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A supramolecular construct for solar energy conversion is developed by covalently bridging the reaction center (RC) from the photosynthetic bacterium Rhodobacter sphaeroides and cytochrome c (Cyt c ) proteins with a tailored organic light harvesting antenna (hCy2). The RC-hCy2-Cyt c biohybrid mimics the working mechanism of biological assemblies located in the bacterial cell membrane to convert sunlight into metabolic energy. hCy2 collects visible light and transfers energy to the RC, increasing the rate of photocycle between a RC and Cyt c that are linked in such a way that enhances proximity without preventing protein mobility. The biohybrid obtained with average 1 RC/10 hCy2/1.5 Cyt c molar ratio features an almost doubled photoactivity versus the pristine RC upon illumination at 660 nm, and ∼10 times higher photocurrent versus an equimolar mixture of the unbound proteins. Our results represent an interesting insight into photoenzyme chemical manipulation, opening the way to new eco-sustainable systems for biophotovoltaics.

Charge recombination kinetics of bacterial photosynthetic protein Reaction Center displays an exquisite sensitivity to the actual occupancy of ubiquinone-10 in its Q B -binding site. Here, we have exploited such phenomenon for assessing the growth and the aggregation/fusion of phosphocholine vesicles embedding RC in their membrane, when treated with sodium oleate.

Following a bottom-up synthetic biology approach it is shown that vesicle-based cell-like systems (shortly "synthetic cells") can be designed and assembled to perform specific function (for biotechnological applications) and for studies in the origin-of-life field. We recently focused on the construction of synthetic cells capable to converting light into chemical energy. Here we first present our approach, which has been realized so far by the reconstitution of photosynthetic reaction centre in the membrane of giant lipid vesicles. Next, the details of our ongoing research program are presented. It involves the use of the reaction centre, the coenzyme Q-cytochrome c oxidoreductase, and the ATP synthase for creating an autonomous synthetic cell. We show experimental results on the chemistry of the first two proteins showing that they can efficiently sustain light-driven chemical oscillations. Moreover, the cyclic pattern has been reproduced in silico by a minimal kinetic model., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP. Herein we show that synthetic protocells, based on giant lipid vesicles embedding an oriented population of reaction centers, are capable of generating a photoinduced proton gradient across the membrane. Under continuous illumination, the protocells generate a gradient of 0.061 pH units per min, equivalent to a proton motive force of 3.6 mV⋅min -1 Remarkably, the facile reconstitution of the photosynthetic reaction center in the artificial lipid membrane, obtained by the droplet transfer method, paves the way for the construction of novel and more functional protocells for synthetic biology.

Ultrasounds are used in many industrial, medical and research applications. Properties and function of proteins are strongly influenced by the interaction with the ultrasonic waves and their bioactivity can be lost because of alteration of protein structure. Surprisingly, to the best of our knowledge no study was carried out on Integral Membrane Proteins (IMPs), which are responsible for a variety of fundamental biological functions. In this work, the photosynthetic Reaction Center (RC) of the bacterium Rhodobacter sphaeroides has been used as a model for the study of the ultrasound-induced IMP denaturation. Purified RCs were suspended in i) detergent micelles, in ii) detergent-free buffer and iii) reconstituted in liposomes, and then treated with ultrasound at 30W and 20kHz at increasing times. The optical absorption spectra showed a progressive and irreversible denaturation in all cases, resulting from the perturbation of the protein scaffold structure, as confirmed by circular dichroism spectra that showed progressive alterations of the RC secondary structure. Charge recombination kinetics were studied to assess the protein photoactivity. The lifetime for the loss of RC photoactivity was 32min in detergent micelles, ranged from 3.8 to 6.5min in the different proteoliposomes formulations, and 5.5min in detergent-free buffer. Atomic force microscopy revealed the formation of large RC aggregates related to the sonication-induced denaturation, in agreement with the scattering increase observed in solution., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The photosynthetic reaction center (RC) from the Rhodobacter sphaeroides bacterium has been covalently bioconjugated with a NIR-emitting fluorophore (AE800) whose synthesis was specifically tailored to act as artificial antenna harvesting light in the entire visible region. AE800 has a broad absorption spectrum with peaks centered in the absorption gaps of the RC and its emission overlaps the most intense RC absorption bands, ensuring a consistent increase of the protein optical cross section. The covalent hybrid AE800-RC is stable and fully functional. The energy collected by the artificial antenna is transferred to the protein via FRET mechanism, and the hybrid system outperforms by a noteworthy 30% the overall photochemical activity of the native protein under the entire range of visible light. This improvement in the optical characteristic of the photoenzyme demonstrates the effectiveness of the bioconjugation approach as a suitable route to new biohybrid materials for energy conversion, photocatalysis, and biosensing.

The reconstitution of the integral membrane protein photosynthetic reaction center (RC) in polymersomes, i.e. artificial closed vesicles, was achieved by the micelle-to-vesicle transition technique, a very mild protocol based on size exclusion chromatography often used to drive the incorporation of proteins contemporarily to liposome formation. An optimized protocol was used to successfully reconstitute the protein in a fully active state in polymersomes formed by the tri-block copolymers PMOXA22-PDMS61-PMOXA22. The RC is very sensitive to its solubilizing environment and was used to probe the positioning of the protein in the vesicles. According to charge-recombination experiments and to the enzymatic activity assay, the RC is found to accommodate in the PMOXA22 region of the polymersome, facing the water bulk solution, rather than in the PDMS61 transmembrane-like region. Furthermore, polymersomes were found to preserve protein integrity efficiently as the biomimetic lipid bilayers but show a much longer temporal stability than lipid based vesicles.

Semiquinone oscillations induced by light pulses in the presence of exogenous electron donors are a valuable source of information on the kinetics and thermodynamics of ubiquinone chemistry relevant to the QB site of the photosynthetic reaction center (RC). In previous attempts to achieve the quantitative interpretation of data, the ubiquinone concentration was considered constant during the experiment since it was much bigger than that of RC. In this work, we extended existing models to low ubiquinone concentrations revealing several hidden processes taking place during the ubiquinone photoreduction and enabling the evaluation of the ubiquinone binding constant K Q at the QB site. The proposed approach provides for the first time the evaluation of K Q without any preliminary treatment of ubiquinone extraction from the binding site, thereby better preserving its native structure.

Liposomes represent a versatile biomimetic environment for studying the interaction between integral membrane proteins and hydrophobic ligands. In this paper, the quinone binding to the QB-site of the photosynthetic reaction centers (RC) from Rhodobacter sphaeroides has been investigated in liposomes prepared with either the zwitterionic phosphatidylcholine (PC) or the negatively charged phosphatidylglycerol (PG) to highlight the role of the different phospholipid polar heads. Quinone binding (K Q) and interquinone electron transfer (L AB) equilibrium constants in the two type of liposomes were obtained by charge recombination reaction of QB-depleted RC in the presence of increasing amounts of ubiquinone-10 over the temperature interval 6-35 °C. The kinetic of the charge recombination reactions has been fitted by numerically solving the ordinary differential equations set associated with a detailed kinetic scheme involving electron transfer reactions coupled with quinone release and uptake. The entire set of traces at each temperature was accurately fitted using the sole quinone release constants (both in a neutral and a charge separated state) as adjustable parameters. The temperature dependence of the quinone exchange rate at the QB-site was, hence, obtained. It was found that the quinone exchange regime was always fast for PC while it switched from slow to fast in PG as the temperature rose above 20 °C. A new method was introduced in this paper for the evaluation of constant K Q using the area underneath the charge recombination traces as the indicator of the amount of quinone bound to the QB-site.

Because of the growing potential of nanoparticles in biological and medical applications, tuning and directing their properties toward a high compatibility with the aqueous biological milieu is of remarkable relevance. Moreover, the capability to combine nanocrystals (NCs) with biomolecules, such as proteins, offers great opportunities to design hybrid systems for both nanobiotechnology and biomedical technology. Here we report on the application of the micelle-to-vesicle transition (MVT) method for incorporation of hydrophobic, red-emitting CdSe@ZnS NCs into the bilayer of liposomes. This method enabled the construction of a novel hybrid proteo-NC-liposome containing, as model membrane protein, the photosynthetic reaction center (RC) of Rhodobacter sphaeroides. Electron microscopy confirmed the insertion of NCs within the lipid bilayer without significantly altering the structure of the unilamellar vesicles. The resulting aqueous NC-liposome suspensions showed low turbidity and kept unaltered the wavelengths of absorbance and emission peaks of the native NCs. A relative NC fluorescence quantum yield up to 8% was preserved after their incorporation in liposomes. Interestingly, in proteo-NC-liposomes, RC is not denatured by Cd-based NCs, retaining its structural and functional integrity as shown by absorption spectra and flash-induced charge recombination kinetics. The outlined strategy can be extended in principle to any suitably sized hydrophobic NC with similar surface chemistry and to any integral protein complex. Furthermore, the proposed approach could be used in nanomedicine for the realization of theranostic systems and provides new, interesting perspectives for understanding the interactions between integral membrane proteins and nanoparticles, i.e., in nanotoxicology studies.

Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c₂ and K₄[Fe(CN)₆]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.

Reconstitution of membrane proteins in phospholipid vesicles allows the investigation of such macromolecules in a biomimetic simplified environment. The often employed micelle-to-vesicle-transition method for proteoliposome preparation is a fast and reproducible technique. In this, communication is shown that the lipid/protein ratio influences the size of the proteoliposomes and the actual protein reconstitution. The results indicate that for photosynthetic reaction centres, the best conditions for ligand-interaction experiments are achieved with a lipid/protein value of 1000:1, while for complete protein incorporation, the 2000:1 ratio should be chosen.

Photophysical properties of tetramethyl-1,4-benzoquinone (TMBQ) and 2,6-dimethoxy-1,4-benzoquinone (DMOBQ) in solution and their interactions with the photosynthetic reaction centre (RC) isolated from the photosynthetic bacterium Rhodobacter sphaeroides have been investigated in this work. For these two benzoquinone derivatives an efficient ISC process which leads to the population of the lowest triplet state of the molecules upon direct excitation was observed. The presence of RC does not alter the properties of the triplet state of DMOBQ suggesting that interactions are negligible; on the other side RC efficiently quenched the T1 state of TMBQ. The behavior is rationalized in terms of redox potentials of quinones and kinetic characteristics of their transients.

The kinetics and thermodynamics of cyclic electron transfer through the isolated reaction center protein of photosynthetic bacterium Rhodobacter sphaeroides were determined in detergent (Triton X-100) solution. The redox reactions between the reducing (ubiquinol-0 or ubiquinol-10) and oxidizing species (ferricenium, ferricytochrome, or ferricyanide) produced chemically or by light excitation of the protein were monitored by absorption changes of the reactants and by acidification of the solution accompanied with the disappearance of the quinol. The bimolecular rate constants of reactions of anionic ubiquinol-0 with different oxidizing agents showed large variation: 5 x 10(8) M(-1) s(-1) for ferricenium, 3.5 x 10(5) M(-1) s(-1) for ferricyanide, and 1.5 x 10(5) M(-1) s(-1) for ferricytochrome. Although the redox partners were created in pairs by the same protein promptly after light excitation, their bimolecular redox reaction was not observed even in the case of the fastest reacting partners of ferricenium and ubiquinol-0. Instead, they equilibrate with the corresponding (donor and acceptor) pools before the electron is transferred. The (logarithms of the) observed rate constants of quinol oxidation showed steep pH-dependence for water soluble ubiquinol-0 (slope +1) and mild pH-dependence for hydrophobic ubiquinol-10 (slope approximately 0.25). Combined with studies of the ionic strength dependence of the rate, it was concluded that the electron-transfer pathways of ubiquinol-0 and ubiquinol-10 oxidation started from their anionic and neutral forms, respectively. The mild pH-dependence of the rate of ubiquinol-10 oxidation came from the electrostatic interactions between ferricenium and the pH-dependent surface charges of the reaction center. The results help to understand, monitor, and design (cyclic) electron flow in bioenergetic proteins.

The thermodynamics and kinetics of light-induced electron transfer in bacterial photosynthetic RCs are sensitive to physiologically important lipids (phosphatidylcholine, cardiolipin and phosphatidylglycerol) in the environment. The analysis of the temperature-dependence of the rate of the P(+)Q(A)(-)Q(B)-->P(+)Q(A)Q(B)(-) interquinone electron transfer revealed high enthalpy change of activation in zwitterionic or neutral micelles and vesicles and low enthalpy change of activation in vesicles constituted of negatively charged phospholipids. The entropy change of activation was compensated by the changes of enthalpy, thus the free energy change of activation ( approximately 500 meV) did not show large variation in vesicles of different lipids.

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.

Reaction centers from the purple bacterium Rhodobacter sphaeroides strain R-26.1 were purified and reconstituted in proteoliposomes formed by the anionic phospholipids phosphatidylglycerol, phosphatidylserine and phosphatidylinositol and by the zwitterionic phospholipid phosphatidylcholine by size-exclusion chromatography in the dark and under illumination. We report the large stabilizing effect induced by anionic phospholipids on the protein charge separated state which results trapped in a long-living (up to tens of minutes) state with a yield up to 80%. This fully reversible state is formed in oxygenic conditions regardless the presence of the secondary quinone QB and its lifetime and relative yield increase at low pH. In proteoliposomes formed with QA-depleted reaction centers (RCs) the resulting protein is very light-sensitive and the long living charge separated state is not observed. The data collected in negatively charged proteoliposomes are discussed in terms of the electrostatic effect on the primary quinone acceptor and compared with similar long living species reported in literature and obtained in anionic, zwitterionic, and non-ionic detergents.

The role of characteristic phospholipids of native membranes, phosphatidylcholine (PC), phosphatidylglycerol (PG), and cardiolipin (CL), was studied in the energetics of the acceptor quinone side in photosynthetic reaction centers of Rhodobacter sphaeroides. The rates of the first, k(AB)(1), and the second, k(AB)(2), electron transfer and that of the charge recombination, k(BP), the free energy levels of Q(A)(-)Q(B) and Q(A)Q(B)(-) states, and the changes of charge compensating protein relaxation were determined in RCs incorporated into artificial lipid bilayer membranes. In RCs embedded in the PC vesicle, k(AB)(1) and k(AB)(2) increased (from 3100 to 4100 s(-1) and from 740 to 3300 s(-1), respectively) and k(BP) decreased (from 0.77 to 0.39 s(-1)) compared to those measured in detergent at pH 7. In PG, k(AB)(1) and k(BP) decreased (to values of 710 and 0.26 s(-1), respectively), while k(AB)(2) increased to 1506 s(-1) at pH 7. The free energy between the Q(A)(-)Q(B) and Q(A)Q(B)(-) states decreased in PC and PG (DeltaG degrees (Q)A-(Q)B(-->)(Q)A(Q)B- = -76.9 and -88.5 meV, respectively) compared to that measured in detergent (-61.8 meV). The changes of the Q(A)/Q(A)(-) redox potential measured by delayed luminescence showed (1) a differential effect of lipids whether RC incorporated in micelles or vesicles, (2) an altered binding interaction between anionic lipids and RC, (3) a direct influence of PC and PG on the free energy levels of the primary and secondary quinones probably through the intraprotein hydrogen-bonding network, and (4) a larger increase of the Q(A)/Q(A)(-) free energy in PG than in PC both in detergent micelles and in single-component vesicles. On the basis of recent structural data, implications of the binding properties of phospholipids to RC and possible interactions between lipids and electron transfer components will be discussed.

Isolated and purified reaction centers (RC) from Rhodobacter sphaeroides R-26.1 were solubilised in detergent with excess quinone and external electron donors and illuminated in the presence of pyranine. The pH change accompanying the reaction center photocycle was monitored by recording the variation of the pyranine fluorescence intensity. Using Q(B)-depleted reaction centers or blocking the photocycle with terbutryne strongly reduced the pH change. The usefulness and limits of this technique in monitoring the pH changes during the RC photocycle are also discussed.

Transmembrane proton translocation in the photosynthetic membranes of the purple bacterium Rhodobacter sphaeroides is driven by light and performed by two transmembrane complexes; the photosynthetic reaction center and the ubiquinol-cytochrome c oxidoreductase complex, coupled by two mobile electron carriers; the cytochrome and the quinone. This paper focuses on the kinetics and thermodynamics of the interaction between the lipophylic electron carrier ubiquinone-10 and the photosynthetic enzyme reconstituted in liposomes. The collected data were simulated with an existing recognized kinetic scheme and the kinetic constants of the uptake (7.2 x 107 M(-1) x s(-1)) and release (40 s(-1)) processes of the ligand were inferred. The results obtained for the quinone release kinetic constant are comparable to the rate of the charge recombination reaction from the state D(+)QA(-). Values for the kinetic constants are discussed as part of the overall photocycle, suggesting that its bottleneck may not be the quinone uptake reaction in agreement with a previous report.

In this paper are reported studies on the detergent role in isolated reaction centers (RC) from Rhodobacter sphaeroides, over a large range of lauryldimethylamino-N-oxide (LDAO) concentrations, in influencing the thermodynamics of the quinone exchange reaction as well as the protein aggregation. The occurrence of the quinone exchange reaction between the QB-binding site (where QB is the second quinone molecule of two in the RC) and the ubiquinone 0 dissolved in the different environments (water, LDAO micelles and detergent phase of the protein-detergent complex) has also been analyzed. Measurements carried out in QB-depleted RC to which exogenous quinone has been added show that the relative amplitudes of the slow and fast phase of the recombination reaction depend on this parameter. The overall amount of the restored QB-functionality is affected by the concentration of the LDAO in solution. Interpolation of the titration curves with a quadratic function obtained by simple considerations allowed the binding constant of UQ0 to the QB-binding site to be calculated. From the fitting procedure, the distribution of the quinone in the different environments present in solution was evaluated, indicating that the exchange reaction can take place only between the QB-site and the detergent phase. The dependence of the quinone pool size upon the volume of the phase in which the interacting quinone is solubilized is also discussed. The increasing difficulty in saturating the QB-pocket above the LDAO critical micellar concentration is finally related to the association of protein-detergent complexes to form large protein clusters.