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Featured researches published by G. Feher.


Biochimica et Biophysica Acta | 2000

Proton and electron transfer in bacterial reaction centers

Melvin Y. Okamura; Mark L. Paddock; M.S. Graige; G. Feher

The bacterial reaction center couples light-induced electron transfer to proton pumping across the membrane by reactions of a quinone molecule Q(B) that binds two electrons and two protons at the active site. This article reviews recent experimental work on the mechanism of the proton-coupled electron transfer and the pathways for proton transfer to the Q(B) site. The mechanism of the first electron transfer, k((1))(AB), Q(-)(A)Q(B)-->Q(A)Q(-)(B), was shown to be rate limited by conformational gating. The mechanism of the second electron transfer, k((2))(AB), was shown to involve rapid reversible proton transfer to the semiquinone followed by rate-limiting electron transfer, H(+)+Q(-)(A)Q(-)(B) ifQ(-)(A)Q(B)H-->Q(A)(Q(B)H)(-). The pathways for transfer of the first and second protons were elucidated by high-resolution X-ray crystallography as well as kinetic studies showing changes in the rate of proton transfer due to site directed mutations and metal ion binding.


Biophysical Journal | 1984

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. III. EPR measurements of the reduced acceptor complex

W.F. Butler; Rafael Calvo; D. R. Fredkin; R.A. Isaacson; Melvin Y. Okamura; G. Feher

Electron paramagnetic resonance (EPR) spectra of the reduced quinone-iron acceptor complex in reaction centers were measured in a variety of environments and compared with spectra calculated from a theoretical model. Spectra were obtained at microwave frequencies of 1, 9, and 35 GHz and at temperatures from 1.4 to 30 K. The spectra are characterized by a broad absorption peak centered at g = 1.8 with wings extending from g approximately equal to 5 to g less than 0.8. The peak is split with the low-field component increasing in amplitude with temperature. The theoretical model is based on a spin Hamiltonian, in which the reduced quinone, Q-, interacts magnetically with Fe2+. In this model the ground manifold of the interacting Q-Fe2+ system has two lowest doublets that are separated by approximately 3 K. Both perturbation analyses and exact numerical calculations were used to show how the observed spectrum arises from these two doublets. The following spin Hamiltonian parameters optimized the agreement between simulated and observed spectra: the electronic g tensor gFe, x = 2.16, gFe, y = 2.27, gFez = 2.04, the crystal field parameters D = 7.60 K and E/D = 0.25, and the antiferromagnetic magnetic interaction tensor, Jx = -0.13 K, Jy = -0.58 K, Jz = -0.58 K. The model accounts well for the g value (1.8) of the broad peak, the observed splitting of the peak, the high and low g value wings, and the observed temperature dependence of the shape of the spectra. The structural implications of the value of the magnetic interaction, J, and the influence of the environment on the spin Hamiltonian parameters are discussed. The similarity of spectra and relaxation times observed from the primary and secondary acceptor complexes Q-AFe2+ and Fe2+Q-B leads to the conclusion that the Fe2+ is approximately equidistant from QA and QB.


Biophysical Journal | 1995

Electrostatic calculations of amino acid titration and electron transfer, Q-AQB-->QAQ-B, in the reaction center

P. Beroza; D. R. Fredkin; Melvin Y. Okamura; G. Feher

The titration of amino acids and the energetics of electron transfer from the primary electron acceptor (QA) to the secondary electron acceptor (QB) in the photosynthetic reaction center of Rhodobacter sphaeroides are calculated using a continuum electrostatic model. Strong electrostatic interactions between titrating sites give rise to complex titration curves. Glu L212 is calculated to have an anomalously broad titration curve, which explains the seemingly contradictory experimental results concerning its pKa. The electrostatic field following electron transfer shifts the average protonation of amino acids near the quinones. The pH dependence of the free energy between Q-AQB and QAQ-B calculated from these shifts is in good agreement with experiment. However, the calculated absolute free energy difference is in severe disagreement (by approximately 230 meV) with the observed experimental value, i.e., electron transfer from Q-A to QB is calculated to be unfavorable. The large stabilization energy of the Q-A state arises from the predominantly positively charged residues in the vicinity of QA in contrast to the predominantly negatively charged residues near QB. The discrepancy between calculated and experimental values for delta G(Q-AQB-->QAQ-B) points to limitations of the continuum electrostatic model. Inclusion of other contributions to the energetics (e.g., protein motion following quinone reduction) that may improve the agreement between theory and experiment are discussed.


Photosynthesis Research | 1998

Identification of proton transfer pathways in the X-ray crystal structure of the bacterial reaction center from Rhodobacter sphaeroides

Edward C. Abresch; Mark L. Paddock; Michael H. B. Stowell; T.M. McPhillips; Herbert L. Axelrod; S.M. Soltis; Douglas C. Rees; Melvin Y. Okamura; G. Feher

Structural features that have important implications for the fundamental process of transmembrane proton transfer are examined in the recently published high resolution atomic structures of the reaction center (RC) from Rhodobacter sphaeroides in the dark adapted state (DQAQB) and the charged separated state (D+QAQB−); the latter is the active state for proton transfer to the semiquinone. The structures have been determined at 2.2 Å and 2.6 Å resolution, respectively, as reported by Stowell et al. (1997) [Science 276: 812–816]. Three possible proton transfer pathways (P1, P2, P3) consisting of water molecules and/or protonatable residues were identified which connect the QB binding region with the cytoplasmic exposed surface at Asp H224 & Asp M240 (P1), Tyr M3 (P2) and Asp M17 (P3). All three represent possible pathways for proton transfer into the RC. P1 contains an uninterrupted chain of water molecules. This path could, in addition, facilitate the exchange of quinone for quinol during the photocycle by allowing water to move into and out of the binding pocket. Located near these pathways is a cluster of electrostatically interacting acid residues (Asp-L213, Glu-H173, Asp-M17, Asp H124, Asp-L210 and Asp H170) each being within 4.5 Å of a neighboring carboxylic acid or a bridging water molecule. This cluster could serve as an internal ‘proton reservoir’ facilitating fast protonation of QB− that could occur at a rate greater than that attainable by proton uptake from solution.


Photosynthesis Research | 1998

Free energy dependence of the direct charge recombination from the primary and secondary quinones in reaction centers from Rhodobacter sphaeroides

James P. Allen; J. C. Williams; M.S. Graige; Mark L. Paddock; A. Labahn; G. Feher; Melvin Y. Okamura

The direct charge recombination rates from the primary quinone, kAD (D+QA− → DQA) and the secondary quinone, kBD (D+QB− → DQB), in reaction centers from Rhodobacter sphaeroides were measured as a function of the free energy differences for the processes, ΔGAD0 and ΔGBD0, respectively. Measurements were performed at 21 °C on a series of mutant reaction centers that have a wide range of dimer midpoint potentials and consequently a large variation in ΔGAD0 and ΔGBD0. As –ΔGAD0 varied from 0.43 to 0.78 eV, kAD varied from 4.6 to 28.6 s−1. The corresponding values for the wild type are 0.52 eV and 8.9 s−1. Observation of the direct charge recombination rate kBD was achieved by substitution of the primary quinone with naphthoquinones in samples in which ubiquinone was present at the secondary quinone site, resulting specifically in an increase in the free energy of the D+QA− state relative to the D+QAQB− state. As –ΔGBD0 varied from 0.37 to 0.67 eV, kBD varied from 0.03 to 1.4 s−1. The corresponding values for the wild type are 0.46 eV and 0.2 s−1. A fit of the two sets of data to the Marcus theory for electron transfer yielded significantly different reorganization energies of 0.82 and 1.3 eV for kAD and kBD, respectively. In contrast, the fitted values for the coupling matrix element, or equivalently the maximum possible rate, were comparable (∼25 s−1) for the two charge recombination processes. These results are in accord with QB having more interactions with dipoles, from both the surrounding protein and bound water molecules, than QA and with the primary determinant of the maximal rate being the quinone-donor distance.


Biochimica et Biophysica Acta | 1997

Light-induced electrogenic events associated with proton uptake upon forming QB− in bacterial wild-type and mutant reaction centers

Peter Brzezinski; Mark L. Paddock; Melvin Y. Okamura; G. Feher

Light-induced voltage changes (electrogenic events) were measured in wild-type and site-directed mutants of reaction centers (RCs) from Rhodobacter sphaeroides oriented in a lipid monolayer adsorbed to a Teflon film. A rapid increase in voltage associated with charge separation was followed by a slower increase attributed to proton transfer from solution to protonatable amino-acid residues in the vicinity of the QB site. In native reaction centers the proton-transfer voltage had a pH-dependent amplitude with two peaks at pH 4.5 and pH 9.7, respectively. In the Glu-L212-->Gln RCs the high-pH peak was absent, whereas in the Asp-L213-->Asn RCs the low-pH peak was absent and the high-pH peak was shifted to lower pH by about 1.3 pH units. The amplitudes of the electrogenic phases as a function of pH follow approximately the measured proton uptake from solution (P.H. McPherson, M.Y. Okamura, G. Feher, Biochim. Biophys. Acta, vol. 934, 1988, pp. 348-368) and are ascribed to proton transfer to amino acid residues upon QB- formation. The peak around pH 9.7 is ascribed to proton uptake predominantly by Glu-L212 and the peak around pH 4.5 to proton uptake predominantly by Asp-L213 or a residue strongly interacting with Asp-L213.


Photosynthesis Research | 1998

Characterization of second site mutations show that fast proton transfer to QB− is restored in bacterial reaction centers of Rhodobacter sphaeroides containing the Asp-L213 → Asn lesion

Mark L. Paddock; M.E. Senft; M.S. Graige; S. H. Rongey; T. Turanchik; G. Feher; Melvin Y. Okamura

The structural basis for proton coupled electron transfer to QB in bacterial reaction centers (RCs) was studied by investigating RCs containing second site suppressor mutations (Asn M44 → Asp, Arg M233 → Cys, Arg H177 → His) that complement the effects of the deleterious Asp L213 → Asn mutation [DN(L213)]. The suppressor RCs all showed an increased proton coupled electron transfer rate kAB(2)(QA−QB− + H+ → QAQBH−) by at least 103 (pH 7.5) and a recombination rate kBD (D+QAQB− → DQAQB) 15–40 times larger than the value found in DN(L213) RCs. Proton transfer was studied by measuring the dependence of kAB(2) on the free energy for electron transfer (δΔGet). kAB(2) was independent of δΔGet in DN(L213) RCs, but dependent on δΔGet in native and all suppressor RCs. This shows that proton transfer limits the kAB(2) reaction with a rate of 0.1s−1 in DN(L213) RCs but is not rate limiting and at least 108-fold faster in native and 105-fold faster in the suppressor RCs. The increased rate of proton transfer by the suppressor mutations are proposed to be due to: (i) a reduction in the barrier to proton transfer by providing a more negative electrostatic potential near QB−; and/or (ii) structural changes that permit fast proton transfer through the network of protonatable residues and water molecules near QB.


Archive | 1988

Structure of the Reaction Center from Rhodobacter sphaeroides R-26 and 2.4.1

James P. Allen; G. Feher; Todd O. Yeates; H. Komiya; Douglas C. Rees

Detailed theories of electron transfer in reaction centers (RCs) require knowledge of their three dimensional structure. We have determined the structure of RCs from Rb. sphaeroides by x-ray diffraction of single crystals. Diffraction data of RCs from both the carotenoidless mutant, R-26, and the wild type strain, 2.4.1 were analyzed at resolutions of 2.8 A and 3.5 A respectively. These structures have been refined to current R-factors of 25% (for the R26 data) and 22% (for the 2.4.1. data). Details concerning data collection and analysis, with descriptions of the structure of the RC from the R-26 strain have been presented elsewhere (1–4). In this report we shall focus on the general features of the structure and compare them with those reported for the RC from R. viridis (5). The structures from these two species have been shown by the molecular replacement method to be homologous (6,7). We shall emphasize the importance of the structural features to the function of electron transfer.


Archive | 1996

Asymmetry of the Binding Sites of QA- and QB- in Reaction Centers of Rb. sphaeroides Probed by Q-Band EPR with 13C-Labeled Quinones

R.A. Isaacson; Edward C. Abresch; F. Lendzian; C. Boullais; Mark L. Paddock; C. Mioskowski; W. Lubitz; G. Feher

Ubiquinone-3 labeled with 13C at either position 1 or 4 was incorporated into Zn-containing reaction centers of Rb. sphaeroides in the mutant His→Cys (M266). Q-band EPR of the primary and secondary quinone anion radicals, Q A - and Q B - , revealed differences in the 13C-hyperfine couplings of the carbons of the two carbonyl groups. The asymmetry of the spin density distribution was particularly pronounced for Q A - and was explained by a strong hydrogen bond from the protein to one of the carbonyl oxygens in accord with the findings of van den Brink et al (FEBS Lett, 1994, 353, 273–276). The binding of (math) is only slightly asymmetric and more similar to that found for the ubiquinone-3 anion radical in an alcoholic solvent matrix. The hydrogen bond donors to Q A - and Q B - of the amino acid framework are identified and consequences for the function of the quinones in the electron transfer process are briefly discussed.


Archive | 1988

ENDOR of Exchangeable Protons of the Reduced Intermediate Acceptor in Reaction Centers from Rhodobacter sphaeroides R-26

G. Feher; R.A. Isaacson; Melvin Y. Okamura; Wolfgang Lubitz

To understand quantitatively the electron transfer kinetics in reaction centers (RCs) one needs to know both the spatial, three-dimensional, structure as well as the electronic structure of the reactants. The advances made in the determination of the three-dimensional structure of RCs in Rp. viridis and Rb. sphaeroides were presented earlier at this Conference by H. Deisenhofer, D. Tiede and our group. In this communication we would like to report on the results of investigations of the electronic structure of the intermediate acceptor I⨪. The acceptor, I, is believed to be a bacteriopheophytin a (Bphe a), that receives an electron from the singlet excited primary donor in ~ 4 picoseconds and passes it on to a quinone acceptor with a characteristic time of ~ 200 ps (for a review see ref. 1). In general, the charge transfer in photosynthesis is a one electron process that results in the formation of donor cation and acceptor anion radicals.

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Douglas C. Rees

California Institute of Technology

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James P. Allen

Arizona State University

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R.A. Isaacson

University of California

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D. R. Fredkin

University of California

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Todd O. Yeates

University of California

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H Komiya

University of California

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S. H. Rongey

University of California

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