Melvin Y. Okamura

Proton and electron transfer in bacterial reaction centers

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.

Electron transfer in reaction centers of Rhodopseudomonas sphaeroides. I. Determination of the charge recombination pathway of D+QAQ−B and free energy and kinetic relations between Q−AQB and QAQ−B

The electron-transfer reactions and thermodynamic equilibria involving the quinone acceptor complex in bacterial reaction centers from R. sphaeroides were investigated. The reactions are described by the scheme: (Formula: see text). We found that the charge recombination pathway of D+QAQ(-)B proceeds via the intermediate state D+Q(-)AQB, the direct pathway contributing less than approx. 5% to the observed recombination rate. The method used to obtain this result was based on a comparison of the kinetics predicted for the indirect pathway (given by the product kAD-times the fraction of reaction centers in the Q-AQB state) with the observed recombination rate, kobsD+----D. The kinetic measurements were used to obtain the pH dependence (6.1 smaller than or equal to pH smaller than or equal to 11.7) of the free energy difference between the states Q(-)AQB and QAQ(-)B. At low pH (less than 9) QAQ(-)B is stabilized relative to Q(-)AQB by 67 meV, whereas at high pH Q(-)AQB is energetically favored. Both Q(-)A and Q(-)B associate with a proton, with pK values of 9.8 and 11.3, respectively. The stronger interaction of the proton with Q(-)B provides the driving force for the forward electron transfer.

Identification of an electron acceptor in reaction centers of Rhodopseudomonas spheroides by EPR spectroscopy

Abstract Photochemically active reaction centers from Rhodopseudomonas spheroides R-26 were prepared in which the electron donor is P(865) and the electron acceptor is ubiquinone. The latter was identified by comparing the EPR characteristics of the light-induced signal with those obtained from a ubiquinone radical.

Spectroscopic and kinetic properties of the transient intermediate acceptor in reaction centers of Rhodopseudomonas sphaeroides

The photoreductive trapping of the transient, intermediate acceptor, I-, in purified reaction centers of Rhodopseudomonas sphaeroides R-26 was investigated for different external conditions. The optical spectrum of I- was found to be similar to that reported for other systems by Shuvalov and Klimov ((1976) Biochim. Biophys. Acta 400, 587--599) and Tiede et al. (P.M. Tiede, R.C. Prince, G.H. Reed and P.L. Dutton (1976) FEBS Lett. 65, 301--304). The optical changes of I- showed characteristics of both bacteriopheophytin (e.g. bleaching at 762, 542 nm and red shift at 400 nm) and bacteriochlorophyll (bleaching at 802 and 590 nm). Two types of EPR signals of I- were observed: one was a narrow singlet at g = 2.0035, deltaH = 13.5 G, the other a doublet with a splitting of 60 G centered around g = 2.00, which was only seen after short illumination times in reaction centers reconstituted with menaquinone. The optical and EPR kinetics of I- on illumination in the presence of reduced cytochrome c and dithionite strongly support the following three-step scheme in which the doublet EPR signal is due to the unstable state DI-Q-Fe2+ and the singlet EPR signal is due to DI-Q2-Fe2+. : formula: (see text), where D is the primary donor (BChl)2+. The above model was supported by the following observations: (1) During the first illumination, sigmoidal kinetics of the formation of I- was observed. This is a direct consequence of the three-sequential reactions. (2) During the second and subsequent illuminations first-order (exponential) kinetics were observed for the formation of I-. This is due to the dark decay, k4, to the state DIQ2-Fe2+ formed after the first illumination. (3) Removal of the quinone resulted in first-order kinetics. In this case, only the first step, k1, is operative. (4) The observation of the doublet signal in reaction centers containing menaquinone but not ubiquinone is explained by the longer lifetime of the doublet species I-(Q-Fe2%) in reaction centers containing menaquinone. The value of tau2 was determined from kinetic measurements to be 0.01 s for ubiquinone and 4 s for menaquinone (T = 20 degrees C). The temperature and pH dependence of the dark electron transfer reaction I-(Q-Fe2+) yields I(Q2-Fe2+) was studied in detail. The activation energy for this process was found to be 0.42 eV for reaction centers containing ubiquinone and 0.67 eV for reaction centers with menaquinone. The activation energy and the doublet splitting were used to calculate the rate of electron transfer from I- to MQ-Fe2+ using Hopfields theory for thermally activated electron tunneling. The calculated rate agrees well with the experimentally determined rate which provides support for electron tunneling as the mechanism for electron transfer in this reaction. Using the EPR doublet splitting and the activation energy for electron transfer, the tunneling matrix element was calculated to be 10(-3) eV. From this value the distance between I- and MQ- was estimated to be 7.5--10 A.

Light-induced proton uptake by photosynthetic reaction centers from Rhodobacter sphaeroides R-26. I: Protonation of the one-electron states D+QA−, DQA−, D+QAQB−, and DQAQB−

Abstract The proton uptake associated with the light-induced transfer of an electron to the acceptor quinones Q A and Q B was investigated in reaction centers from Rhodobacter sphaeroides R-26. The proton uptake was found to be pH dependent with maximum values of approx. 0.5 H + /e − at pH 9 for DQ A − and approx. 0.8 H + /e − at pH 10 for DQ A Q B − . The quinones are not protonated directly. The observed proton uptake is due to shifts in the p K values of amino acid residues that interact with the quinones. The pH-dependences of the proton uptake were fitted with a phenomenological model in which the protons are taken up by four amino acid residues. The deduced p K shifts associated with the reductions of the quinones ranged from 0.4 to 0.8 for Q A and from 0.4 to 1.3 for Q B . The proton uptake by D + Q A − and D + Q A Q B − was less than that by DQ A − and DQ A Q B − , respectively, indicating a release of protons associated with the formation of D + . To calculate the pH-dependence of the redox midpoint potentials of Q A ( E m(Q A ) ) and Q B ( E m(Q B ) ) from the proton uptake, we used a thermodynamic (model-independent) relation. E m(Q A ) decreased approx. 20 mV/pH at 6.0 E (Q B ) decreased approx. 20 mV/pH at 6.0 9.0. The pH dependence of E m(Q A ) in isolated reaction centers is significantly weaker than that determined from redox titrations of Q A in chromatophores of Rb sphaeroides (see for example Prince, R.C. and Dutton, P.L. (1976) Arch. Biochem. Biophys. 172, 329–334). The pH-dependence of the free energy between Q A − Q B and Q A Q B − obtained from the difference of E m(Q A ) and E m(Q B ) is in good agreement with that determined from the measurement of electron-transfer kinetics in isolated RCs from Rb. sphaeroides (Kleinfeld, D., Okamura, M.Y., and Feher, G. (1984) Biochim. Biophys. Acta 766, 126–140). The stronger average interaction of the protons with Q B − provides the driving force for the forward electron transfer. A simplified model was used to calculate the p K shifts from the electrostatic energy between Q A − or Q B − and the charges of the protonatable amino acid residues whose positions were obtained from the three-dimensional structure (Allen, J.P., Feher, G., Yeates, T.O., Komiya, H. and Rees, D.C. (1987) Proc. Natl. Acad. Sci. USA 84, 6162–6166).

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

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.

X-ray structure determination of the cytochrome c2: reaction center electron transfer complex from Rhodobacter sphaeroides.

In the photosynthetic bacterium Rhodobacter sphaeroides, a water soluble cytochrome c2 (cyt c2) is the electron donor to the reaction center (RC), the membrane-bound pigment-protein complex that is the site of the primary light-induced electron transfer. To determine the interactions important for docking and electron transfer within the transiently bound complex of the two proteins, RC and cyt c2 were co-crystallized in two monoclinic crystal forms. Cyt c2 reduces the photo-oxidized RC donor (D+), a bacteriochlorophyll dimer, in the co-crystals in approximately 0.9 micros, which is the same time as measured in solution. This provides strong evidence that the structure of the complex in the region of electron transfer is the same in the crystal and in solution. X-ray diffraction data were collected from co-crystals to a maximum resolution of 2.40 A and refined to an R-factor of 22% (R(free)=26%). The structure shows the cyt c2 to be positioned at the center of the periplasmic surface of the RC, with the heme edge located above the bacteriochlorophyll dimer. The distance between the closest atoms of the two cofactors is 8.4 A. The side-chain of Tyr L162 makes van der Waals contacts with both cofactors along the shortest intermolecular electron transfer pathway. The binding interface can be divided into two domains: (i) A short-range interaction domain that includes Tyr L162, and groups exhibiting non-polar interactions, hydrogen bonding, and a cation-pi interaction. This domain contributes to the strength and specificity of cyt c2 binding. (ii) A long-range, electrostatic interaction domain that contains solvated complementary charges on the RC and cyt c2. This domain, in addition to contributing to the binding, may help steer the unbound proteins toward the right conformation.

Proton transfer pathways and mechanism in bacterial reaction centers

The focus of this minireview is to discuss the state of knowledge of the pathways and rates of proton transfer in the bacterial reaction center (RC) from Rhodobacter sphaeroides. Protons involved in the light driven catalytic reduction of a quinone molecule QB to quinol QBH2 travel from the aqueous solution through well defined proton transfer pathways to the oxygen atoms of the quinone. Three main topics are discussed: (1) the pathways for proton transfer involving the residues: His‐H126, His‐H128, Asp‐L210, Asp‐M17, Asp‐L213, Ser‐L223 and Glu‐L212, which were determined by a variety of methods including the use of proton uptake inhibiting metal ions (e.g. Zn2+ and Cd2+); (2) the rate constants for proton transfer, obtained from a ‘chemical rescue’ study was determined to be 2×105 s−1 and 2×104 s−1 for the proton uptake to Glu‐L212 and QB − , respectively; (3) structural studies of altered proton transfer pathways in revertant RCs that lack the key amino acid Asp‐L213 show a series of structural changes that propagate toward L213 potentially allowing Glu‐H173 to participate in the proton transfer processes.

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

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.

Reaction centers from three herbicide-resistant mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary characterization

Many herbicides that inhibit photosynthesis in plants also inhibit photosynthesis in bacteria. We have isolated three mutants of the photosynthetic bacterium Rhodobacter sphaeroides that were selected for increased resistance to the herbicide terbutryne. All three mutants also showed increased resistance to the known electron transfer inhibitor o-phenanthroline. The primary structures of the mutants were determined by recombinant DNA techniques. All mutations were located on the gene coding for the L-subunit resulting in these changes Ile229 → Met, Ser223 → Pro and Tyr222 → Gly. The mutations of Ser223 is analogous to the mutation of Ser264 in the D1 subunit of photosystem II in green plants, strengthening the functional analogy between D1 and the bacterial L-subunit. The changed amino acids of the mutant strains form part of the binding pocket for the secondary quinone, Qb. This is consistent with the idea that the herbicides are competitive inhibitors for the Qbbinding site. The reaction centers of the mutants were characterized with respect to electron transfer rates, inhibition constants of terbutryne and o-phenanthroline, and binding constants of the quinone UQ0 and the inhibitors. By correlating these results with the three-dimensional structure obtained from x-ray analysis by Allen et al. (1987a, 1987b), the likely positions of o-phenanthroline and terbutryne were deduced. These correspond to the positions deduced by Michel et al. (1986a) for Rhodopseudomonas viridis.