Photosynthetic Reaction Center Of Rhodobacter Sphaeroides Research Articles

Structural and kinetic properties of Rhodobacter sphaeroides photosynthetic reaction centers containing exclusively Zn-coordinated bacteriochlorophyll as bacteriochlorin cofactors

The Zn-BChl-containing reaction center (RC) produced in a bchD (magnesium chelatase) mutant of Rhodobacter sphaeroides assembles with six Zn-bacteriochlorophylls (Zn-BChls) in place of four Mg-containing bacteriochlorophylls (BChls) and two bacteriopheophytins (BPhes). This protein presents unique opportunities for studying biological electron transfer, as Zn-containing chlorins can exist in 4-, 5-, and (theoretically) 6-coordinate states within the RC. In this paper, the electron transfer perturbations attributed exclusively to coordination state effects are separated from those attributed to the presence, absence, or type of metal in the bacteriochlorin at the HA pocket of the RC. The presence of a 4-coordinate Zn2+ ion in the HA bacteriochlorin instead of BPhe results in a small decrease in the rates of the P*→P+HA−→P+QA− electron transfer, and the charge separation yield is not greatly perturbed; however coordination of the Zn2+ by a fifth ligand provided by a histidine residue results in a larger rate decrease and yield loss. We also report the first crystal structure of a Zn-BChl-containing RC, confirming that the HA Zn-BChl was either 4- or 5-coordinate in the two types of Zn-BChl-containing RCs studied here. Interestingly, a large degree of disorder, in combination with a relatively weak anomalous difference electron density was found in the HB pocket. These data, in combination with spectroscopic results, indicate partial occupancy of this binding pocket. These findings provide insights into the use of BPhe as the bacteriochlorin pigment of choice at HA in both BChl- and Zn-BChl-containing RCs found in nature.

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.

Hydrogen Bonding between the QB Site Ubisemiquinone and Ser-L223 in the Bacterial Reaction Center: A Combined Spectroscopic and Computational Perspective

In the Q(B) site of the Rhodobacter sphaeroides photosynthetic reaction center, the donation of a hydrogen bond from the hydroxyl group of Ser-L223 to the ubisemiquinone formed after the first flash is debatable. In this study, we use a combination of spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations to comprehensively explore this topic. We show that ENDOR, ESEEM, and HYSCORE spectroscopic differences between mutant L223SA and the wild-type sample (WT) are negligible, indicating only minor perturbations in the ubisemiquinone spin density for the mutant sample. Qualitatively, this suggests that a strong hydrogen bond does not exist in the WT between the Ser-L223 hydroxyl group and the semiquinone O(1) atom, as removal of this hydrogen bond in the mutant should cause a significant redistribution of spin density in the semiquinone. We show quantitatively, using QM/MM calculations, that a WT model in which the Ser-L223 hydroxyl group is rotated to prevent hydrogen bond formation with the O(1) atom of the semiquinone predicts negligible change for the L223SA mutant. This, together with the better agreement between key QM/MM calculated and experimental hyperfine couplings for the non-hydrogen-bonded model, leads us to conclude that no strong hydrogen bond is formed between the Ser-L223 hydroxyl group and the semiquinone O(1) atom after the first flash. The implications of this finding for quinone reduction in photosynthetic reaction centers are discussed.

Tuning the Midpoint Potential of the Primary Quinone in Rhodobacter Sphaeroides Photosynthetic Reaction Centers by Mutation of Residue M265

The photosynthetic reaction center from Rhodobacter sphaeroides tunes the midpoint potential (Em) of its quinones to allow favorable electron transfer from the primary (QA) to secondary (QB) quinone. Previous work showed that mutation of isoleucine residue 265 of the M subunit (IleM265) to the polar residues serine or threonine lowers the Em by 80 or 100 mV, respectively [1]. We have now solved the x-ray crystal structures of these mutants, and the structures do not support the proposal that the Em drop is caused by lengthening of hydrogen bonds to the quinone. Instead, we hypothesize that the major contribution comes from change to the electrostatic potential at the QA site, and we have characterized additional mutants at this site. Mutation of IleM265 to asparagine shows a faster QA back reaction (kPA=19 s−1 at pH 8.0) and slower QB back reaction (kPB=0.12 s−1 at pH 8.0) than wild type, but similar to the threonine mutant. The estimated equilibrium potential between QA and QB (KAB) for the asparagine mutant is 156, essentially the same as the threonine mutant (157). However, the IleM265 to glutamine mutant has a slower kPA (6.3 s−1 at pH 8.0) and a slower kPB (0.51 s−1 at pH 8.0) than wild type. The estimated KAB of the glutamine mutant is similar to the wild type (11 vs. 10, respectively), indicating no significant change in Em of QA. The anomalous behavior of the glutamine mutant may be due to restricted freedom of rotation of the longer glutamine side chain, likely clashing with the quinone and preventing adoption of a conformation with an electrostatic minimum energy.[1] Wells et al. (2003) Biochemistry 42, 4064-4074

Properties of Rhodobacter sphaeroides photosynthetic reaction center with double amino acid substitution I(L177)H+H(M182)L

Histidine M182 in the reaction center (RC) of Rhodobacter sphaeroides serves as the fifth ligand of the bacteriochlorophyll (BChl) B(B) Mg atom. When this His is substituted by an amino acid that is not able to coordinate Mg, bacteriopheophytin appears in the B(B) binding site instead of BChl (Katilius, E., et al. (1999) J. Phys. Chem. B, 103, 7386-7389). We have shown that in the presence of the additional mutation I(L177)H the coordination of the BChl B(B) Mg atom in the double mutant I(L177)H+H(M182)L RC still remains. Changes in the double mutant RC absorption spectrum attributed to BChl absorption suggest that BChl B(B) Mg atom axial ligation might be realized not from the usual α-side of the BChl macrocycle, but from the opposite, β-side. Weaker coordination of BChl B(B) Mg atom compared to the other mutant RC BChl molecules suggests that not an amino acid residue but a water molecule might be a possible ligand. The results are discussed in the light of the structural changes that occurred in the RC upon Ile/His substitution in the L177 position.

Modeling charge transfer in the photosynthetic reaction center.

In this work, we present a model to elucidate the unidirectionality of the primary charge-separation process in the bacterial reaction centers. We have used a model of three sites/molecules with electron transfer beginning at site 1 with an option to proceed to site 2 or site 3. We used a stochastic model with arbitrary correlation functions. We get the quantum yields of electron escape via the sites 2,3 in two limiting cases that correspond to a spectral density of underdamped and overdamped Brownian oscillator. In the fast modulation limit of an overdamped regime we get the effect, which was named "fear of death," in which for strong enough sink parameters the electron has a tendency to avoid the place with greater sink. The presented model was used to provide a plausible explanation of the temperature dependence of the quantum yields of the Rhodobacter sphaeroides photosynthetic reaction center in the high-temperature regime.

A D-band (130 GHz) EPR study of the primary electron donor triplet state in photosynthetic reaction centers ofRhodobacter sphaeroides R26

A high-field (D-band, 130 GHz) electron spin echo-detected spectrum of the primary electron donor triplet state,3P, in quinone-depleted photosynthetic reaction centers from the bacteriumRhodobacter sphaeroides R26 is obtained. It shows a significantg-anisotropy, which is larger than that of the primary donor oxidized state, P+⋅. Simulation gives the tripletg-tensor principal values of 2.0037, 2.0028, and 2.0022 (precision ±0.0001), assuming that theg-tensor is coaxial to a zerofield splitting tensor. The3P spectral lineshape reveals an orientational anisotropy of the triplet quantum yield. We explain this anisotropy as arising from the difference in the main values and relative orientations between theg-tensors of P+⋅ and I A −⋅ in the primary radical pair (the triplet state’s precursor).

Effects of extraction of the H-subunit from Rhodobacter sphaeroides reaction centers on relaxation processes associated with charge separation.

Effects of extraction of the H-subunit from Rhodobacter sphaeroides photosynthetic reaction centers (RC) on the characteristics of the photoinduced conformational transition associated with electron transfer between photoactive bacteriochlorophyll and primary quinone acceptor were studied. Extraction of the H-subunit (i.e., the subunit that is not directly bound to electron transfer cofactors) was found to have a significant effect on the dynamic properties of the protein--pigment complex of the RC, the effect being mediated by modification of parameters of the relaxation processes associated with charge separation.

Reduction and protonation of the secondary quinone acceptor of Rhodobacter sphaeroides photosynthetic reaction center: kinetic model based on a comparison of wild-type chromatophores with mutants carrying Arg→Ile substitution at sites 207 and 217 in the L-subunit

After the light-induced charge separation in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone Q A, the loosely bound ubiquinone Q B. After two subsequent flashes of light, Q B is reduced to ubiquinol Q BH 2, with a semiquinone anion Q B − formed as an intermediate after the first flash. We studied Q BH 2 formation in chromatophores from Rb. sphaeroides mutants that carried Arg→Ile substitution at sites 207 and 217 in the L-subunit. While Arg-L207 is 17 Å away from Q B, Arg-L217 is closer (9 Å) and contacts the Q B-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated Δ G AB, the free energy difference between the Q A −Q B and Q AQ B − states, and p K 212, the apparent p K of Glu-L212, a residue that is only 4 Å away from Q B. As expected, the replacement of positively charged arginines by neutral isoleucines destabilized the Q B − state in the L217RI mutant to a larger extent than in the L207RI one. Also as expected, p K 212 increased by ∼0.4 pH units in the L207RI mutant. The value of p K 212 in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q A −Q B −→Q AQ BH 2 transition upon the second flash, as monitored by electrometry via the accompanying changes in the membrane potential, was two times faster in the L207RI mutant than in the wild-type, but remained essentially unchanged in the L217RI mutant. To rationalize these findings, we developed and analyzed a kinetic model of the Q A −Q B −→Q AQ BH 2 transition. The model properly described the available experimental data and provided a set of quantitative kinetic and thermodynamic parameters of the Q B turnover. The non-electrostatic, ‘chemical’ affinity of the Q B site to protons proved to be as important for the attracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estimated from the difference between the experimentally established p K 212 shifts and the expected changes in the electrostatic potential at Glu-L212, calculable from the X-ray structure of the RC. Based on functional studies, structural data and kinetic modeling, we suggest a mechanistic scheme of the Q B turnover. The detachment of the formed ubiquinol from its proximal position next to Glu-L212 is considered as the rate-limiting step of the reaction cycle.

The Mechanism of Triplet Energy Transfer from the Special Pair to the Carotenoid in Bacterial Photosynthetic Reaction Centers

The kinetics of triplet energy transfer from the primary electron donor P, a bacteriochlorophyll dimer, to the carotenoid were measured in the M182HL mutant of Rhodobacter sphaeroides photosynthetic reaction centers. In this mutant, the accessory bacteriochlorophyll that is bound in the BM position in wild-type reaction centers is changed to a bacteriopheophytin. By using phosphorescence data for P in the reaction center and for bacteriochlorophyll a and bacteriopheophytin a monomers in simple solvents, the activation energy for triplet energy transfer from the special pair to the bacteriopheophytin at BM in M182HL is predicted to be 910 ± 50 cm-1. Analysis of the decay kinetics of 3P as a function of temperature gives an experimental activation energy of 950 ± 50 cm-1. Thus, the M182HL mutant allows for prediction and quantitative verification of the energetics of triplet energy transfer by a two-step mechanism from the special pair to the carotenoid through the chromophore in the BM binding site.

Effect of temperature and surface potential on the electrogenic proton uptake in the Q B site of the Rhodobacter sphaeroides photosynthetic reaction center: Q A −. B −. → Q A Q B H 2 transition

Direct electrometry was used to study the light-induced voltage changes in the Rhodobacter sphaeroideschromatophores adsorbed to a phospholipid-impregnated nitrocellulose film. After the second laser flash, a fast increase in the voltage associated with charge separation was followed by a slower increase attributed to the proton uptake in the QB site of the photosynthetic reaction centers. Kinetics and relative amplitudes of these voltage changes attributed to the QA QB ! QAQBH2 transition, were measured as a function of pH and temperature between +4 and +40C. The kinetics can be approximated by a single exponent above +23C (100 sa t +25C, pH 7.2), whereas below this temperature, it was a good fit of two exponential approximation (65 s and 360s with similar contributions at +10C, pH 7.2). The faster component diminished with an apparent pK8.5, whereas the slower one was maintained at a constant level until pH9.5 and then decreased. The calculated activation energy from the temperature dependence of the slower component (55 ‐ 65 kJ/mol) was much higher than that of the faster component (< 10 kJ/mol). The two voltage components can be attributed to the transfer of the first (faster component) and the second (slower component) proton from the reaction center surface to QB. We suggested that higher activation energy of the slower component was due to a conformational change in the reaction center kinetically coupled to the second proton transfer to Q BH . The faster component diminished in the presence of 1 M KCl, with an apparent pK7.5. To explain this observation, we assume that: (i) the midpoint potential of the QA/QA redox pair was higher in 1 M KCl because of the reduced surface potential of chromatophores; (ii) the midpoint potential of the Q B /QBH redox pair was insensitive to the surface potential change; (iii) the equilibrium constant of the reaction Q A QB $ QAQBH decreased at high ionic strength.

Temperature dependence of the electrogenic reaction in the Q B site of the Rhodobacter sphaeroides photosynthetic reaction center: the Q A−Q B→Q AQ B− transition

The temperature dependencies for the kinetics and relative amplitudes of electrogenic reaction(s) coupled with the first reduction of the secondary quinone acceptor Q B were measured with dark-adapted chromatophores of Rhodobacter sphaeroides. The kinetics, while acceptably fitted by a single exponent at room temperature, clearly split into two components below 15°C (rise times, 25 μs and 300 μs at pH 7.0 and 10°C) with the slow phase ousting the fast one at pH>9.0. The activation energies of the fast and slow phases were estimated at pH 7.0 as <10 kJ/mol and 60–70 kJ/mol, respectively. To explain the kinetic heterogeneity of the Q B→Q B − transition, we suggest two possible conformations for the neutral oxidized ubiquinone at the Q B site: one with a hydrogen bond between the side chain carboxyl of Glu-L212 and the methoxy oxygen at C 3 of the Q B ring (Q B−H-Glu centers) and the other one, without this bond (Q B:Glu − centers). The fast phase is attributed to Q A −Q B–H–Glu→Q AQ B–H–Glu transition, whereas the slow one to the Q A −Q B:Glu −→Q A −Q B–H–Glu→Q AQ B −–H–Glu transition.

Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron-proton transfer.

High resolution x-ray diffraction data from crystals of the Rhodobacter sphaeroides photosynthetic reaction center (RC) have been collected at cryogenic temperature in the dark and under illumination, and the structures were refined at 2.2 and 2.6 angstrom resolution, respectively. In the charge-separated D+QAQB- state (where D is the primary electron donor (a bacteriochlorophyll dimer), and QA and QB are the primary and secondary quinone acceptors, respectively), QB- is located approximately 5 angstroms from the QB position in the charge-neutral (DQAQB) state, and has undergone a 180 degrees propeller twist around the isoprene chain. A model based on the difference between the two structures is proposed to explain the observed kinetics of electron transfer from QA-QB to QAQB- and the relative binding affinities of the different ubiquinone species in the QB pocket. In addition, several water channels (putative proton pathways) leading from the QB pocket to the surface of the RC were delineated, one of which leads directly to the membrane surface.

An X-ray absorption study of chemically modified bacterial photosynthetic reaction centers

Local structures around metal atoms in Ni and Zn substituted 13 2-hydroxy-bacteriochlorophyll a (Ni-HOB, and Zn-HOB) with and without insertion into the accessory bacteriochlorophyll a (BChla) sites in Rhodobacter sphaeroides photosynthetic reaction centers (RC) are studied using X-ray absorption spectroscopy. The penta-coordination of Ni-HOB and Zn-HOB in the RC is similar to that of BChl a in native RC, where the fifth ligand is from a nearby histidine residue. Average NiN and ZnN distances increase by 0.08 Å in Ni-HOB and decrease by 0.04 Å in Zn-HOB-Et 2O as the modified chlorophylls bind with the RC due to the interaction between the protein and the macrocycle.

Step-Scan FT-IR Spectroscopy of Electron Transfer in the Photosynthetic Bacterial Reaction Center

Step-scan light-induced FT-IR difference spectra (1800–1200 cm−1) of Rhodobacter sphaeroides photosynthetic bacterial reaction centers at low temperature and with a 10-μs time resolution are presented. The molecular changes and relaxation processes accompanying the photoxidation of the primary electron donor ( P) are already present 10 μs after excitation. The photoconversion of P to the triplet state gives rise to characteristic IR absorption changes which decay with a half-time of 130 μs. A negative band at 1695 cm−1 and a positive one at 1670 cm−1 are assigned to the 9-keto carbonyl of bacteriochlorophyll in the states P and 3 P, respectively.