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.

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.

Hybrid complexes of photosynthetic reaction centers and quantum dots in various matrices: resistance to UV irradiation and heating.

The effects of ultraviolet (UV) irradiation (up to 0.6 J/cm2) and heating (65 °C, 20 min) on the absorption spectra and electron transfer in dehydrated film samples of photosynthetic reaction centers (RCs) from purple bacterium Rhodobacter (Rb.) sphaeroides, as well as in hybrid structures consisting of RCs and quantum dots (QDs), have been studied. The samples were placed in organic matrices containing the stabilizers of protein structure-polyvinyl alcohol (PVA) and trehalose. UV irradiation led to partially irreversible oxidation of some RCs, as well as to transformation of some fraction of the bacteriochlorophyll (BChl) molecules into bacteriopheophytin (BPheo) molecules. In addition, UV irradiation causes degradation of some BChl molecules that is accompanied by formation of 3-acetyl-chlorophyll a molecules. Finally, UV irradiation destroys the RCs carotenoid molecules. The incorporation of RCs into organic matrices reduced pheophytinization. Trehalose was especially efficient in reducing the damage to the carotenoid and BChl molecules caused by UV irradiation. Hybrid films containing RC + QD were more stable to pheophytinization upon UV irradiation. However, the presence of QDs in films did not affect the processes of carotenoid destruction. The efficiency of the electronic excitation energy transfer from QD to P865 also did not change under UV irradiation. Heating led to dramatic destruction of the RCs structure and bacteriochlorins acquired the properties of unbound molecules. Trehalose provided strong protection against destruction of the RCs and hybrid (RC + QD) complexes.

pK a of ubiquinone, menaquinone, phylloquinone, plastoquinone, and rhodoquinone in aqueous solution.

Quinones can accept two electrons and two protons, and are involved in electron transfer and proton transfer reactions in photosynthetic reaction centers. To date, the pK a of these quinones in aqueous solution have not been reported. We calculated the pK a of the initial protonation (Q·- to QH·) and the second protonation (QH- to QH2) of 1,4-quinones using a quantum chemical approach. The calculated energy differences of the protonation reactions Q·- to QH· and QH- to QH2 in the aqueous phase for nine 1,4-quinones were highly correlated with the experimentally measured pK a(Q·-/QH·) and pK a(QH-/QH2), respectively. In the present study, we report the pK a(Q·-/QH·) and pK a(QH-/QH2) of ubiquinone, menaquinone, phylloquinone, plastoquinone, and rhodoquinone in aqueous solution.

Redox potentials of ubiquinone, menaquinone, phylloquinone, and plastoquinone in aqueous solution.

Quinones serve as redox active cofactors in bacterial photosynthetic reaction centers: photosystem I, photosystem II, cytochrome bc 1, and cytochrome b 6 f. In particular, ubiquinone is ubiquitous in animals and most bacteria and plays a key role in several cellular processes, e.g., mitochondrial electron transport. Their experimentally measured redox potential values for one-electron reduction E m(Q/Q·-) were already reported in dimethylformamide (DMF) versus saturated calomel electrode but not in water versus normal hydrogen electrode (NHE). We calculated E m(Q/Q·-) of 1,4-quinones using a quantum chemical approach. The calculated energy differences of reduction of Q to Q·- in DMF and water for 1,4-quinone derivatives correlated highly with the experimentally measured E m(Q/Q·-) in DMF and water, respectively. E m(Q/Q·-) were calculated to be -163 mV for ubiquinone, -260 mV for menaquinone and phylloquinone, and -154 mV for plastoquinone in water versus NHE.

Biomimetic Membranes for Multi-Redox Center Proteins.

His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs). An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA) terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM). MRPs, such as the cytochrome c oxidase (CcO) from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs) from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced resonance Raman spectroscopy (SERRS), were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs). The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM) and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

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.

Measurements of the light-induced steady state electric potential generation by photosynthetic pigment-protein complexes.

In this minireview, we consider the methods of measurements of the light-induced steady state transmembrane electric potential (Δψ) generation by photosynthetic systems, e.g. photosystem I (PS I). The microelectrode technique and the detection of electrochromic bandshifts of carotenoid pigments are most appropriate for Δψ measurements in situ and in vivo. Direct electrometrical method and Δψ measurements in the photovoltaic system based on membrane filter (MF) sandwiched between semiconductor indium tin oxide electrodes (ITO) are suitable for studies of isolated pigment-protein complexes and small natural vesicles-chromatophores. In the presence of trehalose, ITO|PS I-MF|ITO system allows to keep a steady state level of ∆ψ after 1 h of illumination. According to preliminary experiments, this system is capable of providing steady state light-induced ∆ψ after several months of storage in the dark at room temperature under controlled humidity in the presence of trehalose. The long-term generation of light-induced ∆ψ in relatively simple system may serve as a source of the solar-to-electric energy conversion.

EPR of Type I photosynthetic reaction centers.

Light-induced reactions in photosynthetic reaction centers are initiated by the absorption of a photon, which results in the transfer of a single electron and the generation of radical ions in the donor and acceptor molecules involved in the charge-separated state. Electron paramagnetic resonance (EPR) spectroscopy is the ideal method for the study of such reactions. In addition to measuring spectra of the electron transfer cofactors in continuous light, reactions can be initiated by brief flashes of light, thereby allowing the kinetics of forward electron transfer as well as recombination reactions to be obtained. Because the donor and acceptor pairs are so closely spaced and because light induced charge separation is so rapid, the donor and early acceptors are in a quantum mechanically spin entangled state, which confers properties such as increased sensitivity, the ability to measure reactions on the nanosecond timescale, and the determination of bond angles between cofactors. Additionally, distances between pairs of cofactors can be measured by detecting the modulation of a phase shifted "out-of-phase" electron spin echo signal. In this methods article, we will describe how continuous wave EPR, time resolved EPR, and pulsed EPR can be used to measure these properties in Type I photosynthetic reaction centers. Methods of analysis are described for the bound electron transfer cofactors in the heterodimeric Photosystem I reaction center of plants and cyanobacteria and in the homodimeric reaction centers found in phototrophic members of the phyla Bacillota, Chlorobiota, and Acidobacteriota.

Novel approaches for the lipid sponge phase crystallization of the Rhodobacter sphaeroides photosynthetic reaction center.

With the recent developments in the field of free-electron-laser-based serial femtosecond crystallography, the necessity to obtain a large number of high-quality crystals has emerged. In this work crystallization techniques were selected, tested and optimized for the lipid mesophase crystallization of the Rhodobacter sphaeroides membrane pigment-protein complex, known as the photosynthetic reaction center (RC). Novel approaches for lipid sponge phase crystallization in comparatively large volumes using Hamilton gas-tight glass syringes and plastic pipetting tips are described. An analysis of RC crystal structures obtained by lipid mesophase crystallization revealed non-native ligands that displaced the native electron-transfer cofactors (carotenoid sphero-idene and a ubi-quinone molecule) from their binding pockets. These ligands were identified and were found to be lipids that are major mesophase components. The selection of distinct co-crystallization conditions with the missing cofactors facilitated the restoration of sphero-idene in its binding site.

A Mechanoresponsive Phase-Changing Electrolyte Enables Fabrication of High-Output Solid-State Photobioelectrochemical Devices from Pigment-Protein Multilayers.

Exploitation of natural photovoltaic reaction center pigment proteins in biohybrid architectures for solar energy harvesting is attractive due to their global abundance, environmental compatibility, and near-unity quantum efficiencies. However, it is challenging to achieve high photocurrents in a device setup due to limitations imposed by low light absorbance by protein monolayers and/or slow long-range diffusion of liquid-phase charge carriers. In an attempt to enhance the photocurrent density achievable by pigment proteins, here, an alternative solid-state device architecture enabled by a mechanoresponsive gel electrolyte that can be applied under nondenaturing conditions is demonstrated. The phase-changing electrolyte gel provides a pervading biocompatible interface for charge conduction through highly absorbing protein multilayers that are fabricated in a simple fashion. Assembled devices exhibit enhanced current stability and a maximal photoresponse of ≈860 µA cm-2 , a fivefold improvement over the best previous comparable devices under standard illumination conditions. Photocurrent generation is enhanced by directional energy transfer through extended layers of light-harvesting complexes, mimicking the modular antenna/transducer architecture of natural photosystems, and by metastable radical pair formation when photovoltaic reaction centers are embedded throughout light-harvesting regions of the device.

Genetic Code Expansion in Rhodobacter sphaeroides to Incorporate Noncanonical Amino Acids into Photosynthetic Reaction Centers.

Photosynthetic reaction centers (RCs) are the membrane proteins responsible for the initial charge separation steps central to photosynthesis. As a complex and spectroscopically complicated membrane protein, the RC (and other associated photosynthetic proteins) would benefit greatly from the insight offered by site-specifically encoded noncanonical amino acids in the form of probes and an increased chemical range in key amino acid analogues. Toward that goal, we developed a method to transfer amber codon suppression machinery developed for E. coli into the model bacterium needed to produce RCs, Rhodobacter sphaeroides. Plasmids were developed and optimized to incorporate 3-chlorotyrosine, 3-bromotyrosine, and 3-iodotyrosine into RCs. Multiple challenges involving yield and orthogonality were overcome to implement amber suppression in R. sphaeroides, providing insights into the hurdles that can be involved in host transfer of amber suppression systems from E. coli. In the process of verifying noncanonical amino acid incorporation, characterization of this membrane protein via mass spectrometry (which has been difficult previously) was substantially improved. Importantly, the ability to incorporate noncanonical amino acids in R. sphaeroides expands research capabilities in the photosynthetic field.