleration of the energy transfer process. These findings should pave the way for understanding the impact of vibronic dynamics on the photo-induced primary processes in the photosynthetic reaction center.

Conditions for EPR detection of chirality-induced spin selectivity in spin-polarized radical pairs in isotropic solution.

Chiral molecules can act as spin filters, preferentially transmitting electrons with spins polarized along their direction of travel, an effect known as chirality-induced spin selectivity (CISS). In a typical experiment, injected electrons tunnel coherently through a layer of chiral material and emerge spin-polarized. It is also possible that spin polarization arises in radical pairs formed photochemically when electrons hop incoherently between donor and acceptor sites. Here we aim to identify the magnetic properties that would optimise the visibility of CISS polarization in time-resolved electron paramagnetic resonance (EPR) spectra of transient radical pairs without the need to orient or align their precursors. By simulating spectra of actual and model systems, we find that CISS contributions to the polarization should be most obvious when at least one of the radicals has small g-anisotropy and an inhomogeneous linewidth larger than the dipolar coupling of the two radicals. Under these conditions there is extensive cancellation of absorptive and emissive enhancements making the spectrum sensitive to small changes in the individual EPR line intensities. Although these cancellation effects are more pronounced at lower spectrometer frequencies, the spectral changes are easier to appreciate with the enhanced resolution afforded by high-frequency EPR. Consideration of published spectra of light-induced radical pairs in photosynthetic bacterial reaction centres reveals no significant CISS component in the polarization generated by the conventional spin-correlated radical pair mechanism.

Lel A. Drachev and the Direct Electrometric Method.

In the bioenergetics studies, the direct electrometric method played an important role. This method is based on measuring the electrical potential difference (Δψ) between two compartments of the experimental cell generated by some membrane proteins. These proteins are incorporated into closed lipid-protein membrane vesicles associated with an artificial lipid membrane that separates the compartments. The very existence of such proteins able to generate Δψ was one of the consequences of Peter Mitchell's chemiosmotic concept. The discovery and investigation of their functioning contributed to the recognition of this concept and, eventually the well-deserved awarding of the Nobel Prize to P. Mitchell. Lel A. Drachev (1926-2022) was one of the main authors of the direct electrometrical method. With his participation, key studies were carried out on the electrogenesis of photosynthetic and respiratory membrane proteins, including bacteriorhodopsin, visual rhodopsin, photosynthetic bacterial reaction centers, cytochrome oxidase and others.

Role of hydrogen-bond networks on the donor side of photosynthetic reaction centers from purple bacteria.

For the last decades, significant progress has been made in studying the biological functions of H-bond networks in membrane proteins, proton transporters, receptors, and photosynthetic reaction centers. Increasing availability of the X-ray crystal and cryo-electron microscopy structures of photosynthetic complexes resolved with high atomic resolution provides a platform for their comparative analysis. It allows identifying structural factors that are ensuring the high quantum yield of the photochemical reactions and are responsible for the stability of the membrane complexes. The H-bond networks are known to be responsible for proton transport associated with electron transfer from the primary to the secondary quinone as well as in the processes of water oxidation in photosystem II. Participation of such networks in reactions proceeding on the periplasmic side of bacterial photosynthetic reaction centers is less studied. This review summarizes the current understanding of the role of H-bond networks on the donor side of photosynthetic reaction centers from purple bacteria. It is discussed that the networks may be involved in providing close association with mobile electron carriers, in light-induced proton transport, in regulation of the redox properties of bacteriochlorophyll cofactors, and in stabilization of the membrane protein structure at the interface of membrane and soluble phases.

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.

Application of amber suppression to study the role of Tyr M210 in electron transfer in R. sphaeroides photosynthetic reaction centers.

The bacterial photosynthetic reaction center (RC) is an important system to understand the structure and chemistry of the biological process of photosynthesis. In wild type reaction centers and at room temperature, the unidirectional electron transfer from the excited-primary donor P∗ to the A-side bacteriopheophytin HA takes place via a two-step mechanism, where P∗ transfers an electron to the A-side bacteriochlorphyll BA in 3 ps making P+BA-, which then transfers an electron in 1 ps to HA to make P+HA-. Even though RCs have a pseudo-C2 symmetry, electron transfer only occurs along the A-branch. Tyrosine M210 has been a key amino acid of particular interest since it is a clear deviation in symmetry between the two sides of RC, and many mutations have been made at this site. One interesting finding was that when M210 is mutated to phenylalanine, the electron transfer from P∗ and HA slows down from ∼4 ps to ∼11 ps and becomes temperature independent (Nagarajan et al. 1990, PNAS). By using amber suppression, we were able to incorporate phenylalanine variants with different electron-withdrawing/donating capabilities at position M210. To our surprise, transient absorption data shows that YM210F RCs do not exhibit temperature independent primary charge separation. In fact, YM210F RCs and all M210 phenylalanine variants RCs exhibit Arrhenius temperature dependence in contrast to WT, wherein the rate increases at low temperature. Significantly, the 1020 nm band, which is characteristic of P+BA-, was not observed at any time for any of the M210 phenylalanine variants RCs nor YM210F RCs, indicating a change to the one-step superexchange mechanism for electron transfer.

Comparison of proton transfer paths to the QA and QB sites of the Rb. sphaeroides photosynthetic reaction centers.

The photosynthetic bacterial reaction centers from purple non-sulfur bacteria use light energy to drive the transfer of electrons from cytochrome c to ubiquinone. Ubiquinone bound in the QA site cycles between quinone, QA, and anionic semiquinone, QA·-, being reduced once and never binding protons. In the QB site, ubiquinone is reduced twice by QA·-, binds two protons and is released into the membrane as the quinol, QH2. The network of hydrogen bonds formed in a molecular dynamics trajectory was drawn to investigate proton transfer pathways from the cytoplasm to each quinone binding site. QA is isolated with no path for protons to enter from the surface. In contrast, there is a complex and tangled network requiring residues and waters that can bring protons to QB. There are three entries from clusters of surface residues centered around HisH126, GluH224, and HisH68. The network is in good agreement with earlier studies, Mutation of key nodes in the network, such as SerL223, were previously shown to slow proton delivery. Mutational studies had also shown that double mutations of residues such as AspM17 and AspL210 along multiple paths in the network presented here slow the reaction, while single mutations do not. Likewise, mutation of both HisH126 and HisH128, which are at the entry to two paths reduce the rate of proton uptake.

Characterizing Protein Protonation Microstates Using Monte Carlo Sampling.

Proteins are polyelectrolytes with acidic and basic amino acids Asp, Glu, Arg, Lys, and His, making up ≈25% of the residues. The protonation state of residues, cofactors, and ligands defines a "protonation microstate". In an ensemble of proteins some residues will be ionized and others neutral, leading to a mixture of protonation microstates rather than in a single one as is often assumed. The microstate distribution changes with pH. The protein environment also modifies residue proton affinity so microstate distributions change in different reaction intermediates or as ligands are bound. Particular protonation microstates may be required for function, while others exist simply because there are many states with similar energy. Here, the protonation microstates generated in Monte Carlo sampling in MCCE are characterized in HEW lysozyme as a function of pH and bacterial photosynthetic reaction centers (RCs) in different reaction intermediates. The lowest energy and highest probability microstates are compared. The ΔG, ΔH, and ΔS between the four protonation states of Glu35 and Asp52 in lysozyme are shown to be calculated with reasonable precision. At pH 7 the lysozyme charge ranges from 6 to 10, with 24 accepted protonation microstates, while RCs have ≈50,000. A weighted Pearson correlation analysis shows coupling between residue protonation states in RCs and how they change when the quinone in the QB site is reduced. Protonation microstates can be used to define input MD parameters and provide insight into the motion of protons coupled to reactions.

Homogeneous Dephasing Time in Photosynthetic Bacterial Reaction Centers

Homogeneous Dephasing Time in Photosynthetic Bacterial Reaction Centers

Correlating structural assemblies of photosynthetic reaction centers on a gold electrode and the photocurrent - potential response

SummaryThe use of biomacromolecules is a nascent development in clean alternative energies. In applications of biosensors and biophotovoltaic devices, the bacterial photosynthetic reaction center (RC) is a protein-pigment complex that has been commonly interfaced with electrodes, in large part to take advantage of the long-lived and high efficiency of charge separation. We investigated assemblies of RCs on an electrode that range from monolayer to multilayers by measuring the photocurrent produced when illuminated by an intensity-modulated excitation light source. In addition, atomic force microscopy and modeling of the photocurrent with the Marcus-Hush-Chidsey theory detailed the reorganization energy for the electron transfer process, which also revealed changes in the RC local environment due to the adsorbed conformations. The local environment in which the RCs are embedded significantly influenced photocurrent generation, which has implications for electron transfer of other biomacromolecules deposited on a surface in sensor and photovoltaic applications employing a redox electrolyte.

In Situ, Protein-Mediated Generation of a Photochemically Active Chlorophyll Analogue in a Mutant Bacterial Photosynthetic Reaction Center.

All possible natural amino acids have been substituted for the native LeuL185 positioned near the B-side bacteriopheophytin (HB) in the bacterial reaction center (RC) from Rhodobacter sphaeroides. Additional mutations that enhance electron transfer to the normally inactive B-side cofactors are present. Approximately half of the isolated RCs with Glu at L185 contain a magnesium chlorin (CB) in place of HB. The chlorin is not the common BChl a oxidation product 3-desvinyl-3-acetyl chlorophyll a with a C-C bond in ring D and a C═C bond in ring B but has properties consistent with reversal of these bond orders, giving 17,18-didehydro BChl a. In such RCs, charge-separated state P+CB- forms in ∼5% yield. The other half of the GluL185-containing RCs have a bacteriochlorophyll a (BChl a) denoted βB in place of HB. Residues His, Asp, Asn, and Gln at L185 yield RCs with ≥85% βB in the HB site, while most other amino acids result in RCs that retain HB (≥95%). To the best of our knowledge, neither bacterial RCs that harbor five BChl a molecules and one chlorophyll analogue nor those with six BChl a molecules have been reported previously. The finding that altering the local environment within a cofactor binding site of a transmembrane complex leads to in situ generation of a photoactive chlorin with an unusual ring oxidation pattern suggests new strategies for amino acid control over pigment type at specific sites in photosynthetic proteins.

Ethylenediammine is not detrimental to the photoactivity of the bacterial photosynthetic reaction center

The effect of the exposure of the photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides to ethylenediamine (EDA) was investigated by transient absorption spectroscopy and UV–Visible-Near Infrared absorption spectroscopy. We show that EDA is not detrimental to the photoactivity of the protein even at pH close to 12. EDA instead appears to inhibit the secondary quinone binding site with an apparent binding constant of 19.05 mM−1.

The Mechanisms of Electrogenic Reactions in Bacterial Photosynthetic Reaction Centers: Studies in Collaboration with Alexander Konstantinov.

In this review, we discuss our studies conducted in 1985-1988 in collaboration with A. A. Konstantinov, one of the top scientists in the field of membrane bioenergetics. Studying fast kinetics of membrane potential generation in photosynthetic reaction centers (RCs) of purple bacteria in response to a laser flash has made it possible to examine in detail the mechanisms of electrogenic reactions at the donor and acceptor sides of RCs. Electrogenesis associated with the intraprotein electron transfer from the exogenous secondary donors, redox dyes, and soluble cytochrome (cyt) c to the photooxidized dimer of bacteriochlorophyll P870 was studied using proteoliposomes containing RCs from the non-sulfur purple bacterium Rhodospirillum rubrum. It was found that reduction of the secondary quinone electron acceptor QB accompanied by its protonation in the chromatophores from R. rubrum in response to every second light flash was electrogenic. Spectral characteristics and redox potentials of the four hemes in the tightly bound cyt c in the RC of Blastochloris viridis and electrogenic reactions associated with the electron transfer within the RC complex were identified. For the first time, relative amplitudes of the membrane potential generated in the course of individual electrogenic reactions were compared with the distances between the redox cofactors determined based on the three-dimensional structure of the Bl. viridis RC.

Photoinduced charge transfer in Zn(II) and Au(III)-ligated symmetric and asymmetric bacteriochlorin dyads: A computational study.

The excited-state properties and photoinduced charge-transfer (CT) kinetics in a series of symmetrical and asymmetrical Zn- and Au-ligated meso-meso-connected bacteriochlorin (BChl) complexes are studied computationally. BChl derivatives, which are excellent near-IR absorbing chromophores, are found to play a central role in bacterial photosynthetic reaction centers but are rarely used in artificial solar energy harvesting systems. The optical properties of chemically linked BChl complexes can be tuned by varying the linking group and involving different ligated metal ions. We investigate charge transfer in BChl dyads that are either directly linked or through a phenylene ring (1,4-phenylene) and which are ligating Zn or Au ions. The directly linked dyads with a nearly perpendicular arrangement of the BChl units bear markedly different properties than phenylene linked dyads. In addition, we find that the dielectric dependence of the intramolecular CT rate is very strong in neutral Zn-ligated dyads, whereas cationic Au-ligated dyads show negligible dielectric dependence of the CT rate. Rate constants of the photo induced CT process are calculated at the semiclassical Marcus level and are compared to fully quantum mechanical Fermi's golden rule based values. The rates are calculated using a screened range separated hybrid functional that offers a consistent framework for addressing environment polarization. We study solvated systems in two solvents of a low and a high scalar dielectric constant.

Charge Recombination Kinetics of Bacterial Photosynthetic Reaction Centres Reconstituted in Liposomes: Deterministic Versus Stochastic Approach

In this theoretical work, we analyse the kinetics of charge recombination reaction after a light excitation of the Reaction Centres extracted from the photosynthetic bacterium Rhodobacter sphaeroides and reconstituted in small unilamellar phospholipid vesicles. Due to the compartmentalized nature of liposomes, vesicles may exhibit a random distribution of both ubiquinone molecules and the Reaction Centre protein complexes that can produce significant differences on the local concentrations from the average expected values. Moreover, since the amount of reacting species is very low in compartmentalized lipid systems the stochastic approach is more suitable to unveil deviations of the average time behaviour of vesicles from the deterministic time evolution.

Unfolding pathway and intermolecular interactions of the cytochrome subunit in the bacterial photosynthetic reaction center

Precise folding of photosynthetic proteins and organization of multicomponent assemblies to form functional entities are fundamental to efficient photosynthetic electron transfer. The bacteriochlorophyll b-producing purple bacterium Blastochloris viridis possesses a simplified photosynthetic apparatus. The light-harvesting (LH) antenna complex surrounds the photosynthetic reaction center (RC) to form the RC-LH1 complex. A non-membranous tetraheme cytochrome (4Hcyt) subunit is anchored at the periplasmic surface of the RC, functioning as the electron donor to transfer electrons from mobile electron carriers to the RC. Here, we use atomic force microscopy (AFM) and single-molecule force spectroscopy (SMFS) to probe the long-range organization of the photosynthetic apparatus from Blc. viridis and the unfolding pathway of the 4Hcyt subunit in its native supramolecular assembly with its functional partners. AFM images reveal that the RC-LH1 complexes are densely organized in the photosynthetic membranes, with restricted lateral protein diffusion. Unfolding of the 4Hcyt subunit represents a multi-step process and the unfolding forces of the 4Hcyt α-helices are approximately 121 picoNewtons. Pulling of 4Hcyt could also result in the unfolding of the RC L subunit that binds with the N-terminus of 4Hcyt, suggesting strong interactions between RC subunits. This study provides new insights into the protein folding and interactions of photosynthetic multicomponent complexes, which are essential for their structural and functional integrity to conduct photosynthetic electron flow.

Dynamics of the Excited State in Photosynthetic Bacterial Reaction Centers.

In the initial charge-separation reaction of photosynthetic bacterial reaction centers, a dimer of strongly interacting bacteriochlorophylls (P) transfers an electron to a third bacteriochlorophyll (BL). It has been suggested that light first generates an exciton state of the dimer and that an electron then moves from one bacteriochlorophyll to the other within P to form a charge-transfer state (PL+PM-), which passes an electron to BL. This scheme, however, is at odds with the most economical analysis of the spectroscopic properties of the reaction center and particularly with the unusual temperature dependence of the long-wavelength absorption band. The present paper explores this conflict with the aid of a simple model in which exciton and charge-transfer states are coupled to three vibrational modes. It then uses a similar model to show that the main experimental evidence suggesting the formation of PL+PM- as an intermediate could reflect pure dephasing of vibrational modes that modulate stimulated emission.

Monte Carlo simulation and thermodynamic integration applied to protein charge transfer.

We introduce a combination of Monte Carlo simulation and thermodynamic integration methods to address a model problem in free energy computations, electron transfer in proteins. The feasibility of this approach is tested using the ferredoxin protein from Clostridium acidurici. The results are compared to numerical solutions of the Poisson-Boltzmann equation and data from recent molecular dynamics simulations on charge transfer in a protein complex, the NrfHA nitrite reductase of Desulfovibrio vulgaris. Despite the conceptual and computational simplicity of the Monte Carlo approach, the data agree well with those obtained by other methods. A link to experiments is established via the cytochrome subunit of the bacterial photosynthetic reaction center of Rhodopseudomonas viridis.