Reaction Centers Of Rhodobacter Sphaeroides Research Articles

Structures and Vibrational Frequencies of the Quinones in Rb. sphaeroides Derived by a Combined Density Functional/Molecular Mechanics Approach

The structures of the ubiquinones QA and QB in the bacterial photosynthetic reaction center of Rhodobacter sphaeroides are calculated by a quantum mechanical/molecular mechanics hybrid technique. T...

Excitation relaxation in the chemically modified photosynthetic reaction center from Rhodobacter sphaeroides 601

The ultrafast excitation relaxation in the sodium borohydride-treated reaction center of Rhodobacter sphaeroides 601 was investigated with selective excitation. From the femtosecond pump-probe measurement at 790 nm, the excitation relaxation demonstrates a biexponential decay with time constants of about 200 fs and 1.4 ps. By comparison with the result from sodium ascorbate-pretreated modified RS601, it could be concluded that the dynamical trace at 790 nm mainly originates from the contribution of accessory bacteriochlorophyll in the active side, and the electrochromic shift arising from the induced positive charge on the special pair primarily affects the absorption band in the red region of the accessory bacteriochlorophyll in RS601. With direct excitation of the special pair, the charge separation and subsequent electron transfer were observed in borohydride-modified RS601. The 2.8 ps component was ascribed to the charge separation and electron transfer from P* to H(A). From the dynamical traces at 790, 800 and 818 nm, the ultrafast energy relaxation from the excited accessory bacteriochlorophyll in the active side is consistent with a two-step energy transfer mechanism. This dynamical observation in modified RS601 is of significance in understanding the physical mechanism of excitation relaxation and energy transfer in the photosynthetic primary process.

The effect of exchange of bacteriopheophytin a with plant pheophytin a on charge separation in Y(M210)W mutant reaction centers of Rhodobacter sphaeroides at low temperature

The bacteriopheophytin a molecules at the H A and H B binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of H A −, presumably by raising the free energy level of the P +B A − state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll B A. Exchange of the bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of H A, inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P +B B − state, where B B is the accessory bacteriochlorophyll in the “inactive” B-branch of the cofactors. The B B molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Q y region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P +B B − state and absorption properties of the B B bacteriochlorophyll in the mutant RCs due to a long-range effect of the Y(M210)W mutation on the protein environment of the B B binding pocket.

Effect of binding of Cd2+ on bacterial reaction center mutants: proton-transfer uses interdependent pathways.

In bacterial reaction center of Rhodobacter sphaeroides, Cd2+ binds in stoichiometric amount to the protein. In the wild type, this results into a notable decrease of the rates of electron-transfer between the two quinone acceptors after the first (kAB(1)) and second flash (kAB(2)). We have studied these effects in two single mutants, L209PY and L209PF. L209Pro is situated in a protein region rich in hydrogen-bond networks involving water molecules. We show that (1) the combined effects of Cd2+ binding and point mutations have a cumulative consequence in the two mutants, decreasing very substantially the observed rates of electron-transfer. Interestingly, the [Cd2+] titration curves of kAB(2) in the L209PY and L209PF mutants are nearly superimposable to those previously reported for the M17DN and L210DN mutants (Paddock, M. L., Feher, G., and Okamura, M. Y. (2000) Proc. Natl. Acad. Sci U.S.A. 97, 1548-1553). These observations suggest a common effect of all of these mutations (L209, M17, L210) on the protonation state of the histidine cluster to which Cd2+ binds; (2) in the L209PY mutant, the pH titration curves of kAB(1), kAB(2), and k(H)(+), the proton-transfer rate at the second flash, are systematically downshifted by 1.5-2 pH units in the presence of 300 microM Cd2+, similarly to the wild type RCs (Gerencser, L., and Maroti, P. (2001) Biochemistry 40, 1850-1860). We propose that Cd2+ binding influences the electrostatics of interdependent ways of proton penetration within the protein, involving at least, directly or indirectly, L209P, L210D, and M17D, probably in conjunction with hydrogen-bonded connected water molecules.

Quantum chemical study of π–π stacking interactions of the bacteriochlorophyll dimer in the photosynthetic reaction center of Rhodobacter sphaeroides

Intermolecular π–π stacking interactions of the bacteriochlorophyll dimer in the photosynthetic reaction center of the purple bacterium Rhodobacter sphaeroides were analyzed by the second order Møller–Plesset perturbation method using the modified 6-31G*(0.25) basis set with diffuse d-polarization by Hobza and co-workers. MP2/6-31G*(0.25) calculations yield an intermolecular interaction energy of −21.50 kcal/mol for the bacteriochlorophyll dimer. Thus, the attractive nature of the π–π stacking interaction of the bacteriochlorophyll dimer in the photosynthetic reaction center from Rhodobacter sphaeroides is, for the first time, firmly established.

(13)C MAS NMR and photo-CIDNP reveal a pronounced asymmetry in the electronic ground state of the special pair of Rhodobacter sphaeroides reaction centers.

Reaction centers of wild-type Rhodobacter sphaeroides were selectively (13)C-isotope labeled in bacteriochlorophyll and bacteriopheophytin. (13)C solid-state CP/MAS NMR and photo-CIDNP were used to provide insight into the electronic structure of the primary electron donor and acceptor on the atomic scale. The first 2-dimensional photochemically induced dynamic nuclear polarization (photo-CIDNP) (13)C-(13)C solid-state MAS NMR spectra reveal that negative charging of the two BChl rings of the primary donor is involved in ground-state tuning of the oxidation potential of these cofactors in the protein via local electrostatic interactions. In particular, the (13)C shifts show moderate differences in the electronic structure between the two BChl molecules of the special pair in the electronic ground state, which can be attributed to hydrogen bonding of one of the BChl molecules. The major fraction of the electron spin density is strongly delocalized over the two BChl molecules of the special pair and the photochemically active BPhe. A small fraction of the pi-spin density is distributed over a fourth component, which is assigned to the accessory BChl. Comparison of the photo-CIDNP data with "dark" NMR spectra obtained in ultra high field indicates a rigid special pair environment upon photoreaction and suggests that structural changes of the aromatic macrocycles of the two BChl molecules of the special pair do not significantly contribute to the reorganization energy associated with the charge-transfer process.

Electrostatic interactions in an integral membrane protein.

The effects of charge-charge interactions on the midpoint reduction potential (E(m)()) of the primary electron donor (P) in the photosynthetic reaction center of Rhodobacter sphaeroides were investigated by introducing mutations of ionizable amino acids at selected sites. The mutations were designed to alter the electrostatic environment of P, a bacteriochlorophyll dimer, without greatly affecting its structure or molecular orbitals. Two arginine residues at homologous positions in the L and M subunits [residues (L135) and (M164)], Asp (L155), Tyr (L164), and Cys (L247) were changed independently. Arginine (L135) was replaced by Lys, Leu, Gln, or Glu; Arg (M164), by Leu or Glu; Asp (L155), by Asn; Tyr (L164), by Phe; and Cys (L247), by Lys or Asp. The R(L135)E/C(L247)K double mutant also was made. The shift in the E(m)() of P/P(+) was measured in each mutant and was compared with the effect predicted by electrostatics calculations using several different computational approaches. A simple distance-dependent dielectric screening factor reproduced the effects remarkably well. By contrast, microscopic methods that considered the reaction field in the protein and solvent but did not include explicit counterions overestimated the changes in the E(m)() considerably. Including counterions for the charged residues reduced the calculated effects of the mutations in molecular dynamics calculations. The results show that electrostatic interactions of P with ionizable amino acid residues are strongly screened, and suggest that counterions make major contributions to this screening. The screening also could reflect penetration of water or other relaxations not taken into account because of incomplete sampling of configurational space.

Double mutant studies identify electrostatic interactions that are important for docking cytochrome c2 onto the bacterial reaction center.

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.

Selective perturbation of the second electron transfer step in mutant bacterial reaction centers

In order to specifically perturb the primary electron acceptor B A — a monomeric bacteriochlorophyll (BChl) a — involved in bacterial photosynthetic charge separation (CS), the protein environment of B A in the reaction center (RC) of Rhodobacter sphaeroides was modified by site-directed mutagenesis. Isolated RCs were characterized by redox titrations, low temperature optical spectroscopy, ENDOR/TRIPLE resonance spectroscopy and femtosecond time-resolved spectroscopy. Two mutations were studied: In the GS(M203) mutant a serine is introduced near the ring E keto group of B A, while in FY(L146) a phenylalanine near the ring A acetyl group of B A is replaced by tyrosine. In all mutations the oxidation potential of the primary electron donor P as well as the electronic structure of both the P + radical cation and the radical anion of the secondary electron acceptor, H A − , are not significantly altered compared to the wild type (WT), while changes of the optical absorption spectra at 77 K in the BChl Q X and Q Y regions are observed. The GS(M203) mutation only leads to a minor retardation of the CS reactions at room temperature, whereas for FY(L146) significant deviations from the native electron transfer (ET) rates could be detected: In addition to a faster first (2.9 ps) and a slower second (1 ps) ET step, a new 8-ps time constant was found in the FY(L146) mutant, which can be ascribed to a fraction of RCs with slowed down secondary ET. The results allow us to address the functional role of the acetyl group of B A and question the role of the free energy changes as the main determining factor of ET rates in RCs. It is concluded that structural rearrangements alter the electronic coupling between the pigments and thereby influence the rate of fast CS.

Protein Conformational Gate Controlling Binding Site Preference and Migration for Ubiquinone-B in the Photosynthetic Reaction Center of Rhodobacter sphaeroides

An abundance of X-ray-derived structural data exists for the photosynthetic reaction center of the purple bacteria Rhodobacter sphaeroides; yet, the preferred binding site of the secondary electron...

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to Q B in Rhodobacter sphaeroides reaction centers

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q A −Q B→Q AQ B − ( k AB (1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k AB (1), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q B [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4–29 ms following a flash showed peaks at 1670 (−), 1601 (−) and 1467 (+) cm −1, characteristic of Q A reduction. The time evolution of the spectra shows reoxidation of Q A − and concomitant reduction of Q B with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q B − occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm −1 [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q B in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q A − to Q B and the identification of Glu-L212 as the main proton acceptor in the state Q AQ B −.

Comparison of M-Side Electron Transfer in Rb. sphaeroides and Rb. capsulatus Reaction Centers

Subpicosecond time-resolved absorption and steady-state resonance Raman studies are reported for Rhodobacter sphaeroides reaction centers (RCs) that incorporate the G(M203)D/L(M214)H double mutatio...

B Side Electron Transfer in a Rhodobacter sphaeroides Reaction Center Mutant in Which the B Side Monomer Bacteriochlorophyll Is Replaced with Bacteriopheophytin: Low-Temperature Study and Energetics of Charge-Separated States

The mutation, HL(M182), in the Rhodobacter sphaeroides reaction center (RC) results in the replacement of the monomer bacteriochlorophyll (BChl) on the inactive side (B side) of the RC with a bacteriopheophytin (BPheo; the new cofactor is referred to as φB). In φB-containing RCs, the first excited state of the primary donor (P*) decays with an accelerated time constant of 2.6 ± 0.1 ps at room temperature as compared to 3.1 ± 0.2 ps in wild-type (WT) RCs. At low temperatures, P* decay is essentially the same in the HL(M182) mutant and WT RCs: 1.4 ± 0.1 ps at 77 K and 1.1 ± 0.1 ps at 9 K. The yield of the charge-separated P+φB- state decreases from 35% at room temperature to 12% at 77 and 9 K. The decreased P+φB- yield is apparently due to the fact that the rate of the charge separation along the A side of the RC at low temperature increases, while the rate along the B side remains essentially unchanged. From measurements of the long-lived fluorescence decay at room temperature, the standard free energy of the P+φB- state is estimated to be about 0.16 ± 0.04 eV below P*. Given a difference between the midpoint potentials of BChl and BPheo of 0.26 ± 0.03 V, the standard free energy of the P+BB- state in WT RC is estimated to be 0.1 ± 0.07 eV above P*.

Theoretical studies on the mechanism of primary electron transfer in the photosynthetic reaction center of Rhodobacter sphaeroides.

The mechanism of the primary electron transfer (ET) process in the photosynthetic reaction center (PRC) of Rhodobacter sphaeroides has been studied with quantum chemistry method of ab initio density functional theory (DFT) (B3LYP/6-31G) based on the optimized X-ray crystallographic structure. The calculation was carried out on different structural levels. The electronic structure of pigment molecules was first studied, and then the influence of the neighboring protein was taken into account at three approximation levels: (a) the surrounding proteins were treated as a homogeneous medium with a uniform dielectric constant (SCRF); (b) both the influence of axial coordination of His to the special pair P and ABChl as, and the hydrogen bonds between related residues and P and also BPhas were included; and (c) the influence of the electronic structure of the protein subunit chains as a whole was studied. The results suggest that: (1) according to the composition of the HOMO and LUMO of P, there might be a charge-separated state of (BChl(L) (+)BChl(M) (-)) for the excited state of P; (2) to treat the protein surroundings as a homogeneous medium is not sufficient. Different interactions between pigment molecules and related residues play different roles in the ET process; (3) the axial coordination of His to P raises the E (LUMO) of P greatly, and it is very important for the ET process to occur in the PRC of wild-type bacterium; the axial coordination of His to ABChl as also raises their E (LUMO) significantly; (4) the hydrogen-bonds between amino acid residues and P and also BPh as depress the E (LUMO) of the pigment molecules to some extent, which makes the E (LUMO) of P lower than those of ABChlas, and the E (LUMO) of BPh a (L) lower than that of BPh a (M). Consequently, the ET process from P to BPh a (L) does not, according to our calculation model, occur via ABChl a (L). The possibility of the ET pathway from P to BPh a (L) via ABChl a (L) was discussed; (5) the frontier orbitals of protein subunit chains L and M are localized at the random coil area and the alpha-helix areas, respectively. Results mentioned above support the fact that the ET process proceeds in favourable circumstances along the branch L.

A mathematical model for quinone-herbicide competition in the reaction centres of Rhodobacter sphaeroides.

A quantitative model describing the amplitude of semiquinone absorption in photosynthetic reaction centres after successive flashes in the presence of increasing inhibitor concentration is presented. By using relatively simple algebra, the semiquinone signals can be calculated and fitted to the oscillation pattern by optimizing only two parameters; the electron and quinone equilibrium constants, Ke and Kq, respectively. In this work we expand our earlier model [Tandori et al. (1991) Photosynthetica 25, 159-166] by introducing the inhibitor equilibrium constant, Ki, describing the best fit of the model to the measured oscillation pattern. We found that there are characteristic parameters of the measured and normalized signal, and of those calculated from the mathematical model, which fit well with competitive Michaelis-Menten kinetics.

Contribution of the processes of solvation of nonequilibrium states of cofactors to charge separation and electron transfer in reaction centers of Rhodobacter sphaeroides.

Contribution of the processes of solvation of nonequilibrium states of cofactors to charge separation and electron transfer in reaction centers of Rhodobacter sphaeroides.

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis.

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, beta, for H(A) [Kirmaier et al. (1991) Science 251: 922-927] with two mutations, G(M203)D and Y(M210)W, near B(A), we have created a double and a triple mutant with long lifetimes of the excited state P(*) of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P(+)Q(A) (-) formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P(+)H(B) (-) formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P(*) lifetime of 15 ps [Heller et al. (1995) Science 269: 940-945]. We conclude that the lifetime of P(*) is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q(B), was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q(A) does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9-26]. It is shown that B-branch electron transfer leading to P(+)Q(B) (-) formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P(+)Q(B) (-) formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P(+)Q(B) (-) state under continuous illumination were not successful. Charge recombination of P(+)Q(B) (-) formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P(+)Q(B) (-) trapped in wildtype RCs (10(5) s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780-5786]. This difference is discussed in light of the different binding sites for Q(B) and Q(B) (-) that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812-816]. We present the first low-temperature absorption difference spectrum due to P(+)Q(B) (-).

Effects of oxygen on the dark recombination between photoreduced secondary quinone and oxidized bacteriochlorophyll in Rhodobacter sphaeroides reaction centers.

The influence of duration of exposure to actinic light (from 1 sec to 10 min) and temperature (from 3 to 35 degrees C) on the temporary stabilization of the photomobilized electron in the secondary quinone acceptor (QB) locus of Rhodobacter sphaeroides reaction centers (RC) was studied under aerobic or anaerobic conditions. Optical spectrophotometry and ESR methods were used. The stabilization time increased significantly upon increasing the exposure duration under aerobic conditions. The stabilization time decreased under anaerobic conditions, its dependence on light exposure duration being significantly less pronounced. Generation of superoxide radical in photoactivated aerobic samples was revealed by the ESR method. Possible interpretation of the effects is suggested in terms of interaction between the semiquinone QB with oxygen, the interaction efficiency being determined by the conformational transitions in the structure of RC triggered by actinic light on and off.

Effect of isotope substitution and controlled dehydration on the photoinduced electron transport reactions of quinone acceptors and multiheme cytochrome C in bacterial photosynthetic reaction center.

Isotope substitution of H2O by 2H2O causes an increase in the rate of dark recombination between photooxidized bacteriochlorophyll (P+) and reduced primary quinone acceptor in Rhodobacter sphaeroides reaction centers (RC) at room temperature. The isotopic effect declines upon decreasing the temperature. Dehydration of RC complexes of Ectothiorhodospira shaposhnikovii chromatophores containing multiheme cytochrome c causes a decrease in the efficiency of transfer of a photomobilized electron between the primary and secondary quinone acceptors and from cytochrome to P+. In the case of H2O medium these effects are observed at a lower hydration than in 2H2O-containing medium. In the E. shaposhnikovii chromatophores subjected to dehydration in H2O, the rate of electron transfer from the nearest high-potential cytochrome heme to P+ is virtually independent of hydration within the P/P0 range from 0.1 to 0.5. In samples hydrated in 2H2O this rate is approximately 1.5 times lower than in H2O. However, the isotopic effect of this reaction disappears upon dehydration. The intramolecular electron transfer between two high-potential hemes of cytochrome c in samples with 2H2O is inhibited within this range of P/P0, whereas in RC samples with H2O there is a trend toward gradual inhibition of the interheme electron transfer with dehydration. The experimental results are discussed in terms of the effects of isotope substitution and dehydration on relaxation processes and charge state of RC on implementation of the reactive states of RC providing electron transfer control.

Role Played by Water–Protein Surrounding of Cofactors in Picosecond Electron Transport Steps in Photosynthesis Reaction Centers

Effect of the water–protein environment of cofactors on the rates and efficiency of conversion of light energy into the energy of a photochemical potential in reaction centers of purple bacterium Rhodobacter sphaeroides is studied. The environment is modified by isotopic replacement D2O → H2O or by adding glycerol and dimethyl sulfoxide (DMSO). The replacement D → H is shown to makes no impact on the midpoint potential Em of the electron donor, whereas addition of 70 vol % of glycerol or 35 vol % of DMSO raises Em by 30 and 45 mV, respectively. Rate constants of charge separation ke and electron transport onto quinone kQ remained unchanged following glycerol addition, while deuteration and DMSO addition diminished ke and kQ by two to three times. In addition to the known component with a characteristic time of about 10 ns, a component with a duration of 0.5–0.8 ns appears in the recombination kinetics of charges of deuterated and DMSO-treated preparations of reaction centers. The mechanism of the environment response on the emergence of nonequilibrium states of cofactors is analyzed theoretically. The energy model proposed for primary processes of photosynthesis accounts for the contribution made by the environment in the realization of a highly effective electron transport.