Reaction Centers Of Rhodobacter Sphaeroides Research Articles

Identification of the Intermediate Charge-Separated State P +βL− in a Leucine M214 to Histidine Mutant of the Rhodobacter sphaeroides Reaction Center Using Femtosecond Midinfrared Spectroscopy

Energy and electron transfer in a Leu M214 to His (LM214H) mutant of the Rhodobacter sphaeroides reaction center (RC) were investigated by applying time-resolved visible pump/midinfrared probe spectroscopy at room temperature. This mutant replacement of the Leu at position M214 resulted in the incorporation of a bacteriochlorophyll (BChl) in place of the native bacteriopheophytin in the L-branch of cofactors (denoted β L). Purified LM214H RCs were excited at 600 nm (unselective excitation), at 800 nm (direct excitation of the monomeric BChl cofactors B L and B M), and at 860 nm (direct excitation of the primary donor (P) BChl pair (P L/P M)). Absorption changes associated with carbonyl (C=O) stretch vibrational modes (9-keto, 10a-ester, and 2a-acetyl) of the cofactors and of the protein were recorded in the region between 1600 cm −1 and 1770 cm −1, and the data were subjected to both a sequential analysis and a simultaneous target analysis. After photoexcitation of the LM214H RC, P ∗ decayed on a timescale of ∼6.3 ps to P +B L −. The decay of P +B L − occurred with a lifetime of ∼2 ps, ∼3 times slower than that observed in wild-type and R-26 RCs (∼0.7 ps). Further electron transfer to the β L BChl resulted in formation of the P + β L − state, and its infrared absorbance difference spectrum is reported for the first time, to our knowledge. The fs midinfrared spectra of P +B L − and P + β L − showed clear differences related to the different environments of the two BChls in the mutant RC.

Monitoring and Interpretation of Photoinduced Biochemical Processes by Rapid-Scan FTIR Difference Spectroscopy and Hybrid Hard and Soft Modeling

Natural photochemical processes often require special instrumentation to monitor them at a suitable time scale. Rapid-scan FTIR difference spectroscopy is one of the preferred techniques to obtain rich structural information in the scale of milliseconds about photochemical processes of complex natural systems. The difference spectra obtained by this technique enhance the fine spectroscopic changes undergone during the process but require powerful data analysis methodologies to take full advantage of the information provided. Hybrid hard- and soft-modeling methodologies allow for coping with difficulties linked to the nature of the time-resolved measurement and to the complexity of the kinetic model describing the natural photochemical process. Thus, this methodology presents the following advantages: (a) handles difference spectra, taking into account the consequences of the lack of measurement about the initial stage of the process, (b) models events of the process that may be defined by a kinetic model (by hard modeling) and events that do not obey a mechanistic behavior (by soft modeling), (c) adapts to the photoaccumulation/relaxation stages of reversible photochemical processes, and (d) works simultaneously with series of experiments performed in different conditions and showing different kinetic behavior. The results of this data treatment provide complete kinetic information on the photochemical processes, e.g., rate constants, and a global picture of the difference spectra and the concentration profiles linked to each of the events (hard or soft modeled) contributing to the measured signal. The performance of the combination of time-resolved differential FTIR and hybrid hard and soft modeling is shown in a complex case study related to the photosynthetic activity of the reaction center of the purple bacteria Rhodobacter sphaeroides.

Properties of mutant reaction centers of Rhodobacter sphaeroides with substitutions of histidine L153, the axial Mg2+ ligand of bacteriochlorophyll BA

Mutant reaction centers (RC) from Rhodobacter sphaeroides have been studied in which histidine L153, the axial ligand of the central Mg atom of bacteriochlorophyll B(A) molecule, was substituted by cysteine, methionine, tyrosine, or leucine. None of the mutations resulted in conversion of the bacteriochlorophyll B(A) to a bacteriopheophytin molecule. Isolated H(L153)C and H(L153)M RCs demonstrated spectral properties similar to those of the wild-type RC, indicating the ability of cysteine and methionine to serve as stable axial ligands of the Mg atom of bacteriochlorophyll B(A). Because of instability of mutant H(L153)L and H(L153)Y RCs, their properties were studied without isolation of these complexes from the photosynthetic membranes. The most prominent effect of the mutations was observed with substitution of histidine by tyrosine. According to the spectral data and the results of pigment analysis, the B(A) molecule is missing in the H(L153)Y RC. Nevertheless, being associated with the photosynthetic membrane, this RC can accomplish photochemical charge separation with quantum yield of approximately 7% of that characteristic of the wild-type RC. Possible pathways of the primary electron transport in the H(L153)Y RC in absence of photochemically active chromophore are discussed.

The nature of oscillations in the kinetics of electron transfer in the reaction center of purple bacteria

87 The reaction centers (RCs) of purple bacteria are natural nanostructures able to transform electron excitation energy into the energy of separated charges with a high efficiency (~100%). The RC main unit is formed of two protein subunits, L and M, with four bacteriochlorophyll (BCl) molecules, two bacteriopheophytin ( H A and H B ) molecules, and two quinone ( Q A and Q B ) molecules attached to them. An iron atom is localized between the quinones. In turn, two of the four BCl molecules form a special pair, the primary electron donor P. Spatial organization of the Rhodobacter sphaeroides RC has been determined with a resolution of 2.65 A [1]. Study of the interaction between the excited states and the charge transfer states of RC cofactors provided for discovering oscillations in the kinetics of stimulated luminescence in the R. sphaeroides RC at the excitation at Q y absorption band of the special pair [2, 3]. Shuvalov et al. studied the oscillations in the absorption band of the reduced intermediate acceptor [4, 5]. These oscillations were explained by a wave packet formed on the potential energy surface for the interaction between the special pair and BCl in the active photosynthesis chain during electron transfer. The oscillation data were described by the Redfield super

Proton-Transfer Pathways in Photosynthetic Reaction Centers Analyzed by Profile Hidden Markov Models and Network Calculations

In the bacterial reaction center (bRC) of Rhodobacter sphaeroides, the key residues of proton transfer to the secondary quinone (Q(B)) are known. Also, several possible proton entry points and proton-transfer pathways have been proposed. However, the mechanism of the proton transfer to Q(B) remains unclear. The proton transfer to Q(B) in the bRC of Blastochloris viridis is less explored. To analyze whether the bRCs of different species use the same key residues for proton transfer to Q(B), we determined the conservation of these residues. We performed a multiple-sequence alignment based on profile hidden Markov models. Residues involved in proton transfer but not located at the protein surface are conserved or are only exchanged to functionally similar amino acids, whereas potential proton entry points are not conserved to the same extent. The analysis of the hydrogen-bond network of the bRC from R. sphaeroides and that from B. viridis showed that a large network connects Q(B) with the cytoplasmic region in both bRCs. For both species, all non-surface key residues are part of the network. However, not all proton entry points proposed for the bRC of R. sphaeroides are included in the network in the bRC of B. viridis. From our analysis, we could identify possible proton entry points. These proton entry points differ between the two bRCs. Together, the results of the conservation analysis and the hydrogen-bond network analysis make it likely that the proton transfer to Q(B) is not mediated by distinct pathways but by a large hydrogen-bond network.

Mutant reaction centers of Rhodobacter sphaeroides I(L177)H with strongly bound bacteriochlorophyll a: Structural properties and pigment-protein interactions

Methods of photoinduced Fourier transform infrared (FTIR) difference spectroscopy and circular dichroism were employed for studying features of pigment-protein interactions caused by replacement of isoleucine L177 by histidine in the reaction center (RC) of the site-directed mutant I(L177)H of Rhodobacter sphaeroides. A functional state of pigments in the photochemically active cofactor branch was evaluated with the method of photo-accumulation of reduced bacteriopheophytin H(A)(-). The results are compared with those obtained for wild-type RCs. It was shown that the dimeric nature of the radical cation of the primary electron donor P was preserved in the mutant RCs, with an asymmetric charge distribution between the bacteriochlorophylls P(A) and P(B) in the P(+) state. However, the dimers P in the wild-type and mutant RCs are not structurally identical due probably to molecular rearrangements of the P(A) and P(B) macrocycles and/or alterations in their nearest amino acid environment induced by the mutation. Analysis of the electronic absorption and FTIR difference P(+)Q(-)/PQ spectra suggests the 17(3)-ester group of the bacteriochlorophyll P(A) to be involved in covalent interaction with the I(L177)H RC protein. Incorporation of histidine into the L177 position does not modify the interaction between the primary electron acceptor bacteriochlorophyll B(A) and the bacteriopheophytin H(A). Structural changes are observed in the monomer bacteriochlorophyll B(B) binding site in the inactive chromophore branch of the mutant RCs.

Calcium Ions Are Required for the Enhanced Thermal Stability of the Light-harvesting-Reaction Center Core Complex from Thermophilic Purple Sulfur Bacterium Thermochromatium tepidum

Thermochromatium tepidum is a thermophilic purple sulfur photosynthetic bacterium collected from the Mammoth Hot Springs, Yellowstone National Park. A previous study showed that the light-harvesting-reaction center core complex (LH1-RC) purified from this bacterium is highly stable at room temperature (Suzuki, H., Hirano, Y., Kimura, Y., Takaichi, S., Kobayashi, M., Miki, K., and Wang, Z.-Y. (2007) Biochim. Biophys. Acta 1767, 1057-1063). In this work, we demonstrate that thermal stability of the Tch. tepidum LH1-RC is much higher than that of its mesophilic counterparts, and the enhanced thermal stability requires Ca2+ as a cofactor. Removal of the Ca2+ from Tch. tepidum LH1-RC resulted in a complex with the same degree of thermal stability as that of the LH1-RCs purified from mesophilic bacteria. The enhanced thermal stability can be restored by addition of Ca2+ to the Ca2+-depleted LH1-RC, and this process is fully reversible. Interchange of the thermal stability between the two forms is accompanied by a shift of the LH1 Qy transition between 915 nm for the native and 880 nm for the Ca2+-depleted LH1-RC. Differential scanning calorimetry measurements reveal that degradation temperature of the native LH1-RC is 15 degrees C higher and the enthalpy change is about 28% larger than the Ca2+-depleted LH1-RC. Substitution of the Ca2+ with other metal cations caused a decrease in thermal stability of an extent depending on the properties of the cations. These results indicate that Ca2+ ions play a dual role in stabilizing the structure of the pigment-membrane protein complex and in altering its spectroscopic properties, and hence provide insight into the adaptive strategy of this photosynthetic organism to survive in extreme environments using natural resources.

Unusual Temperature Dependence of Photosynthetic Electron Transfer due to Protein Dynamics

The initial electron transfer rate and protein dynamics in wild type and five mutant reaction centers from Rhodobacter sphaeroides have been studied as a function of temperature (10-295 K). Detailed kinetic measurements of initial electron transfer in Rhodobacter sphaeroides reaction centers can be quantitatively described by a reaction diffusion formalism at all temperatures from 10 to 295 K. In this model, the time course of electron transfer is determined by the ability of the protein to interconvert between conformations until one is found where the activation energy for electron transfer is near zero. In reaction centers with a free energy for electron transfer similar to wild type, the reaction proceeds at least as fast at cryogenic temperatures as at room temperature. This may be because interconversion between conformations at low temperature is restricted to conformations with near zero activation energy (it is not possible to diffuse away from this region of conformational space). In contrast, mutants with a decreased free energy initially find themselves in conformations unfavorable for electron transfer and require more extensive conformational diffusion to achieve a low activation energy conformation. They therefore undergo electron transfer more slowly at 10 K vs 295 K.

High-precision determination of quantum yields of photoelectric excitation conversion in reaction centers of purple bacteria Rhodospirillum rubrum and Rhodobacter sphaeroides R-26

Using an original model of primary events in bacterial photosynthetic reaction centers (RCs), a complete set of kinetic and energy characteristics, and temperature dependences of nanosecond luminescence component(s) emitted by the RCs, upper limits to the quantum yield of primary transmembrane charge separation in RCs of the purple bacterium Rhodospirillum rubrum and the carotenoidless mutant Rhodobacter sphaeroides R-26 have been estimated. The calculations have given the values of 0.915 ± 0.045 and 0.978 ± 0.006, respectively. In the case of Rb. sphaeroides R-26, the quantum yield partly overlapped with that reported earlier (1.02 ± 0.04) by Wraight and Clayton (BBA, 1974. vol. 333, pp. 246–260).

Identification of the First Steps in Charge Separation in Bacterial Photosynthetic Reaction Centers of Rhodobacter sphaeroides by Ultrafast Mid-Infrared Spectroscopy: Electron Transfer and Protein Dynamics

Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm −1 was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q A, were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct excitation of the dimer of primary donor ( P) BChls ( P L/ P M)). The region between 1600 and 1800 cm −1 encompasses absorption changes associated with carbonyl (C O) stretch vibrational modes of the cofactors and protein. After photoexcitation of the RC the primary electron donor P excited singlet state ( P*) decayed on a timescale of 3.7 ps to the state P + B L − (where B L is the accessory BChl electron acceptor). This is the first report of the mid-IR absorption spectrum of P + B L − ; the difference spectrum indicates that the 9-keto C O stretch of B L is located around 1670–1680 cm −1. After subsequent electron transfer to the bacteriopheophytin H L in ∼1 ps, the state P + H L − was formed. A sequential analysis and simultaneous target analysis of the data showed a relaxation of the P + H L − radical pair on the ∼20 ps timescale, accompanied by a change in the relative ratio of the P L + and P M + bands and by a minor change in the band amplitude at 1640 cm −1 that may be tentatively ascribed to the response of an amide C O to the radical pair formation. We conclude that the drop in free energy associated with the relaxation of P + H L − , is due to an increased localization of the electron hole on the P L half of the dimer and a further consequence is a reduction in the electrical field causing the Stark shift of one or more amide C O oscillators.

Stabilization of the electron in the quinone acceptor part of the Rhodobacter sphaeroides reaction centers

The evolution of the light-induced absorption difference spectrum (380–500 nm) of the reaction centers from photosynthetic purple bacteria Rhodobacter sphaeroides has been examined over 200 μs. The observed changes are interpreted as the effects of proton movement along the H-bond between the primary quinone acceptor and its protein surroundings. A theoretical analysis of the spectral evolution, considering the proton tunneling kinetics, corroborates this interpretation. The electronic state of the primary quinone is stabilized within tens of microseconds; the process is retarded upon deuteration of the reaction center as well as in 90% glycerol, and accelerated upon nondestructive heating to 40°C.

Energetics and Kinetics of Primary Charge Separation in Bacterial Photosynthesis

We report the results of molecular dynamics (MD) simulations and formal modeling of the free-energy surfaces and reaction rates of primary charge separation in the reaction center of Rhodobacter sphaeroides. Two simulation protocols were used to produce MD trajectories. Standard force-field potentials were employed in the first protocol. In the second protocol, the special pair was made polarizable to reproduce a high polarizability of its photoexcited state observed by Stark spectroscopy. The charge distribution between covalent and charge-transfer states of the special pair was dynamically adjusted during the simulation run. We found from both protocols that the breadth of electrostatic fluctuations of the protein/water environment far exceeds previous estimates, resulting in about 1.6 eV reorganization energy of electron transfer in the first protocol and 2.5 eV in the second protocol. Most of these electrostatic fluctuations become dynamically frozen on the time scale of primary charge separation, resulting in much smaller solvation contributions to the activation barrier. While water dominates solvation thermodynamics on long observation times, protein emerges as the major thermal bath coupled to electron transfer on the picosecond time of the reaction. Marcus parabolas were obtained for the free-energy surfaces of electron transfer by using the first protocol, while a highly asymmetric surface was obtained in the second protocol. A nonergodic formulation of the diffusion-reaction electron-transfer kinetics has allowed us to reproduce the experimental results for both the temperature dependence of the rate and the nonexponential decay of the population of the photoexcited special pair.

Average Electron Tunneling Route of the Electron Transfer in Protein Media

We present a new theoretical method to determine and visualize the average tunneling route of the electron transfer (ET) in protein media. In this, we properly took into account the fluctuation of the tunneling currents and the quantum-interference effect. The route was correlated with the electronic factor <TDA(2)> in the case of ET by the elastic tunneling mechanism. We expanded <TDA(2)> by the interatomic tunneling currents <Jab(2)>'s. Incorporating the quantum-interference effect into the mean-square interatomic tunneling currents, denoted as <Jab(2)>, we could express <TDA(2)> as a sum of variant Planck's over 2pi(2)<Jab(2)>. Drawing the distribution of <Jab(2)> on the protein structure, we obtain the <Jab(2)> map which visually represents which parts of bonds and spaces most significantly contribute to <TDA(2)>. We applied this method to the ET from the bacteriopheophytin anion to the primary quinone in the bacterial photosynthetic reaction center of Rhodobacter sphaeroides. We obtained <Jab(2)>'s by a combined method of molecular dynamics simulations and quantum chemical calculations. In calculating <Jab(2)>, we found that much destructive interference works among the interatomic tunneling currents even after taking the average. We drew the <Jab(2)> map by a pipe model where atoms a and b are connected by a pipe with width proportional to the magnitude of <Jab(2)>. We found that two groups of <Jab(2)>'s, which are mutually coupled with high correlation in each group, have broad pipes and form the average tunneling routes, called Trp route and Met route. Each of the two average tunneling routes is composed of a few major pathways in the Pathways model which are fused at considerable part to each other. We also analyzed the average tunneling route for the ET by the inelastic tunneling mechanism.

Protein−Matrix Coupling/Uncoupling in “Dry” Systems of Photosynthetic Reaction Center Embedded in Trehalose/Sucrose: The Origin of Trehalose Peculiarity

Trehalose is a nonreducing disaccharide of glucose found in organisms, which can survive adverse conditions such as extreme drought and high temperatures. Furthermore, isolated structures, as enzymes or liposomes, embedded in trehalose are preserved against stressing conditions [see, e.g., Crowe, L. M. Comp. Biochem. Physiol. A 2002, 131, 505-513]. Among other hypotheses, such protective effect has been suggested to stem, in the case of proteins, from the formation of a water-mediated, hydrogen bond network, which anchors the protein surface to the water-sugar matrix, thus coupling the internal degrees of freedom of the biomolecule to those of the surroundings [Giuffrida, S.; et al. J. Phys. Chem. B 2003, 107, 13211-13217]. Analogous protective effect is also accomplished by other saccharides, although with a lower efficiency. Here, we studied the recombination kinetics of the primary, light-induced charge separated state (P(+)Q(A)(-)) and the thermal stability of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides in trehalose-water and in sucrose-water matrixes of decreasing water content. Our data show that, in sucrose, at variance with trehalose, the system undergoes a "nanophase separation" when the water/sugar mole fraction is lower than the threshold level approximately 0.8. We rationalize this result assuming that the hydrogen bond network, which anchors the RC surface to its surrounding, is formed in trehalose but not in sucrose. We suggest that both the couplings, in the case of trehalose, and the nanophase separation, in the case of sucrose, start at low water content when the components of the system enter in competition for the residual water.

S2.8 Substitution of ILE L177 by His in Rhodobacter sphaeroides reaction center affects interaction of BCHL molecule with the surrounding protein environment

S2.8 Substitution of ILE L177 by His in Rhodobacter sphaeroides reaction center affects interaction of BCHL molecule with the surrounding protein environment

Influence of the energy of H-bond protons on the electron transfer rate in photosynthetic reaction centers

We present here a theoretical interpretation of the temperature dependence of the rate of dark recombination between a primary quinone (QA) and a bacteriochlorophyll dimer in the reaction center of Rhodobacter sphaeroides. We were able to describe qualitatively the nonmonotonous character of this dependence using the energy of interaction between an excess electron and H-bond protons. We considered a molecular model of QA and two reaction center fragments that make H-bonds with QA: His(M219) and Asn(M259)-Ala(M260). We used the two-center approach with regard for electron-phonon interaction in order to calculate the characteristic time of electron tunneling during the recombination reaction. The energy of the phonon emitted/ absorbed during the electron tunneling was determined by the relative shift of donor and acceptor energy levels, the detuning of levels. The detuning was shown to depend on temperature nonmonotonously for H-bonds with double-well potential energy surface. The characteristic time (or the reaction rate) depended on temperature parametrically. The computed dependence was in qualitative agreement with the experimental one.

Kinetics and yields of bacteriochlorophyll fluorescence: redox and conformation changes in reaction center of Rhodobacter sphaeroides

Induction of the bacteriochlorophyll fluorescence under rectangular shape of intense laser diode illumination (1 W cm(-2), 808 nm) was measured over wide time range from 10 microseconds to 4 s in whole cells, chromatophore and isolated reaction center protein of wild type and carotenoid-less mutant (R-26.1) of purple photosynthetic bacterium Rhodobacter sphaeroides. While the antenna-containing species showed large and positive variable fluorescence (Fv) to initial fluorescence (F0) (Fv/F0 approximately 4.5 in whole cells), the isolated RC had negative change (Fv/F0 approximately -0.6) during photochemistry. In chromatophore from R-26.1, only seven times higher rate was measured than in isolated reaction center under identical experimental conditions. The enhancement effect of large antenna on the rate of photochemistry in chromatophore was partially compensated by the favorable pigment absorption properties in isolated RC. The transition from membrane bound to isolated form of the reaction center was probed by titration of zwitterionic detergent LDAO in chromatophore, and at 0.03% LDAO concentration, sharp change of the variable fluorescence was observed. The sudden drop was explained by the formation of LDAO micelles. After the photochemical phase, additional change of fluorescence yield could be observed in isolated RC considered as manifestation of long-living conformations of the trapped redox states of the protein characterized by non-exponential kinetics. Strong support was provided for use of the fluorescence induction to track structural and conformation changes at their earliest phases in chromatophores and isolated reaction centers.

Singlet oxygen generation in the reaction centers of Rhodobacter sphaeroides

Singlet oxygen (1O2) generation in the reaction centers (RCs) of Rhodobacter sphaeroides wild type was characterized by luminescent emission in the near infrared region (time resolved transients and emission spectra) and quantified to have quantum yield of 0.03 +/- 0.005. 1O2 emission was measured as a function of temperature, ascorbate, urea and potassium ferricyanide concentrations and as a function of incubation time in H2O:D2O mixtures. 1O2 was shown to be affected by the RC dynamics and to originate from the reaction of molecular oxygen with two sources of triplets: photoactive dimer formed by singlet-triplet mixing and bacteriopheophytin formed by direct photoexcitation and intersystem crossing.

Low-Temperature Pulsed EPR Study at 34 GHz of the Triplet States of the Primary Electron Donor P865and the Carotenoid in Native and Mutant Bacterial Reaction Centers ofRhodobacter sphaeroides

The photosynthetic charge separation in bacterial reaction centers occurs predominantly along one of two nearly symmetric branches of cofactors. Low-temperature EPR spectra of the triplet states of the chlorophyll and carotenoid pigments in the reaction center of Rhodobacter sphaeroides R-26.1, 2.4.1 and two double-mutants GD(M203)/AW(M260) and LH(M214)/AW(M260) have been recorded at 34 GHz to investigate the relative activities of the "A" and "B" branches. The triplet states are found to derive from radical pair and intersystem crossing mechanisms, and the rates of formation are anisotropic. The former mechanism is operative for Rb. sphaeroides R-26.1, 2.4.1, and mutant GD(M203)/AW(M260) and indicates that A-branch charge separation proceeds at temperatures down to 10 K. The latter mechanism, derived from the spin polarization and operative for mutant LH(M214)/AW(M260), indicates that no long-lived radical pairs are formed upon direct excitation of the primary donor and that virtually no charge separation at the B-branch occurs at low temperatures. When the temperature is raised above 30 K, B-branch charge separation is observed, which is at most 1% of A-branch charge separation. B-branch radical pair formation can be induced at 10 K with low yield by direct excitation of the bacteriopheophytin of the B-branch at 590 nm. The formation of a carotenoid triplet state is observed. The rate of formation depends on the orientation of the reaction center in the magnetic field and is caused by a magnetic field dependence of the oscillation frequency by which the singlet and triplet radical pair precursor states interchange. Combination of these findings with literature data provides strong evidence that the thermally activated transfer step on the B-branch occurs between the primary donor, P865, and the accessory bacteriochlorophyll, whereas this step is barrierless down to 10 K along the A-branch.

Structure of the Charge Separated State in the Photosynthetic Reaction Centers ofRhodobactersphaeroidesby Quantum Beat Oscillations and High-Field Electron Paramagnetic Resonance: Evidence for Light-Induced Reorientation

The structure of the secondary radical pair, P865(+)Q(A)-, in fully deuterated and Zn-substituted reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides R-26 has been determined by high-time resolution and high-field electron paramagnetic resonance (EPR). A computer analysis of quantum beat oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the orientation of the various magnetic tensors of P865(+)Q(A)- with respect to a magnetic reference frame. The orientation of the g-tensor of P865(+) in an external reference system is adapted from a single-crystal W-band (95 GHz) EPR study [Klette, R.; Törring, J. T.; Plato, M.; Möbius, K.; Bönigk, B.; Lubitz, W. J. Phys. Chem. 1993, 97, 2015-2020]. Thus, we obtain the three-dimensional structure of the charge separated state P865(+)Q(A)- on a nanosecond time scale after light-induced charge separation. Comparison with crystallographic data reveals that the position of the quinone is essentially the same as that in the X-ray structure. However, the head group of Q(A)- has undergone a 60 degrees rotation in the ring plane relative to its orientation in the crystal structure. Analysis suggests that the two different QA conformations are functionally relevant states which control the electron-transfer kinetics from Q(A)- to the secondary quinone acceptor QB. It appears that the rate-limiting step of this reaction is a reorientation of Q(A)- in its binding pocket upon light-induced reduction. The new kinetic model accounts for striking observations by Kleinfeld et al. who reported that electron transfer from Q(A)- to QB proceeds in RCs cooled to cryogenic temperature under illumination but does not proceed in RCs cooled in the dark [Kleinfeld, D.; Okamura, M. Y.; Feher, G. Biochemistry 1984, 23, 5780-5786].