Bacterial Photosynthetic Reaction Centers Research Articles

Quinone Binding in Bacterial Photosynthetic Reaction Center

Quinones play a crucial role in transmembrane electron and proton transfer in proteins, thereby creating a gradient that is utilized for energy conversion. The medicinal properties of these molecules render them important for drug design. The quinone binding site varies considerably across different proteins and any strict binding motif is not defined yet. The bacterial photosynthetic reaction center (BRC) is one such protein with two quinone binding sites - QA and QB. Kinetic studies have shown differently substituted foreign quinones to be good binders at the QA site, however there are no protein structures available with the foreign quinones. The goal of this computational study is to derive a BRC-quinone interaction signature, with different quinones docked at the QA site. The derived signature could pave the way for future computer aided drug design, targeted at quinone binding proteins.Sets of substituted foreign quinones were built and optimized in Gaussian09 and docked into the BRC QA site with AutoDock4.2. The protein-ligand vanderwaals overlap was calculated in Chimera. Analyses of the van der waals interaction of the complexes brought out a pattern of favorable interactions of the ligand with the protein, that directly correlate with high binding affinity. The benzoquinone strong binders showed favorable interaction with TrpM252, MetM256 and MetM262 of the BRC. The methyl and methoxy flanking a carbonyl formed a benzoquinone ligand-motif for strong binding. LeuM215, TrpM252, Met256, PheM258, MetM262, and IleM265 of the receptor favorably interacted with the strong naptho binders. Future work includes Molecular dynamics studies that will involve the FEP method on the docked complexes in NAMD2.9.

“Garnishing” the photosynthetic bacterial reaction center for bioelectronics

Chemical manipulations of the photosynthetic bacterial reaction center for the implementation of this photoenzyme into bioelectronic devices are overviewed.

Horizontal transfers of two types of puf operons among phototrophic members of the Roseobacter clade.

The Roseobacter clade represents one of the most important bacterial groups in marine environments. While some of its members are heterotrophs, many Roseobacter clade members contain bacterial photosynthetic reaction centers. We investigated the phylogeny of pufL and pufM genes encoding the L and M subunits of reaction centers using available genomic data and our own cultured species. Interestingly, phylogeny of pufL and pufM genes largely deviated from 16S rRNA-based phylogeny. The sequences split into two clearly distinct clades. While most of the studied species contained pufL and pufM sequences related to those found in Roseobacter litoralis, some of the marine species contained sequences related to the freshwater Rhodobacter species. In addition, genomic data documents that Roseobacter-type centers contain cytochrome c subunits (pufC gene product), whereas Rhodobacter-type centers incorporate PufX proteins. This indicates that the two forms of the reaction centers are not only distinct phylogenetically, but also structurally. The large deviation of pufL and pufM phylogeny from 16S phylogeny indicates multiple horizontal transfers of the puf operon among members of the order Rhodobacterales.

Modeling gating charge and voltage changes in response to charge separation in membrane proteins.

Measurements of voltage changes in response to charge separation within membrane proteins can offer fundamental information on mechanisms of charge transport and displacement processes. A recent example is provided by studies of cytochrome c oxidase. However, the interpretation of the observed voltage changes in terms of the number of charge equivalents and transfer distances is far from being trivial or unique. Using continuum approaches to describe the voltage generation may involve significant uncertainties and reliable microscopic simulations are not yet available. Here, we attempt to solve this problem by using a coarse-grained model of membrane proteins, which includes an explicit description of the membrane, the electrolytes, and the electrodes. The model evaluates the gating charges and the electrode potentials (c.f. measured voltage) upon charge transfer within the protein. The accuracy of the model is evaluated by a comparison of measured voltage changes associated with electron and proton transfer in bacterial photosynthetic reaction centers to those calculated using our coarse-grained model. The calculations reproduce the experimental observations and thus indicate that the method is of general use. Interestingly, it is found that charge-separation processes with different spatial directions (but the same distance perpendicular to the membrane) can give similar observed voltage changes, which indicates that caution should be exercised when using simplified interpretation of the relationship between charge displacement and voltage changes.

The 2-Methoxy Group Orientation Regulates the Redox Potential Difference between the Primary (QA) and Secondary (QB) Quinones of Type II Bacterial Photosynthetic Reaction Centers.

Recent studies have shown that only quinones with a 2-methoxy group can act simultaneously as the primary (QA) and secondary (QB) electron acceptors in photosynthetic reaction centers from purple bacteria such as Rb. sphaeroides. 13C HYSCORE measurements of the 2-methoxy group in the semiquinone states, SQA and SQB, were compared with DFT calculations of the 13C hyperfine couplings as a function of the 2-methoxy dihedral angle. X-ray structure comparisons support 2-methoxy dihedral angle assignments corresponding to a redox potential gap (ΔEm) between QA and QB of 175–193 mV. A model having a methyl group substituted for the 2-methoxy group exhibits no electron affinity difference. This is consistent with the failure of a 2-methyl ubiquinone analogue to function as QB in mutant reaction centers with a ΔEm of ∼160–195 mV. The conclusion reached is that the 2-methoxy group is the principal determinant of electron transfer from QA to QB in type II photosynthetic reaction centers with ubiquinone serving as both acceptor quinones.

Role of Electronic-Vibrational Mixing in Enhancing Vibrational Coherences in the Ground Electronic States of Photosynthetic Bacterial Reaction Center

We describe polarization controlled two-color coherence photon echo studies of the reaction center complex from a purple bacterium Rhodobacter sphaeroides. Long-lived oscillatory signals that persist up to 2 ps are observed in neutral, oxidized, and mutant (lacking the special pair) reaction centers, for both (0°,0°,0°,0°) and (45°,-45°,90°,0°) polarization sequences. We show that the long-lived signals arise via vibronic coupling of the bacteriopheophytin (H) and accessory bacteriochlorophyll (B) pigments that leads to vibrational wavepackets in the B ground electronic state. Fourier analysis of the data suggests that the 685 cm(-1) mode of B may play a key role in the H to B energy transfer.

Problems of the theory of electron transfer in biological systems

Based on comparative analysis, it is shown that the electron transfer theory traditionally used in biophysics is often unable to explain the electron transfer regularities observed in biological molecular systems. The data for seven electron transfer reactions (direct and reverse) that occur in bacterial photosynthetic reaction centers (mainly, purple bacteria Rhodobacter sphaeroides) have been analyzed. Conceivable reasons for the discrepancy between the theoretical and experimental data are discussed and some approaches to overcoming this contradiction are offered.

Structural and dynamical characteristics of trehalose and sucrose matrices at different hydration levels as probed by FTIR and high-field EPR

Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, particularly trehalose and sucrose. Trehalose is most effective also in protecting isolated in vitro biostructures. In an attempt to clarify the molecular mechanisms of disaccharide bioprotection, we compared the structure and dynamics of sucrose and trehalose matrices at different hydration levels by means of high-field W-band EPR and FTIR spectroscopy. The hydration state of the samples was characterized by FTIR spectroscopy and the structural organization was probed by EPR using a nitroxide radical dissolved in the respective matrices. Analysis of the EPR spectra showed that the structure and dynamics of the dehydrated matrices as well as their evolution upon re-hydration differ substantially between trehalose and sucrose. The dehydrated trehalose matrix is homogeneous in terms of distribution of the residual water and spin-probe molecules. In contrast, dehydrated sucrose forms a heterogeneous matrix. It is comprised of sucrose polycrystalline clusters and several bulk water domains. The amorphous form was found only in 30% (volume) of the sucrose matrix. Re-hydration leads to a structural homogenization of the sucrose matrix, whilst in the trehalose matrix several domains develop differing in the local water/radical content and radical mobility. The molecular model of the matrices provides an explanation for the different protein-matrix dynamical coupling observed in dried ternary sucrose and trehalose matrices, and accounts for the superior efficacy of trehalose as a bioprotectant. Furthermore, for bacterial photosynthetic reaction centers it is shown that at low water content the protein-matrix coupling is modulated by the sugar/protein molar ratio in sucrose matrices only. This effect is suggested to be related to the preference for sucrose, rather than trehalose, as a bioprotective disaccharide in some anhydrobiotic organisms.

Tuning Cofactor Redox Potentials: The 2-Methoxy Dihedral Angle Generates a Redox Potential Difference of >160 mV between the Primary (QA) and Secondary (QB) Quinones of the Bacterial Photosynthetic Reaction Center

Only quinones with a 2-methoxy group can act simultaneously as the primary (QA) and secondary (QB) electron acceptors in photosynthetic reaction centers from Rhodobacter sphaeroides. (13)C hyperfine sublevel correlation measurements of the 2-methoxy in the semiquinone states, SQA and SQB, were compared with quantum mechanics calculations of the (13)C couplings as a function of the dihedral angle. X-ray structures support dihedral angle assignments corresponding to a redox potential gap (ΔEm) between QA and QB of ~180 mV. This is consistent with the failure of a ubiquinone analogue lacking the 2-methoxy to function as QB in mutant reaction centers with a ΔEm of ≈160-195 mV.

On the Nature of the Hydrogen Bonds to Neutral Ubiquinone in the QABinding Site in Purple Bacterial Photosynthetic Reaction Centers

The nature of hydrogen bonding to pigments in protein complexes is currently a topic of some debate. The debate centers on whether hydrogen bonds can be understood on purely electrostatic grounds or whether they need to be considered quantum mechanically. This distinction is of current relevance primarily because of the application of QM/MM computational methods to the study of biological problems. To address this problem we have used QM/MM methods to study the neutral state of the hydrogen bonded ubiquinone molecule termed QA that functions as an electron transfer cofactor in purple bacterial photosynthetic reaction centers. In these calculations we have treated the hydrogen bonding amino acids either quantum mechanically or using molecular mechanics methods. As a specific metric for comparing the different computational methods isotope edited FTIR difference spectra are calculated. The calculated spectra are in remarkable agreement with experimental spectra, and it is found that the calculated spectra are very similar when hydrogen bonding amino acids are treated using either QM or MM methods. The latter result suggests that hydrogen bonding to neutral ubiquinone in purple bacterial reaction centers can be considered in purely electrostatic terms, which is contrary to the widely held belief that the hydrogen bonding amino acids should be treated quantum mechanically. Natural bond orbital analysis is used to further verify that the hydrogen bonds are predominantly electrostatic in nature. Calculated bond lengths and vibrational frequencies of the N-H groups involved in hydrogen bonding are used to estimate the relative strengths of the hydrogen bonds to either ubiquinone carbonyl group.

Utilizing the Dynamic Stark Shift as a Probe for Dielectric Relaxation in Photosynthetic Reaction Centers During Charge Separation

In photosynthetic reaction centers, the electric field generated by light-induced charge separation produces electrochromic shifts in the transitions of reaction center pigments. The extent of this Stark shift indirectly reflects the effective field strength at a particular cofactor in the complex. The dynamics of the effective field strength near the two monomeric bacteriochlorophylls (BA and BB) in purple photosynthetic bacterial reaction centers has been explored near physiological temperature by monitoring the time-dependent Stark shift during charge separation (dynamic Stark shift). This dynamic Stark shift was determined through analysis of femtosecond time-resolved absorbance change spectra recorded in wild type reaction centers and in four mutants at position M210. In both wild type and the mutants, the kinetics of the dynamic Stark shift differ from those of electron transfer, though not in the same way. In wild type, the initial electron transfer and the increase in the effective field strength near the active-side monomer bacteriochlorophyll (BA) occur in synchrony, but the two signals diverge on the time scale of electron transfer to the quinone. In contrast, when tyrosine is replaced by aspartic acid at M210, the kinetics of the BA Stark shift and the initial electron transfer differ, but transfer to the quinone coincides with the decay of the Stark shift. This is interpreted in terms of differences in the dynamics of the local dielectric environment between the mutants and the wild type. In wild type, comparison of the Stark shifts associated with BA and BB on the two quasi-symmetric halves of the reaction center structure confirm that the effective dielectric constants near these cofactors are quite different when the reaction center is in the state P(+)QA(-), as previously determined by Steffen et al. at 1.5 K (Steffen, M. A.; et al. Science 1994, 264, 810-816). However, it is not possible to determine from static, low-temperature measurments if the difference in the effective dielectric constant between the two sides of the reaction center is manifest on the time scale of initial electron transfer. By comparing directly the Stark shift dynamics of the ground-state spectra of the two monomer bacteriochlorophylls, it is evident that there is, in fact, a large dielectric difference between protein environments of the two quasi-symmetric electron-transfer branches on the time scale of initial electron transfer and that the effective dielectric constant in the region continues to evolve on a time scale of hundreds of picoseconds.

Affinity and activity of non-native quinones at the QB site of bacterial photosynthetic reaction centers

Purple, photosynthetic reaction centers from Rhodobacter sphaeroides bacteria use ubiquinone (UQ10) as both primary (Q(A)) and secondary (Q(B)) electron acceptors. Many quinones reconstitute Q(A) function, while a few will act as Q(B). Nine quinones were tested for their ability to bind and reconstitute Q(A) and Q(B) functions. Only ubiquinone (UQ) reconstitutes both functions in the same protein. The affinities of the non-native quinones for the Q(B) site were determined by a competitive inhibition assay. The affinities of benzoquinones, naphthoquinone (NQ), and 2-methyl-NQ for the Q(B) site are 7 ± 3 times weaker than that at Q(A) site. However, di-ortho-substituted NQs and anthraquinone bind tightly to the Q(A) site (K d ≤ 200 nM), and ≥1,000 times more weakly to the Q(B) site, perhaps setting a limit on the size of the site. With a low-potential electron donor, 2-methyl, 3-dimethylamino-1,4-NQ, (Me-diMeAm-NQ) at Q(A), Q(B) reduction is 260 meV, more favorable than with UQ as Q(A). Electron transfer from Me-diMeAm-NQ at the Q(A) site to NQ at the Q(B) site can be detected. In the Q(B) site, the NQ semiquinone is estimated to be ≈60-100 meV higher in energy than the UQ semiquinone, while in the Q(A) site, the semiquinone energy level is similar or lower with NQ than with UQ. Thus, the NQ semiquinone is more stable in the Q(A) than in the Q(B) site. In contrast, the native UQ semiquinone is ≈60 meV lower in energy in the Q(B) than in the Q(A) site, stabilizing forward electron transfer from Q(A) to Q(B).

Protein Influence on Charge-Asymmetry of the Primary Donor in Photosynthetic Bacterial Reaction Centers Containing a Heterodimer: Effects on Photophysical Properties and Electron Transfer

The substantial electronic distinctions between bacteriochlorophyll (BChl) and its Mg-free analogue bacteriopheophytin (BPh) are exploited in two sets of Rhodobacter capsulatus reaction center (RC) mutants that contain a heterodimeric BChl-BPh primary electron donor (D). The BPh component of the M-heterodimer (Mhd) or L-heterodimer (Lhd) obtains from substituting a Leu for His M200 or for His L173, respectively. Lhd-β and Mhd-β RCs serve as the initial templates in the two mutant sets, where β denotes that the L-side BPh acceptor (HL) has been replaced by a BChl (due to substituting His for Leu M212). Three variants each of Lhd-β and Mhd-β mutants were constructed: (1) a swap (denoted YF) of the native Phe (L181) and Tyr (M208) residues, which flank D and the nearby M- and L-side monomeric BChl cofactors, respectively, giving Tyr (L181) and Phe (M208); (2) addition of a hydrogen bond (denoted L131LH) to the ring V keto group of the L-macrocycle of D, via replacing the native Leu at L131 with His; (3) the combination of 1 and 2. A low yield of electron transfer (ET) to the M-side BPh (HM) is observed in all four Lhd-containing RCs. Comparison with the yield of ET to β on the L-side shows that electron density on the L-macrocycle of D* favors ET to the M-side cofactors and vice versa. Increasing or decreasing the electronic asymmetry of D* via the YF, L131LH mutations or the combination results in consistent trends in the characteristics of the long-wavelength ground state absorption band of D, the rate constant of internal conversion of D* to the ground state, and the rate constants for ET to both the L- and M-side cofactors. A surprising correlation is that an increase in the charge asymmetry in D* not only increases the D* internal-conversion rate constant, but also the rate constants for ET to both the L- and M-side cofactors, spanning time scales of tens of picoseconds to several nanoseconds. The YF swap has a previously unrecognized effect on the electronic asymmetry of D*, resulting in increased charge asymmetry for the Mhd and decreased charge asymmetry for the Lhd. This result indicates that the native Tyr (M208) and Phe (L181) in the wild-type RC promote an electron distribution in P* that is the reverse of that favorable for ET to the photoactive L-branch. This conclusion reinforces the view that the native configuration of these residues promotes ET to the L branch primarily by poising the free energies of the charge-separated states. Overall, this work addresses the extent to which electronic couplings complement energetics in underpinning the directionality of ET in the bacterial RC.

Rate constants of primary reversible reactions of electron transfer do not depend on temperature. Numerical experiment

The process of irreversible photochemical charge separation in photosynthetic bacterial reaction centers is proposed to be characterized by the effective rate constant. A formula to compute this effective rate constant is derived. Similar rate constant was previously considered by R.A. Marcus (Marcus R.A. 1956. J. Chem. Phys.24, 966–978) in order to describe nonphotochemical intermolecular electron transfer. The effective rate constant of the irreversible charge separation in photosynthetic bacterial reaction centers is shown to depend on the temperature. In contrast, rate constants of the forward electron transfer from the excited singlet primary donor to the bacteriochlorophyll and from its ion-radical to the bacteriopheophytin acceptor do not depend on temperature.

Protein dynamics to optimize and control bacterial photosynthesis

Proteins function by sampling conformational sub-states within a given fold. How this configurational flexibility and the associated protein dynamics affect the rates of chemical reactions are open questions. The difficulty in exploring this issue arises in part from the need to identify the relevant nuclear modes affecting the reaction rate for each characteristic time-scale of the reaction. Proteins as reaction media display a hierarchy of such nuclear modes, of increasingly collective character, that produce both a broad spectrum of static fluctuations and a broad spectrum of relaxation times. In order to understand the effect of protein dynamics on reaction rates, we have chosen to study a sub-nanosecond electron transfer reaction between the bacteriopheophytin and primary quinone cofactors of the photosynthetic bacterial reaction center. We show that dynamics affects the activation barrier of the reaction through a dynamical restriction of the configurational space sampled by the protein–water solvent on the reaction time-scale. The modes which become dynamically arrested on the reaction time-scale of hundreds of picoseconds are related to elastic motions of the protein that are strongly coupled to the hydration layer of water. Several mechanistic consequences for protein electron transfer emerge from this picture. Importantly, energy parameters used to define the activation barrier of electron transfer reactions lose their direct connection to equilibrium thermodynamics and become dependent in a very direct way on the relative magnitudes of the reaction and nuclear reorganization time-scales. As a result, the energetics of protein electron transfer need to be defined on each specific reaction time-scale. This perspective offers a mechanism to optimize protein electron transfer by tuning the reaction rate to the relaxation spectrum of the reaction coordinate.

B-side Electron Transfer in Bacterial Photosynthetic Reaction Centers Revealed by a Few-Cycle Pulse Laser

B-side electron transfer in wide-type reaction centers from Rhodobacter sphaeroides 2.4.1 was investigated by broadband real-time vibrational spectroscopy. HB − was found to be generated in less than 50 fs after the excitation of 6.3 fs laser pulse, and its decay lifetime was determined to be ~280fs and 1.4ps, which is extremely fast compared with the one occurring in A-side. Considering the ultrafast of the generation time of HB − , BB rather than P was found to be the initial electron donor to the H. Hence, the initial charge-separated state is determined to be BB + HB − state.

Comparison of calculated and experimental isotope edited FTIR difference spectra for purple bacterial photosynthetic reaction centers with different quinones incorporated into the QA binding site

Previously we have shown that ONIOM type (QM/MM) calculations can be used to simulate isotope edited FTIR difference spectra for neutral ubiquinone in the QA binding site in Rhodobacter sphaeroides photosynthetic reaction centers. Here we considerably extend upon this previous work by calculating isotope edited FTIR difference spectra for reaction centers with a variety of unlabeled and 18O labeled foreign quinones incorporated into the QA binding site. Isotope edited spectra were calculated for reaction centers with 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone (MQ0), 2,3,5,6-tetramethyl-1, 4-benzoquinone (duroquinone, DQ), and 2,3-dimethyl-l,4-naphthoquinone (DMNQ) incorporated, and compared to corresponding experimental spectra. The calculated and experimental spectra agree well, further demonstrating the utility and applicability of our ONIOM approach for calculating the vibrational properties of pigments in protein binding sites. The normal modes that contribute to the bands in the calculated spectra, their composition, frequency, and intensity, and how these quantities are modified upon 18O labeling, are presented. This computed information leads to a new and more detailed understanding/interpretation of the experimental FTIR difference spectra. Hydrogen bonding to the carbonyl groups of the incorporated quinones is shown to be relatively weak. It is also shown that there is some asymmetry in hydrogen bonding, accounting for 10–13 cm−1 separation in the frequencies of the carbonyl vibrational modes of the incorporated quinones. The extent of asymmetry in H-bonding could only be established by considering the spectra for various types of quinones incorporated into the QA binding site. The quinones listed above are “tail-less.” Spectra were also calculated for reaction centers with corresponding “tail” containing quinones incorporated, and it is found that replacement of the quinone methyl group by a phytyl or prenyl chain does not alter ONIOM calculated spectra.

Comparative ENDOR study at 34 GHz of the triplet state of the primary donor in bacterial reaction centers of Rb. sphaeroides and Bl. viridis

The primary electron donor (P) in the photosynthetic bacterial reaction center of Rhodobacter sphaeroides and Blastochloris viridis consists of a dimer of bacteriochlorophyll a and b cofactors, respectively. Its photoexcited triplet state in frozen solution has been investigated by time resolved ENDOR spectroscopy at 34 GHz. The observed ENDOR spectra for (3)P865 and (3)P960 are essentially the same, indicating very similar spin density distributions. Exceptions are the ethylidene groups unique to the bacteriochlorophyll b dimer in (3)P960. Strikingly, the observed hyperfine coupling constants of the ethylidene groups are larger than in the monomer, which speaks for an asymmetrically delocalized wave function over both monomer halves in the dimer. The latter observation corroborates previous findings of the spin density in the radical cation states P 865 (•+) (Lendzian et al. in Biochim Biophys Acta 1183:139-160, 1993) and P 960 (•+) (Lendzian et al. in Chem Phys Lett 148:377-385, 1988). As compared to the bacteriochlorophyll monomer, the hyperfine coupling constants of the methyl groups 2(1) and 12(1) are reduced by at least a factor of two, and quantitative analysis of these couplings gives rise to a ratio of approximately 3:1 for the spin density on the halves PL:PM. Our findings are discussed in light of the large difference in photosynthetic activity of the two branches of cofactors present in the bacterial reaction center proteins.

The Role of Gold-Adsorbed Photosynthetic Reaction Centers and Redox Mediators in the Charge Transfer and Photocurrent Generation in a Bio-Photoelectrochemical Cell

Bacterial photosynthetic reaction centers (RCs) are promising materials for solar energy harvesting, due to their high quantum efficiency. A simple approach for making a photovoltaic device is to apply solubilized RCs and charge carrier mediators to the electrolyte of an electrochemical cell. However, the adsorption of analytes on the electrodes can affect the charge transfer from RCs to the electrodes. In this work, photovoltaic devices were fabricated incorporating RCs from purple bacteria, ubiquinone-10 (Q2), and cytochrome c (Cyt c) (the latter two species acting as redox mediators). The adsorption of each of these three species on the gold working electrode was investigated, and the roles of adsorbed species in the photocurrent generation and the cycle of charge transfer were studied by a series of photochronoamperometric, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and cyclic voltammetry (CV) tests. It was shown that both redox mediators were required for photocurrent generation; hence, the RC itself is likely unable to inject electrons into the gold electrode directly. The reverse redox reactions of mediators at the electrodes generates electrical current. Cyclic voltammograms for the RC-exposed gold electrode revealed a redox couple due to the adsorbed RC at ∼ +0.5 V (vs NHE), which confirmed that the RC was still redox active, upon adsorption to the gold. Photochronoamperometric studies also indicated that RCs adsorb, and are strongly bound to the surface of the gold, retaining functionality and contributing significantly to the process of photocurrent generation. Similar experiments showed the adsorption of Q2 and Cyt c on unmodified gold surfaces. It was indicated by the photochronoamperometric tests that the photocurrent derives from Q2-mediated charge transfer between the RCs and the gold electrode, while solubilized Cyt c mediates charge transfer between the P-side of adsorbed RC and the Pt counter electrode. Also, the stability of the adsorbed RCs and mediators was evaluated by measuring the photocurrent response over a period of 1 week. It is found that ∼46% of the adsorbed RCs remain active after a week under aerobic conditions. A significantly extended lifetime is expected by removing oxygen from the electrolyte and sealing the device.

Mechanism by which a single water molecule affects primary charge separation kinetics in a bacterial photosynthetic reaction center of Rhodobacter sphaeroides

Using quantum-chemical methods, we have studied the role played by water molecules W-A and W-B that are bound by hydrogen bonds to accessory bacteriochlorophyll molecules BA and BB in the process of primary charge separation in the reaction center of Rhodobacter Sphaeroides. We have found that the occurrence of a rotational mode of the W-A molecule at 32 cm−1 and/or its harmonics in stimulated emission of an electron donor P* and the dynamics of population of the states P+BA− and P+HA− may be related to the structural heterogeneity of the reaction center and the existence of a conformation in which the W-A molecule is predominantly involved in one hydrogen bond (with BA). Based on the calculated redox potentials BA and P, it has been shown that the appearance of the W-A molecule in the reaction center reduces the energy of the P+BA− state by ∼600 cm−1. This is somewhat smaller than the influence of the amino-acid residue TyrM210 (∼870 cm−1) and correlates well with a substantial decrease in the electron transfer rate in mutant forms of reaction centers GM203L (which do not contain W-A molecules) and YM210F (in which TyrM210 is replaced with Phe). The data obtained allow us to suggest that rotation of the water molecule with a fixed position of its H atom that is involved in a hydrogen bond with the keto carbonyl group of BA is initiated due to the charge separation between the halves of special pair P and the formation of the state PA+PB−. The large effect of this rotation on the kinetics of population of the states P+BA− and P+HA− after the excitation of P is quite consistent with its influence on the energy of the state P+BA−.