Reaction Centers ofRhodobacter Research Articles

Histidine is involved in coupling proton uptake to electron transfer in photosynthetic proteins

In photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake and release. Despite intense investigations of different photosynthetic protein complexes, including the photosystem II (PS II) in plants and the reaction center (RC) in bacteria, the molecular details of this fundamental process remain incompletely understood. In the RC of Rhodobacter (Rb.) sphaeroides, fast formation of the charge separated state, P +Q A −, is followed by a slower electron transfer from the primary acceptor, Q A, to the secondary acceptor, Q B, and the uptake of a proton from the cytoplasm. The proton transfer to Q B takes place via a protonated water chain. Mutation of the amino acid AspL210 to Asn (L210DN mutant) near the entry of the proton pathway can disturb this water chain and consequently slow down proton uptake. Time-resolved step-scan Fourier transform infrared (FTIR) measurements revealed an intermediate X in the Q A −Q B to Q AQ B − transition. The nature of this transition remains a matter of debate. In this study, we investigated the role of the iron–histidine complex located between Q A and Q B. We used time-resolved fast-scan FTIR spectroscopy to characterize the Rb. sphaeroides L210DN RC mutant marked with isotopically labeled histidine. FTIR marker bands of the intermediate X between 1120 cm −1 and 1050 cm −1 are assigned to histidine vibrations and indicate the protonation of a histidine, most likely HisL190, during the disappearance of the intermediate. Based on these results we propose a novel mechanism of the coupling of electron and proton transfer in photosynthesis.

Spin-labeled photosynthetic reaction centers fromRhodobacter sphaeroides studied by electron paramagnetic resonance spectroscopy and molecular dynamics simulations

A new strategy has been applied that combines molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy to study the structure and conformational dynamics of the spin-labeled photosynthetic reaction center (RC) ofRhodobacter sphaeroides. This protein serves here as a model system to demonstrate the applicability of this new methodology. The RC contains five native cysteines and EPR experiments show that only one cysteine, located on the H subunit, is accessible for spin labeling. The EPR spectra calculated from MD simulation trajectories of spin labels bound to the native cysteines C156 and C234 in subunit H reveal that only the spin label side chain at position 156 provides a spectrum which agrees with the experimental EPR spectrum.

A short note on orientation selection in the DEER experiments on a native cofactor and a spin label in the reaction center ofRhodobacter sphaeroides

The analysis of the two-frequency pulsed electron paramagnetic resonance (EPR) (double electron-electron spin resonance, DEER) investigation on the coupling between the semiquinone anion state of the primary acceptor (QA) and the spin label at the cysteine 156 in the H-subunit in the photosynthetic reaction center (RC) fromRhodobacter sphaerodes (R26) (I. V. Borovykh, S. Ceola, P. Gajula, P. Gast, H. J. Steinhoff, M. Huber: J. Magn. Reson. 180, 178–185, 2006) is reinvestigated to include orientation selection. The combination of the EPR properties of the two radicals and the pump and observer frequencies suggests that such an effect could play a role even at the X-band (9 GHz) EPR fields and frequencies employed. The magnitude of the effect is estimated from the structures obtained from the molecular-dynamics (MD) simulations from the previous study: the distance change is small (around 2%) and the distance of 3.05 nm obtained then is possibly underestimated by 0.06 nm. Thus, the difference of at least 0.2 nm between the measured distance and the average distance of 2.8 nm found by the MD simulation remains, suggesting a significant difference between the measurement and the X-ray structure of the RC, as discussed previously.

Theoretical investigation of Q A −· -ligand interactions in bacterial reaction centers ofRhodobacter sphaeroides

Density functional theory was used to calculate magnetic resonance parameters for the primary stable electron acceptor anion radical (QA−·) in its binding site in the bacterial reaction center (bRC) ofRhodobacter sphaeroides. The models used for the calculations of the QA−· binding pocket included all short-range interactions of the ubiquinone with the protein surroundings in a gradual manner and thus allowed a decomposition and detailed analysis of the different specific interactions. Comparison of the obtained hyperfine and quadrupole couplings with experimental data demonstrates the feasibility and reliability of calculations on such complex biologically relevant systems. With these results, the interpretation of previously published 3-pulse electron spin echo envelope modulation data could be extended and an assignment of the observed double quantum peak to a specific amino acid is proposed. The computations provide evidence for a slightly altered binding site geometry for the QA ground state as investigated by X-ray crystallography with respect to the QAt-· anion radical state as accessible via EPR spectroscopy. This new geometry leads to improved fits of the W-band correlated-coupled radical pair spectra of QA−·-P865+· compared to orientation data from the crystal structure. Finally, a correlation of the14N quadrupole parameters of His219 with the hydrogen bond geometry and a comparison with previous systematic studies on the influence of hydrogen bond geometry on quadrupole coupling parameters (J. Fritscher: Phys. Chem. Chem. Phys. 6, 4950–4956, 2004) is presented.

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).

Models of Ultrafast Energy and Electron Transfers in Bacterial Reaction Centers

AbstractIn this paper, we present a microscopic model to describe the spectroscopy and dynamics for photosynthetic bacterial reaction centers (RCs). In this model, we propose eight vibrational modes and their Huang‐Rhys factors for different electronic energy states, and the couplings between the electronic states. As applications, we have constructed steady state and ultrafast time‐resolved spectra for the mutant RC of Rhodobacter (Rb.)sphaeroides R26. Our model can also treat the temperature effect on the spectroscopy and dynamics of RCs.

Excitation energy trapping by the reaction center ofRhodobacter Sphaeroides

The excitation energy transfer between light-harvesting complex I (LH-I) and the photosynthetic reaction center (RC) of the purple bacterium Rhodobacter (Rb.) sphaeroides is investigated on the basis of the atomic level structures of the two proteins, assuming a ring-shaped model for LH-I. Rates of excitation energy transfer are calculated, based on Förster theory. The LH-I and RC electronic excitations are described through effective Hamiltonians established previously, with parameters derived from quantum chemistry calculations by Cory and co-workers. We also present an effective Hamiltonian description with parameters based on spectroscopic properties. We study two extreme models of LH-I excitations: electronic excitations delocalized over the entire LH-I ring and excitations localized on single bacteriochlorophylls. The role of accessory bacteriochlorophylls in bridging the excitation energy transfer is investigated. The rates of back-transfer, i.e., RC → LH-I excitation energy transfer, are determined, too. © 2000 John Wiley & Sons, Inc. Int J Quant Chem 77: 139–151, 2000

In bacterial reaction centers, a key residue suppresses mutational blockage of two different proton transfer steps.

In reaction centers of Rhodobacter (Rb.) capsulatus, the M43Asn --> Asp substitution is capable of restoring rapid rates for delivery of the second proton to QB in a mutant that lacks L212Glu. Flash-induced absorbance spectroscopy was used to show a nearly native rate for transfer of the second proton to QB (approximately 700 s-1) in the L212Gln+M43Asp double-mutant reaction center; this rate was shown to decrease more than 1000-fold in the photoincompetent L212Glu --> Gln mutant [Miksovska, J., Kálmán, L., Maróti, P., Schiffer, M., Sebban, P., and Hanson, D.K. (1997) Biochemistry 36, 12216-12226]. In Rb. sphaeroides, the equivalent M44Asn --> Asp mutation was reported to restore the rate of transfer of the first proton to a wild-type level when it is added to the L213Asp --> Asn photoincompetent mutant [Rongey, S.H., Paddock, M.L., Feher, G., and Okamura, M.Y. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 1325-1329]. It is remarkable that the same second-site mutation can compensate for both of these mutations which severely impair reaction center function by blocking two different proton-transfer reactions. We suggest that residue M43Asp is situated in a key position which can link pathways for delivery of both the first and second protons (involving structured water molecules) to QB.

The spectroscopic and photochemical properties of locked-15,15′-cis-spheroidene in solution and incorporated into the reaction center of Rhodobacter sphaeroides R-26.1

The spectroscopic and photochemical properties of the synthetic carotenoid, locked-15,15′-cis-spheroidene, were studied by absorption, fluorescence, circular dichroism, fast transient absorption and electron spin resonance spectroscopies in solution and after incorporation into the reaction center of Rhodobacter (Rb.) sphaeroides R-26.1. HPLC purification of the synthetic molecule reveals the presence of several di-cis geometric isomers in addition to the mono-cis isomer of locked-15,15′-cis-spheroidene. In solution, the absorption spectrum of the purified mono-cis sample was red-shifted and showed a large cis-peak at 351 nm compared to unlocked all-trans spheroidene. Molecular modeling and semi-empirical calculations reveal how geometric isomerization and structural factors affect the room temperature spectra. The spectroscopic studies of the purified locked-15,15′-mono-cis molecule in solution reveal a more stable manifold of excited states compared to the unlocked spheroidene. Reaction centers of Rb. sphaeroides R-26.1 in which the locked-15,15′-cis-spheroidene was incorporated show no difference in either the spectroscopic properties or photochemistry compared to reaction centers in which unlocked spheroidene was incorporated or to Rb. sphaeroides wild type strain 2.4.1 reaction centers which naturally contain spheroidene. The data suggest that the natural selection of a cis-isomer of spheroidene for incorporation into native reaction centers of Rb. sphaeroides wild type strain 2.4.1 is more determined by the structure or assembly of the reaction center protein than by any special quality of the cis-isomer of the carotenoid that would affect its ability to participate in triplet energy transfer or carry out photoprotection.

Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB <==> QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.

Time resolved ADMR applied to the triplet state of the primary donor of bacterial photosynthetic reaction centers

The population and depopulation kinetics of the triplet sublevels of reaction centers ofRhodobacter (Rb.) sphaeroides strains R26 and GA were determined at 9 K by time resolved absorption detected magnetic resonance (ADMR) using the RF pulse method. The comparison of the saturation and pulse methods of time resolved ADMR proofs the latter to be superior. The kinetic behavior of the 2|E| signal was investigated for the first time. It exhibits an unexpected slow decaying component with a rate comparable to thez-level decay. Using a simple kinetic 4-level model for the triplet dynamics we conclude that this slow component as well as the sign of the ADMR signal of the 2|E| transition can be explained by a selective spin lattice relaxation channel connecting the triplet sublevelsx andz.

Directed mutations affecting spectroscopic and electron transfer properties of the primary donor in the photosynthetic reaction center

Oligonucleotide-mediated mutagenesis has been used to change the histidine residues that act as axial ligands to the central Mg(2+) ions of the "special pair" bacteriochlorophylls in the reaction center of Rhodobacter capsulatus. Histidine-173 of the L subunit has been replaced with glutamine, while histidine-200 of the M subunit has been replaced with glutamine, leucine, or phenylalanine. When leucine or phenylalanine is introduced at M200, one of the special pair bacteriochlorophylls is converted to bacteriopheophytin, which generates a heterodimer at the special pair binding site. The pigment composition of the reaction center is unaltered when either histidine is replaced with glutamine. All of these mutant reaction centers are photochemically active, although the electron transfer properties of heterodimer-containing reaction centers are altered. These mutations begin to define the structural parameters that determine whether bacteriochlorophyll or bacteriopheophytin will be incorporated into the tetrapyrrole binding sites of the photosynthetic reaction center. Our results demonstrate that the properties of the photosynthetic reaction center can be changed by directed mutagenesis, which makes this complex an excellent model for testing theories of electron transfer in biological systems.