Cu2+ site in photosynthetic bacterial reaction centers from Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis

The interaction of metal ions with isolated photosynthetic reaction centers (RCs) from the purple bacteria Rhodobacter sphaeroides, Rhodobacter capsulatus, and Rhodopseudomonas viridis has been investigated with transient optical and magnetic resonance techniques. In RCs from all species, the electrochromic response of the bacteriopheophytin cofactors associated with Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer is slowed in the presence of Cu(2+). This slowing is similar to the metal ion effect observed for RCs from Rb. sphaeroides where Zn(2+) was bound to a specific site on the surface of the RC [Utschig et al. (1998) Biochemistry 37, 8278]. The coordination environments of the Cu(2+) sites were probed with electron paramagnetic resonance (EPR) spectroscopy, providing the first direct spectroscopic evidence for the existence of a second metal site in RCs from Rb. capsulatus and Rps. viridis. In the dark, RCs with Cu(2+) bound to the surface exhibit axially symmetric EPR spectra. Electron spin echo envelope modulation (ESEEM) spectral results indicate multiple weakly hyperfine coupled (14)N nuclei in close proximity to Cu(2+). These ESEEM spectra resemble those observed for Cu(2+) RCs from Rb. sphaeroides [Utschig et al. (2000) Biochemistry 39, 2961] and indicate that two or more histidines ligate the Cu(2+) at the surface site in each RC. Thus, RCs from Rb. sphaeroides, Rb. capsulatus, and Rps. viridis each have a structurally analogous Cu(2+) binding site that is involved in modulating the Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer process. Inspection of the Rps. viridis crystal structure reveals four potential histidine ligands from three different subunits (M16, H178, H72, and L211) located beneath the Q(B) binding pocket. The location of these histidines is surprisingly similar to the grouping of four histidine residues (H68, H126, H128, and L211) observed in the Rb. sphaeroides RC crystal structure. Further elucidation of these Cu(2+) sites will provide a means to investigate localized proton entry into the RCs of Rb. capsulatus and Rps. viridis as well as locate a site of protein motions coupled with electron transfer.     

Affinity and activity of non-native quinones at the QB site of bacterial photosynthetic reaction centers

Purple, photosynthetic reaction centers from Rhodobacter sphaeroides bacteria use ubiquinone (UQ₁₀) as both primary (Q_A) and secondary (Q_B) electron acceptors. Many quinones reconstitute Q_A function, while a few will act as Q_B. Nine quinones were tested for their ability to bind and reconstitute Q_A and Q_B functions. Only ubiquinone (UQ) reconstitutes both functions in the same protein. The affinities of the non-native quinones for the Q_B site were determined by a competitive inhibition assay. The affinities of benzoquinones, naphthoquinone (NQ), and 2-methyl-NQ for the Q_B site are 7 ± 3 times weaker than that at Q_A site. However, di-ortho-substituted NQs and anthraquinone bind tightly to the Q_A site (K _d ≤ 200 nM), and ≥1,000 times more weakly to the Q_B site, perhaps setting a limit on the size of the site. With a low-potential electron donor, 2-methyl, 3-dimethylamino-1,4-NQ, (Me-diMeAm-NQ) at Q_A, Q_B reduction is 260 meV, more favorable than with UQ as Q_A. Electron transfer from Me-diMeAm-NQ at the Q_A site to NQ at the Q_B site can be detected. In the Q_B site, the NQ semiquinone is estimated to be ≈60–100 meV higher in energy than the UQ semiquinone, while in the Q_A site, the semiquinone energy level is similar or lower with NQ than with UQ. Thus, the NQ semiquinone is more stable in the Q_A than in the Q_B site. In contrast, the native UQ semiquinone is ≈60 meV lower in energy in the Q_B than in the Q_A site, stabilizing forward electron transfer from Q_A to Q_B.     

An x-ray absorption study of the iron site in bacterial photosynthetic reaction centers.

Measurements were made of the extended x-ray absorption fine structure (EXAFS) of the iron site in photosynthetic reaction centers from the bacterium Rhodopseudomonas sphaeroides. Forms with two quinones, two quinones with added o-phenanthroline, and one quinone were studied. Only the two forms containing two quinones maintained their integrity and were analyzed. The spectra show directly that the added o-phenanthroline does not chelate the iron atom. Further analysis indicates that the iron is octahedrally coordinated by nitrogen and/or oxygen atoms located at various distances, with the average value of about 2.14 A. The analysis suggests that most of the ligands are nitrogens and that three of the nitrogen ligands belong to histidine rings. This interpretation accounts for several unusual features of the EXAFS spectrum. We speculate that the quinones are bound to the histidine rings in some manner. Qualitative features of the absorption edge spectra also are discussed and are related to the Fe-ligand distance.     

Electron transport dynamics at the quinone acceptor site of bacterial photosynthetic reaction centers as probed using fast temperature changes

Methods of laser-induced temperature jumps and fast freezing were used for testing the rates of thermoinduced conformational transitions of reaction center (RC) complexes in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria. An electron transfer reaction from primary to secondary quinone acceptors was used as a probe of electron transport efficiency. The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied. To investigate the dynamics of spontaneous decay of the RC state induced by the thermal pulse, the thermal pulse was applied either before or during photoinduced activation of electron transport reactions in the RC acceptor complex. The maximum effect was observed if the thermal pulse was applied against the background of steady-state photoactivation of the RC. It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds. Independent support of the estimates was obtained from experiments with varied cooling rates of the samples tested.     

Multiple scattering x-ray absorption studies of Zn2+ binding sites in bacterial photosynthetic reaction centers

Binding of transition metal ions to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides has been previously shown to slow light-induced electron and proton transfer to the secondary quinone acceptor molecule, Q(B). On the basis of x-ray diffraction at 2.5 angstroms resolution a site, formed by AspH124, HisH126, and HisH128, has been identified at the protein surface which binds Cd(2+) or Zn(2+). Using Zn K-edge x-ray absorption fine structure spectroscopy we report here on the local structure of Zn(2+) ions bound to purified RC complexes embedded into polyvinyl alcohol films. X-ray absorption fine structure data were analyzed by combining ab initio simulations and multiparameter fitting; structural contributions up to the fourth coordination shell and multiple scattering paths (involving three atoms) have been included. Results for complexes characterized by a Zn to RC stoichiometry close to one indicate that Zn(2+) binds two O and two N atoms in the first coordination shell. Higher shell contributions are consistent with a binding cluster formed by two His, one Asp residue, and a water molecule. Analysis of complexes characterized by approximately 2 Zn ions per RC reveals a second structurally distinct binding site, involving one O and three N atoms, not belonging to a His residue. The local structure obtained for the higher affinity site nicely fits the coordination geometry proposed on the basis of x-ray diffraction data, but detects a significant contraction of the first shell. Two possible locations of the second new binding site at the cytoplasmic surface of the RC are proposed.     

Design of a redox-linked active metal site: manganese bound to bacterial reaction centers at a site resembling that of photosystem II

Metals bound to proteins perform a number of crucial biological reactions, including the oxidation of water by a manganese cluster in photosystem II. Although evolutionarily related to photosystem II, bacterial reaction centers lack both a strong oxidant and a manganese cluster for mediating the multielectron and proton transfer needed for water oxidation. In this study, carboxylate residues were introduced by mutagenesis into highly oxidizing reaction centers at a site homologous to the manganese-binding site of photosystem II. In the presence of manganese, light-minus-dark difference optical spectra of reaction centers from the mutants showed a lack of the oxidized bacteriochlorophyll dimer, while the reduced primary quinone was still present, demonstrating that manganese was serving as a secondary electron donor. On the basis of these steady-state optical measurements, the mutant with the highest-affinity site had a dissociation constant of approximately 1 microM. For the highest-affinity mutant, a first-order rate with a lifetime of 12 ms was observed for the reduction of the oxidized bacteriochlorophyll dimer by the bound manganese upon exposure to light. The dependence of the amplitude of this component on manganese concentration yielded a dissociation constant of approximately 1 muM, similar to that observed in the steady-state measurements. The three-dimensional structure determined by X-ray diffraction of the mutant with the high-affinity site showed that the binding site contains a single bound manganese ion, three carboxylate groups (including two groups introduced by mutagenesis), a histidine residue, and a bound water molecule. These reaction centers illustrate the successful design of a redox active metal center in a protein complex.     

Primary quinone (QA) binding site of bacterial photosynthetic reaction centers: mutations at residue M265 probed by FTIR spectroscopy

In the primary quinone (Q(A)) binding site of Rb. sphaeroides reaction centers (RCs), isoleucine M265 is in extensive van der Waals contact with the ubiquinone headgroup. Substitution of threonine or serine for this residue (mutants M265IT and M265IS), but not valine (mutant M265IV), lowers the redox midpoint potential of Q(A) by about 100 mV (Takahashi et al. (2001) Biochemistry 40, 1020-1028). The unexpectedly large effect of the polar substitutions is not due to reorientation of the methoxy groups as similar redox potential changes are seen for these mutants with either ubiquinone or anthraquinone as Q(A). Using FTIR spectroscopy to compare Q(A)(-)/Q(A) IR difference spectra for wild type and the M265 mutant RCs, we found changes in the polar mutants (M265IT and M265IS) in the quinone C[double bond]O and C[double bond]C stretching region (1600-1660 cm(-1)) and in the semiquinone anion band (1440-1490 cm(-1)), as well as in protein modes. Modeling the mutations into the X-ray structure of the wild-type RC indicates that the hydroxyl group of the mutant polar residues, Thr and Ser, is hydrogen bonded to the peptide C[double bond]O of Thr(M261). It is suggested that the mutational effect is exerted through the extended backbone region that includes Ala(M260), the hydrogen bonding partner to the C1 carbonyl of the quinone headgroup. The resulting structural perturbations are likely to include lengthening of the hydrogen bond between the quinone C1[double bond]O and the peptide NH of Ala(M260). Possible origins of the IR spectroscopic and redox potential effects are discussed.     

Picosecond kinetics of the initial photochemical electron-transfer reaction in bacterial photosynthetic reaction centers

The absorption changes that occur in reaction centers of the photosynthetic bacterium Rhodopseudomonas sphaeroides during the initial photochemical electron-transfer reaction have been examined. Measurements were made between 740 and 1300 nm at 295 and 80 K by using a pulse-probe technique with 610-nm, 0.8-ps flashes. An excited singlet state of the bacteriochlorophyll dimer P* was found to give rise to stimulated emission with a spectrum similar to that determined previously for fluorescence from reaction centers. The stimulated emission was used to follow the decay of P*; its lifetime was 4.1 +/- 0.2 ps at 295 K and 2.2 +/- 0.1 ps at 80 K. Within the experimental uncertainty, the absorption changes associated with the formation of a bacteriopheophytin anion, Bph-, develop in concert with the decay of P* at both temperatures, as does the absorption increase near 1250 nm due to the formation of the cation of P, P+. No evidence was found for the formation of a bacteriochlorophyll anion, Bchl-, prior to the formation of Bph-. This is surprising, because in the crystal structure of the Rhodopseudomonas viridis reaction center [Deisenhofer, J., Epp, O., Miki, K., Huber, R., & Michel, H. (1984) J. Mol. Biol. 180, 385-398] a Bchl is located approximately in between P and the Bph. It is possible that Bchl- (or Bchl+) is formed but, due to kinetic or thermodynamic constraints, is never present at a sufficient concentration for us to observe. Alternatively, a virtual charge-transfer state, such as P+Bchl-Bph or PBchl+Bph-, could serve to lower the energy barrier for direct electron transfer between P* and the Bph.     

Modeling binding kinetics at the Q(A) site in bacterial reaction centers

Bacterial reaction centers (RCs) catalyze a series of electron-transfer reactions reducing a neutral quinone to a bound, anionic semiquinone. The dissociation constants and association rates of 13 tailless neutral and anionic benzo- and naphthoquinones for the Q(A) site were measured and compared. The K(d) values for these quinones range from 0.08 to 90 microM. For the eight neutral quinones, including duroquinone (DQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ(0)), the quinone concentration and solvent viscosity dependence of the association rate indicate a second-order rate-determining step. The association rate constants (k(on)) range from 10(5) to 10(7) M(-)(1) s(-)(1). Association and dissociation rate constants were determined at pH values above the hydroxyl pK(a) for five hydroxyl naphthoquinones. These negatively charged compounds are competitive inhibitors for the Q(A) site. While the neutral quinones reach equilibrium in milliseconds, anionic hydroxyl quinones with similar K(d) values take minutes to bind or dissociate. These slow rates are independent of ionic strength, solvent viscosity, and quinone concentration, indicating a first-order rate-limiting step. The anionic semiquinone, formed by forward electron transfer at the Q(A) site, also dissociates slowly. It is not possible to measure the association rate of the unstable semiquinone. However, as the protein creates kinetic barriers for binding and releasing anionic hydroxyl quinones without greatly increasing the affinity relative to neutral quinones, it is suggested that the Q(A) site may do the same for anionic semiquinone. Thus, the slow semiquinone dissociation may not indicate significant thermodynamic stabilization of the reduced species in the Q(A) site.     

Electron-conformation transitions in the complex of bacterial photosynthetic reaction centers with cytochromes

A theoretical model of conformation--regulated electron transfer from multihaem cytochrome c to bacteriochlorophyll of the reaction centre (RC) is considered. The theoretical data are compared with the experimental ones on the basis of temperature dependence of laser-induced electron transfer from high-potential cytochrome Ch bacheriochlorophyll of RC in Ectothiohodospira shaposhnikovii chromatophores. From this comparison there were calculated the thermodynamic characteristics of cytochrome Ch transfer from the configuration without electron transfer to RC bacteriochlorophyll into the coordinated configuration with an effective transfer. The values obtained are: H = 7,1 kJ/M; S = --(30,2--36,9) J/grad. M. Possible regulatory role of such conformation transitions is discussed.     

Reorganization energy of the initial electron-transfer step in photosynthetic bacterial reaction centers.

The reorganization energy (lambda) for electron transfer from the primary electron donor (P*) to the adjacent bacteriochlorophyll (B) in photosynthetic bacterial reaction centers is explored by molecular-dynamics simulations. Relatively long (40 ps) molecular-dynamics trajectories are used, rather than free energy perturbation techniques. When the surroundings of the reaction center are modeled as a membrane, lambda for P* B --> P+ B- is found to be approximately 1.6 kcal/mol. The results are not sensitive to the treatment of the protein's ionizable groups, but surrounding the reaction center with water gives higher values of lambda (approximately 6.5 kcal/mol). In light of the evidence that P+ B- lies slightly below P* in energy, the small lambda obtained with the membrane model is consistent with the speed and temperature independence of photochemical charge separation. The calculated reorganization energy is smaller than would be expected if the molecular dynamics trajectories had sampled the full conformational space of the system. Because the system does not relax completely on the time scale of electron transfer, the lambda obtained here probably is more pertinent than the larger value that would be obtained for a fully equilibrated system.     

Demonstration of a conserved histidine and two water ligands at the Mn2+ site in Diocleinae lectins by pulsed EPR spectroscopy

Lectins from the Diocleinae subtribe, including Canavalia brasiliensis, Canavalia bonariensis, Canavalia grandiflora, Cratylia floribunda, Dioclea grandiflora, Dioclea guianensis, Dioclea rostrata, Dioclea violacea, and Dioclea virgata, have been recently isolated and characterized in terms of their carbohydrate binding specificities. Although all of the lectins are Man/Glc specific, they possess different biological activities. In the present study, electron paramagnetic resonance (EPR) spectroscopy demonstrates that all nine Diocleinae lectins contain Mn2+. The spectra of C. floribunda and D. rostrata suggest Mn2+ site symmetry different from that of the other seven lectins. However, electron spin-echo envelope modulation (ESEEM) spectroscopy indicates that all nine lectins are coordinated to a histidyl imidazole, with similar electron-nuclear coupling to the Mn2+-bound imidazole nitrogen. ESEEM also demonstrates ligation of two water molecules to Mn2+ in all nine Diocleinae lectins. Thus, the EPR and ESEEM data indicate the presence of a Mn2+ binding site in the above Diocleinae lectins with a conserved histidine residue and two water ligands.     

Electron paramagnetic resonance investigation of photosynthetic reaction centers from Rhodobacter sphaeroides R-26 in which Fe2+ was replaced by Cu2+. Determination of hyperfine interactions and exchange and dipole-dipole interactions between Cu2+ and QA-.

We report electron paramagnetic resonance (EPR) experiments in frozen solutions of unreduced and reduced photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides R-26 in which Fe2+ has been chemically replaced by the isotope 65Cu2+. Samples in which the primary quinone acceptor QA is unreduced (Cu2+QA:RCs) give a powder EPR spectrum typical for Cu2+ having axial symmetry, corresponding to a d(x2 - y2) ground state orbital, with g values g parallel = 2.314 +/- 0.001 and g perpendicular = 2.060 +/- 0.003. The spectrum shows a hyperfine structure for the nuclear spin of copper (65I = 3/2) with A parallel = (-167 +/- 1) x 10(-4) cm-1 and /A perpendicular/ = (16 +/- 2) x 10(-4) cm-1, and hyperfine couplings with three nitrogen ligands. This has been verified in samples containing the naturally occurring 14N isotope (l = 1), and in samples where the nitrogen ligands to copper were replaced by the isotope 15N (l = 1/2). We introduce a model for the electronic structure at the position of the metal ion which reflects the recently determined three-dimensional structure of the RCs of Rb. sphaeroides (Allen, J. P., G. Feher, T. O. Yeates, H. Komiya, and D. C. Rees. 1987. Proc. Natl. Acad. Sci. USA. 84:5730: Allen, J. P., G. Feher, T. O. Yeates, H. Komiya, and D. C. Rees. 1988. Proc. Natl. Acad. Sci. USA, 85:8487) as well as our EPR results. In this model the copper ion is octahedrally coordinated to three nitrogens from histidine residues and to one carboxylate oxygen from a glutamic acid, forming a distorted square in the plane of the d(x2 = y2) ground state orbital. It is also bound to a nitrogen of another histidine and to the other carboxylate oxygen of the same glutamic acid residue, in a direction approximately normal to this plane. The EPR spectrum changes drastically when the quinone acceptor QA is chemically reduced (Cu2+QA-:RCs); the change is due to the exchange and dipole-dipole interactions between the Cu2+ and QA- spins. A model spin Hamiltonian proposed for this exchange coupled cooper-quinone spin dimer accounts well for the observed spectra. From a comparison of the EPR spectra of the Cu2+QA:RC and CU2+QA-:RC complexes we obtain the values /J0/ = (0.30 +/- 0.02) K for the isotropic exchange coupling, and /d/ = (0.010 +/- 0.002) K for the projection of the dipole-dipole interaction tensor on the symmetry axis of the copper spin. From the EPR experiments only the relative signs of J0 and d can be deduced; it was determined that they have the same sign. The magnitude of the exchange coupling calculated for Cu2+QA-:RC is similar to that observed for the Fe2+QA-:RC complex (J0 = -0.43K). The exchange coupling is discussed in terms of the superexchange paths connecting the Cu2+ ion and the quinone radical using the structural data for the RCs of Rb. sphaeroides. From the value of the dipole-dipole interaction, d, we determined R approximately 8.4 A for the weighted distance between the metal ion and the quinone in reduced RCs, which is to be compared with 10 A obtained from x-ray analysis of unreduced RCs. This points to a shortening of the Cu2+ -QA- distance upon reduction of the quinone, as has been proposed by Allen et al. (1988).     

The fe2+ site of photosynthetic reaction centers probed by multiple scattering x-ray absorption fine structure spectroscopy: improving structure resolution in dry matrices

We report on the x-ray absorption fine structure of the Fe(2+) site in photosynthetic reaction centers from Rhodobacter sphaeroides. Crystallographic studies show that Fe(2+) is ligated with four N(epsilon) atoms from four histidine (His) residues and two O(epsilon) atoms from a Glu residue. By considering multiple scattering contributions to the x-ray absorption fine structure function, we improved the structural resolution of the site: His residues were split into two groups, characterized by different Fe-N(epsilon) distances, and two distinct Fe-O(epsilon) bond lengths resolved. The effect of the environment was studied by embedding the reaction centers into a polyvinyl alcohol film and into a dehydrated trehalose matrix. Incorporation into trehalose caused elongation in one of the two Fe-N(epsilon) distances, and in one Fe-O(epsilon) bond length, compared with the polyvinyl alcohol film. The asymmetry detected in the cluster of His residues and its response to incorporation into trehalose are ascribed to the hydrogen bonds between two His residues and the quinone acceptors. The structural distortions observed in the trehalose matrix indicate a strong interaction between the reaction-centers surface and the water-trehalose matrix, which propagates deeply into the interior of the protein. The absence of matrix effects on the Debye-Waller factors is brought back to the static heterogeneity and rigidity of the ligand cluster.     

Structure of a bacterial photosynthetic membrane: integrity of reaction centers following proteolysis and detergent solubilization

cDNAs were molecularly cloned for proteins specifically expressed in embryo as well as in a chemically induced rat pancreatic B cell tumor in which virally related oncogenes such as v-myc, v-src, v-yes, v-mos and v-kis were previously demonstrated not to be expressed. A plasmid cDNA library consisting of 48,000 independent colonies was constructed from poly(A) containing cytoplasmic RNA isolated from 12 day rat embryo. The library was screened by hybridization with 32p-labelled cDNA synthesized from poly(A) containing RNA of rat pancreatic B cell tumor or normal islet B cells. Two clones were obtained which showed a clearly positive reaction only with tumor probe. Nucleotide sequence of one of them harboring insert of 615 nucleotides was determined and its amino acid sequence of 119 residues was deduced, which showed that the protein encoded by this mRNA is highly basic, basic residues/acidic residues being 1.63.     

Orientation of the primary quinone of bacterial photosynthetic reaction centers contined in chromatophore and reconstituted membranes

The orientation of the reaction center bacteriochlorophyll dimer, (BChl)₂, and primary quinone, Q_I, has been studied by EPR in chromatophores of Rhodopseudomonas sphaeroides R26 and Chromatium vinosum and in the reconstituted membrane multilayers of the isolated Rps. sphaeroides reaction center protein. The similarity in the angular dependence of the (BChl)₂ triplet and Q_I?Fe²⁺ signals in the chromatophore and reconstituted reaction center membrane multilayers indicates that the reaction center is similarly oriented in both native and model membranes. The principle magnetic axes of the (BChl)₂ triplet are found to lie with the x direction approximately parallel to the plane of the membrane surface, and the z and y directions approx. 10–20° away from the plane of the membrane surface and membrane normal, respectively. The Q_I?Fe²⁺ signals are found to have the g 1.82 component positioned perpendicular to the plane of the membrane surface, with an orthogonal low-field transition (at g 1.68 in Rps. Sphaeroides and at g 1.62 in C. vinosum) lying parallel to the plane of the membrane surface. The orientation of Q_I was determined by the angular dependence of this signal in Fe²⁺-depleted reaction center reconstituted membrane multilayers, and it was found to be situated most likely with the plane of the quinone ring perpendicular to the plane of the membrane surface.     

Spectroscopic characterization of a semi-stable, charge-separated state in Cu(2+)-substituted reaction centers from Rhodobacter sphaeroides

In reaction centers from Rhodobacter sphaeroides exposed to continuous illumination in the presence of an inhibitor of the Q(A)(-) to Q(B) electron transfer, a semi-stable, charge-separated state was formed with halftimes of formation and decay of several minutes. When the non-heme iron was replaced by Cu(2+), the decay of the semi-stable, charge-separated state became much slower than in centers with bound Fe(2+) with about the same rate constant for formation. In Cu(2+)-substituted reaction centers, the semi-stable state was associated with an EPR signal, significantly different from that observed after chemical reduction of the acceptor-side quinone or after illumination at low temperature, but similar to that of an isolated Cu(2+) in the absence of magnetic interaction. The EPR results, obtained with Cu(2+)-substituted reaction centers, suggest that the slow kinetics of formation and decay of the charge-separated, semi-stable state is associated with a structural rearrangement of the acceptor side and the immediate environment of the metal-binding site.     

Electron acceptors of bacterial photosynthetic reaction centers II. H+ binding coupled to secondary electron transfer in the quinone acceptor complex

The photoreduction of ubiquinone in the electron acceptor complex (Q₁Q₁₁) of photosynthetic reaction centers from Rhodopseudomonas sphaeroides, R26, was studied in a series of short, saturating flashes. The specific involvement of H⁺ in the reduction was revealed by the pH dependence of the electron transfer events and by net H⁺ binding during the formation of ubiquinol, which requires two turnovers of the photochemical act. On the first flash Q₁₁ receives an electron via Q₁ to form a stable ubisemiquinone anion (Q?^?₁₁); the second flash generates Q?^?₁. At low pH the two semiquinones rapidly disproportionate with the uptake of 2 H⁺, to produce Q₁₁H₂. This yields out-of-phase binary oscillations for the formation of anionic semiquinone and for H⁺ uptake. Above pH 6 there is a progressive increase in H⁺ binding on the first flash and an equivalent decrease in binding on the second flash until, at about pH 9.5, the extent of H⁺ binding is the same on all flashes. The semiquinone oscillations, however, are undiminished up to pH 9. It is suggested that a non-chromophoric, acid-base group undergoes a pK shift in response to the appearance of the anionic semiquinone and that this group is the site of protonation on the first flash. The acid-base group, which may be in the reaction center protein, appears to be subsequently involved in the protonation events leading to fully reduced ubiquinol. The other proton in the two electron reduction of ubiquinone is always taken up on the second flash and is bound directly to Q?^?₁₁. At pH values above 8.0, it is rate limiting for the disproportionation and the kinetics, which are diffusion controlled, are properly responsive to the prevailing pH. Below pH 8, however, a further step in the reaction mechanism was shown to be rate limiting for both H⁺ binding electron transfer following the second flash.