Proton and electron transfer in the acceptor quinone complex of Rhodobacter sphaeroides reaction centers: characterization of site-directed mutants of the two ionizable residues, GluL212 and AspL213, in the QB binding site.

Proton and electron transfer events in reaction centers (RCs) from Rhodobacter sphaeroides were investigated by site-directed mutagenesis of glutamic acid at position 212 and aspartic acid at 213 in the secondary quinone (QB) binding domain of the L subunit. These residues were mutated singly to the corresponding amides (mutants L212EQ and L213DN) and together to give the double mutant (L212EQ/L213DN). In the double mutant RCs, the rate of electron transfer from the primary (QA) to the secondary (QB) acceptor quinones is fast (tau approximately 300 microseconds) and is pH independent from pH 5 to 11. The rate of recombination between the oxidized primary donor, P+, and QB- is also pH independent and much slower (tau approximately 10 s) than in the wild type (Wt), indicating a significant stabilization of the QB- semiquinone. In the double mutant, and in L213DN mutant RCs at low pH, the P+QB- decay is suggested to occur significantly via a direct recombination rather than by repopulating the P+QA- state, as in the Wt. Comparison of the behavior of Wt and the three mutant RC types leads to the following conclusions: the pK of AspL213 in the Wt is approximately 4 for the QAQB state (pKQB) and approximately 5 for the QAQB-state (pKQB-); for GluL212, pKQB approximately 9.5 and pKQB- approximately 11. In L213DN mutant RCs, pKQB of GluL212 is less than or equal to 7, indicating that the high pK values of GluL212 in the Wt are due largely to electrostatic interaction with the ionized AspL213 which contributes a shift of at least 2.5 pH units. Transfer of the second electron and all associated proton uptake to form QBH2 is drastically inhibited in double mutant and L213DN mutant RCs. At pH greater than or equal to 8, the rates are at least 10(4)-fold slower than in Wt RCs. In L212EQ mutant RCs the second electron transfer and proton uptake are biphasic. The fast phase of the electron transfer is similar to that of the Wt, but the extent of rapid transfer is pH dependent, revealing the pH dependence of the equilibrium QA(-)QB- in equilibrium with QAQBH-. The estimated limits on the pK values--pKQA-QB-less than or equal to 7.3, pKQAQB2- greater than or equal to 10.4--are similar to those derived earlier for Wt RCs [Kleinfeld et al. (1985) Biochim. Biophys. Acta 809, 291-310] and may pertain to the quinone head group, per se.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for delocalized anticooperative flash induced proton binding as revealed by mutants at the M266His iron ligand in bacterial reaction centers

Bacterial reaction centers (RCs) convert light energy into chemical free energy via the double reduction and protonation of the secondary quinone electron acceptor, QB, to the dihydroquinone QBH2. Two RC mutants (M266His --> Leu and M266His --> Ala) with a modified ligand of the non-heme iron have been studied by flash-induced absorbance change spectroscopy. No important changes were observed for the rate constants of the first and second electron transfers between the first quinone electron acceptor, QA, and QB. However, in the M266HL mutant a destabilization of approximately 40 meV of the free energy level of QA- was observed, at variance with the M266HA mutant. The superposition of the three-dimensional X-ray structures of the three proteins in the QA region provides no obvious explanation for the energy modification in the M266HL mutant. The shift of the midpoint redox potential of QA/QA- in M266HL caused accelerated recombination of the charges in the P+ QA- state of the RCs where the native QA was replaced by a low potential anthraquinone (AQA). As previously reported for the native RCs, in the M266HL we observed a biphasicity of the P+ AQA- --> P AQA charge recombination. Interestingly, both phases present a similar acceleration in the M266HL mutant with respect to the wild type. The pH dependencies of the proton uptake upon QA- and QB- formations are superimposable in both mutants but very different from those of native RCs. The data measured in mutants are similar to those that we previously obtained on strains modified at various sites of the cytoplasmic region. The similarity of the response to these different mutations is puzzling, and we propose that it arises from a collective behavior of multiple acidic residues resulting in strongly anticooperative proton binding. The unspecific disappearance of the high pH band of proton uptake observed in all these mutants appears as the natural consequence of removing any member of an interactive proton cluster. This long range interaction also accounts for the similar responses to mutations of the proton uptake pattern induced by either QA- or QB-. We surmise that the presence of an extended protonated water H-bond network providing protons to QB is responsible for these effects.     

The unusually strong hydrogen bond between the carbonyl of Q(A) and His M219 in the Rhodobacter sphaeroides reaction center is not essential for efficient electron transfer from Q(A)(-) to Q(B)

In native reaction centers (RCs) from photosynthetic purple bacteria the primary quinone (QA) and the secondary quinone (QB) are interconnected via a specific His-Fe-His bridge. In Rhodobacter sphaeroides RCs the C4=O carbonyl of QA forms a very strong hydrogen bond with the protonated Npi of His M219, and the Ntau of this residue is in turn coordinated to the non-heme iron atom. The second carbonyl of QA is engaged in a much weaker hydrogen bond with the backbone N-H of Ala M260. In previous work, a Trp side chain was introduced by site-directed mutagenesis at the M260 position in the RC of Rb. sphaeroides, resulting in a complex that is completely devoid of QA and therefore nonfunctional. A photochemically competent derivative of the AM260W mutant was isolated that contains a Cys side chain at the M260 position (denoted AM260(W-->C)). In the present work, the interactions between the carbonyl groups of QA and the protein in the AM260(W-->C) suppressor mutant have been characterized by light-induced FTIR difference spectroscopy of the photoreduction of QA. The QA-/QA difference spectrum demonstrates that the strong interaction between the C4=O carbonyl of QA and His M219 is lost in the mutant, and the coupled CO and CC modes of the QA- semiquinone are also strongly perturbed. In parallel, a band assigned to the perturbation of the C5-Ntau mode of His M219 upon QA- formation in the native RC is lacking in the spectrum of the mutant. Furthermore, a positive band between 2900 and 2400 cm-1 that is related to protons fluctuating within a network of highly polarizable hydrogen bonds in the native RC is reduced in amplitude in the mutant. On the other hand, the QB-/QB FTIR difference spectrum is essentially the same as for the native RC. The kinetics of electron transfer from QA- to QB were measured by the flash-induced absorption changes at 780 nm. Compared to native RCs the absorption transients are slowed by a factor of about 2 for both the slow phase (in the hundreds of microseconds range) and fast phase (microseconds to tens of microseconds range) in AM260(W-->C) RCs. We conclude that the unusually strong hydrogen bond between the carbonyl of QA and His M219 in the Rb. sphaeroides RC is not obligatory for efficient electron transfer from QA- to QB.     

Trapped conformational states of semiquinone (D+*QB-*) formed by B-branch electron transfer at low temperature in Rhodobacter sphaeroides reaction centers

The reaction center (RC) from Rhodobacter sphaeroides captures light energy by electron transfer between quinones QA and QB, involving a conformational gating step. In this work, conformational states of D+*QB-* were trapped (80 K) and studied using EPR spectroscopy in native and mutant RCs that lack QA in which QB was reduced by the bacteriopheophytin along the B-branch. In mutant RCs frozen in the dark, a light induced EPR signal due to D+*QB-* formed in 30% of the sample with low quantum yield (0.2%-20%) and decayed in 6 s. A small signal with similar characteristics was also observed in native RCs. In contrast, the EPR signal due to D+*QB-* in mutant RCs illuminated while freezing formed in approximately 95% of the sample did not decay (tau >107 s) at 80 K (also observed in the native RC). In all samples, the observed g-values were the same (g = 2.0026), indicating that all active QB-*'s were located in a proximal conformation coupled with the nonheme Fe2+. We propose that before electron transfer at 80 K, the majority (approximately 70%) of QB, structurally located in the distal site, was not stably reducible, whereas the minority (approximately 30%) of active configurations was in the proximal site. The large difference in the lifetimes of the unrelaxed and relaxed D+*QB-* states is attributed to the relaxation of protein residues and internal water molecules that stabilize D+*QB-*. These results demonstrate energetically significant conformational changes involved in stabilizing the D+*QB-* state. The unrelaxed and relaxed states can be considered to be the initial and final states along the reaction coordinate for conformationally gated electron transfer.     

Iron-depleted reaction centers from Rhodopseudomonas sphaeroides R-26.1: characterization and reconstitution with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+

Reaction centers (RCs) from the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26.1 were depleted of Fe by a simple procedure involving reversible dissociation of the H subunit. The resulting intact Fe-depleted RCs contained 0.1-0.2 Fe per RC as determined from atomic absorption and electron paramagnetic resonance (EPR) spectroscopy. Fe-depleted RCs that have no metal ion occupying the Fe site differed from native RCs in the following respects: (1) the rate of electron transfer from QA- to QB exhibited nonexponential kinetics with the majority of RCs having a rate constant slower by only a factor of approximately 2, (2) the efficiency of light-induced charge separation (DQA----D+QA-) produced by a saturating flash decreased to 63%, and (3) QA appeared readily reducible to QA2-. Various divalent metal ions were subsequently incorporated into the Fe site. The electron transfer characteristics of Fe-depleted RCs reconstituted with Fe2+, Mn2+, Co2+, Ni2+, Cu2+, and Zn2+ were essentially the same as those of native RCs. These results demonstrate that neither Fe2+ nor any divalent metal ion is required for rapid electron transfer from QA- to QB. However, the presence of a metal ion in the Fe site is necessary to establish the characteristic, native, electron-transfer properties of QA. The lack of a dominant role of Fe2+ or other divalent metals in the observed rate of electron transfer from QA- to QB suggests that a rate-limiting step (for example, a protonation event or a light-induced structural change) precedes electron transfer.     

Coupling of light-induced electron transfer to proton uptake in photosynthesis

Light energy is transformed into chemical energy in photosynthesis by coupling a light-induced electron transfer to proton uptake. The resulting proton gradient drives ATP synthesis. In this study, we monitored the light-induced reactions in a 100-kDa photosynthetic protein from 30 ns to 35 s by FTIR difference spectroscopy. The results provide detailed mechanistic insights into the electron and proton transfer reactions of the QA to QB transition: reduction of QA in picoseconds induces protonation of histidines, probably of His126 and His128 in the H subunit at the entrance of the proton uptake channel, and of Asp210 in the L subunit inside the channel at 12 micros and 150 micros. This seems to be a prerequisite for the reduction of QB, mainly at 150 micros. QA- is reoxidized at 1.1 ms, and a proton is transferred from Asp210 to Glu212 in the L subunit, the proton donor to QB-. Notably, our data indicate that QB is not reduced directly by QA- but presumably through an intermediary electron donor.     

Calculated protein and proton motions coupled to electron transfer: electron transfer from QA- to QB in bacterial photosynthetic reaction centers.

Reaction centers from Rhodobacter sphaeroides were subjected to Monte Carlo sampling to determine the Boltzmann distribution of side-chain ionization states and positions and buried water orientation and site occupancy. Changing the oxidation states of the bacteriochlorophyll dimer electron donor (P) and primary (QA) and secondary (QB) quinone electron acceptors allows preparation of the ground (all neutral), P+QA-, P+QB-, P0QA-, and P0QB- states. The calculated proton binding going from ground to other oxidation states and the free energy of electron transfer from QA-QB to form QAQB- (DeltaGAB) compare well with experiment from pH 5 to pH 11. At pH 7 DeltaGAB is measured as -65 meV and calculated to be -80 meV. With fixed protein positions as in standard electrostatic calculations, DeltaGAB is +170 meV. At pH 7 approximately 0.2 H+/protein is bound on QA reduction. On electron transfer to QB there is little additional proton uptake, but shifts in side chain protonation and position occur throughout the protein. Waters in channels leading from QB to the surface change site occupancy and orientation. A cluster of acids (GluL212, AspL210, and L213) and SerL223 near QB play important roles. A simplified view shows this cluster with a single negative charge (on AspL213 with a hydrogen bond to SerL233) in the ground state. In the QB- state the cluster still has one negative charge, now on the more distant AspL210. AspL213 and SerL223 move so SerL223 can hydrogen bond to QB-. These rearrangements plus other changes throughout the protein make the reaction energetically favorable.     

Proton uptake in the reaction center mutant L210DN from Rhodobacter sphaeroides via protonated water molecules

The reaction center (RC) of Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, QB (the secondary quinone electron acceptor), to form quinol, QBH2. Asp210 in the L-subunit has been shown to be a catalytic residue in this process. Mutation of Asp210 to Asn leads to a deceleration of reoxidation of QA- in the QA-QB --> QAQB- transition. Here we determined the structure of the Asp210 to Asn mutant to 2.5 A and show that there are no major structural differences as compared to the wild-type protein. We found QB in the distal position and a chain of water molecules between Asn210 and QB. Using time-resolved Fourier transform infrared (trFTIR) spectroscopy, we characterized the molecular reaction mechanism of this mutant. We found that QB- formation precedes QA- oxidation even more pronounced than in the wild-type reaction center. Continuum absorbance changes indicate deprotonation of a protonated water cluster, most likely of the water chain between Asn210 and QB. A detailed analysis of wild-type structures revealed a highly conserved water chain between Asp210 or Glu210 and QB in Rb. sphaeroides and Rhodopseudomonas viridis, respectively.     

Conformational heterogeneity of the bacteriopheophytin electron acceptor HA in reaction centers from Rhodopseudomonas viridis revealed by Fourier transform infrared spectroscopy and site-directed mutagenesis.

The light-induced Fourier transform infrared (FTIR) difference spectra corresponding to the photoreduction of either the HA bacteriopheophytin electron acceptor (HA-/HA spectrum) or the QA primary quinone (QA-/QA spectrum) in photosynthetic reaction centers (RCs) of Rhodopseudomonas viridis are reported. These spectra have been compared for wild-type (WT) RCs and for two site-directed mutants in which the proposed interactions between the carbonyls on ring V of HA and the RC protein have been altered. In the mutant EQ(L104), the putative hydrogen bond between the protein and the 9-keto C=O of HA should be affected by changing Glu L104 to a Gln. In the mutant WF(M250), the van der Waals interactions between Trp M250 and the 10a-ester C=O of HA should be modified. The characteristic effects of both mutations on the FTIR spectra support the proposed interactions and allow the IR modes of the 9-keto and 10a-ester C=O of HA and HA- to be assigned. Comparison of the HA-/HA and QA-/QA spectra leads us to conclude that the QA-/QA IR signals in the spectral range above 1700 cm-1 are largely dominated by contributions from the electrostatic response of the 10a-ester C=O mode of HA upon QA photoreduction. A heterogeneity in the conformation of the 10a-ester C=O mode of HA in WT RCs, leading to three distinct populations of HA, appears to be related to differences in the hydrogen-bonding interactions between the carbonyls of ring V of HA and the RC protein. The possibility that this structural heterogeneity is related to the observed multiexponential kinetics of electron transfer and the implications for primary processes are discussed. The effect of 1H/2H exchange on the QA-/QA spectra of the WT and mutant RCs shows that neither Glu L104 nor any other exchangeable carboxylic residue changes appreciably its protonation state upon QA reduction.     

ENDOR spectroscopy reveals light induced movement of the H-bond from Ser-L223 upon forming the semiquinone (Q(B)(-)(*)) in reaction centers from Rhodobacter sphaeroides

Proton ENDOR spectroscopy was used to monitor local conformational changes in bacterial reaction centers (RC) associated with the electron-transfer reaction DQB --> D+*QB-* using mutant RCs capable of photoreducing QB at cryogenic temperatures. The charge separated state D+*QB-* was studied in mutant RCs formed by either (i) illuminating at low temperature (77 K) a sample frozen in the dark (ground state protein conformation) or (ii) illuminating at room temperature prior to and during freezing (charge separated state protein conformation). The charge recombination rates from the two states differed greatly (>10(6) fold) as shown previously, indicating a structural change (Paddock et al. (2006) Biochemistry 45, 14032-14042). ENDOR spectra of QB-* from both samples (35 GHz, 77 K) showed several H-bond hyperfine couplings that were similar to those for QB-* in native RCs indicating that in all RCs, QB-* was located at the proximal position near the metal site. In contrast, one set of hyperfine couplings were not observed in the dark frozen samples but were observed only in samples frozen under illumination in which the protein can relax prior to freezing. This flexible H-bond was assigned to an interaction between the Ser-L223 hydroxyl and QB-* on the basis of its absence in Ser L223 --> Ala mutant RCs. Thus, part of the protein relaxation, in response to light induced charge separation, involves the formation of an H-bond between the OH group of Ser-L223 and the anionic semiquinone QB-*. These results show the flexibility of the Ser-L223 H-bond, which is essential for its function in proton transfer to reduced QB.     

Excitation pressure regulates the activation energy for recombination events in the photosystem II reaction centres of Chlamydomonas reinhardtii.

Using in vivo thermoluminescence, we examined the effects of growth irradiance and growth temperature on charge recombination events in photosystem II reaction centres of the model green alga Chlamydomonas reinhardtii. We report that growth at increasing irradiance at either 29 or 15 degrees C resulted in comparable downward shifts in the temperature peak maxima (T(M)) for S2QB- charge pair recombination events, with minimal changes in S2QA- recombination events. This indicates that such growth conditions decrease the activation energy required for S2QB- charge pair recombination events with no concomitant change in the activation energy for S2QA- recombination events. This resulted in a decrease in the DeltaT(M) between S2QA- and S2QB- recombination events, which was reversible when shifting cells from low to high irradiance and back to low irradiance at 29 degrees C. We interpret these results to indicate that the redox potential of QB was modulated independently of QA, which consequently narrowed the redox potential gap between QA and QB in photosystem II reaction centres. Since a decrease in the DeltaT(M) between S2QA- and S2QB- recombination events correlated with growth at increasing excitation pressure, we conclude that acclimation to growth under high excitation pressure narrows the redox potential gap between QA and QB in photosystem II reaction centres, enhancing the probability for reaction center quenching in C. reinhardtii. We discuss the molecular basis for the modulation of the redox state of QB, and suggest that the potential for reaction center quenching complements antenna quenching via the xanthophyll cycle in the photoprotection of C. reinhardtii from excess light.     

Formation of a semiquinone at the QB site by A- or B-branch electron transfer in the reaction center from Rhodobacter sphaeroides

In Rhodobacter sphaeroides reaction centers containing the mutation Ala M260 to Trp (AM260W), transmembrane electron transfer along the A-branch of cofactors is prevented by the loss of the QA ubiquinone. Reaction centers that contain this AM260W mutation are proposed to photoaccumulate the P(+)QB- radical pair following transmembrane electron transfer along the B-branch of cofactors (Wakeham, M. C., Goodwin, M. G., McKibbin, C., and Jones, M. R. (2003) Photoaccumulation of the P(+)QB- radical pair state in purple bacterial reaction centers that lack the QA ubiquinone. FEBS Lett. 540, 234-240). The yield of the P(+)QB- state appears to depend upon which additional mutations are present. In the present paper, Fourier transform infrared (FTIR) difference spectroscopy was used to demonstrate that photooxidation of the reaction center's primary donor in QA-deficient reaction centers results in formation of a semiquinone at the QB site by B-branch electron transfer. Reduction of QB by the B-branch pathway still occurs at 100 K, with a yield of approximately 10% relative to that at room temperature, in contrast to the QA- to QB reaction in the wild-type reaction center, which is not active at cryogenic temperatures. These FTIR results suggest that the conformational changes that "gate" the QA- to QB reaction do not necessarily have the same influence on QB reduction when the electron donor is the HB anion, at least in a minority of reaction centers.     

Electron nuclear double resonance of semiquinones in reaction centers of Rhodopseudomonas sphaeroides

Replacement of Fe2+ by Zn2+ in reaction centers of Rhodopseudomonas sphaeroides enabled us to perform ENDOR (electron nuclear double resonance) experiments on the anion radicals of the primary and secondary ubiquinone acceptors (QA- and QB-. Differences between the QA and QB sites, hydrogen bonding to the oxygens, interactions with the protons of the proteins and some symmetry properties of the binding sites were deduced from an analysis of the ENDOR spectra.     

The interaction of quinone and detergent with reaction centers of purple bacteria. I. Slow quinone exchange between reaction center micelles and pure detergent micelles.

The kinetics of light-induced electron transfer in reaction centers (RCs) from the purple photosynthetic bacterium Rhodobacter sphaeroides were studied in the presence of the detergent lauryldimethylamine-N-oxide (LDAO). After the light-induced electron transfer from the primary donor (P) to the acceptor quinone complex, the dark re-reduction of P+ reflects recombination from the reduced acceptor quinones, QA- or QB-. The secondary quinone, QB, which is loosely bound to the RC, determines the rate of this process. Electron transfer to QB slows down the return of the electron to P+, giving rise to a slow phase of the recovery kinetics with time tau P approximately 1 s, whereas charge recombination in RCs lacking QB generates a fast phase with time tau AP approximately 0.1 s. The amount of quinone bound to RC micelles can be reduced by increasing the detergent concentration. The characteristic time of the slow component of P+ dark relaxation, observed at low quinone content per RC micelle (at high detergent concentration), is about 1.2-1.5 s, in sharp contrast to expectations from previous models, according to which the time of the slow component should approach the time of the fast component (about 0.1 s) when the quinone concentration approaches zero. To account for this large discrepancy, a new quantitative approach has been developed to analyze the kinetics of electron transfer in isolated RCs with the following key features: 1) The exchange of quinone between different micelles (RC and detergent micelles) occurs more slowly than electron transfer from QB- to P+; 2) The exchange of quinone between the detergent "phase" and the QB binding site within the same RC micelle is much faster than electron transfer between QA- and P+; 3) The time of the slow component of P+ dark relaxation is determined by (n) > or = 1, the average number of quinones in RC micelles, calculated only for those RC micelles that have at least one quinone per RC (in excess of QA). An analytical function is derived that relates the time of the slow component of P+ relaxation, tau P, and the relative amplitude of the slow phase. This provides a useful means of determining the true equilibrium constant of electron transfer between QA and QB (LAB), and the association equilibrium constant of quinone binding at the QB site (KQ+). We found that LAB = 22 +/- 3 and KQ = 0.6 +/- 0.2 at pH 7.5. The analysis shows that saturation of the QB binding site in detergent-solubilized RCs is difficult to achieve with hydrophobic quinones. This has important implications for the interpretation of apparent dependencies of QB function on environmental parameters (e.g. pH) and on mutational alterations. The model accounts for the effects of detergent and quinone concentration on electron transfer in the acceptor quinone complex, and the conclusions are of general significance for the study of quinone-binding membrane proteins in detergent solutions.     

Absence of a bicarbonate-depletion effect in electron transfer between quinones in chromatophores and reaction centers of Rhodobacter sphaeroides

Higher plants, algae, and cyanobacteria are known to require bicarbonate ions for electron flow from the first stable electron acceptor quinone QA to the second electron acceptor quinone QB, and to the intersystem quinone pool. It has been suggested that in Photosystem II of oxygenic photosynthesis, bicarbonate ion functions to maintain the reaction center in a proper conformation and, perhaps, to provide the protons needed to stabilize the semiquinone (QB-). In this paper, we show that bicarbonate ions do not influence the electron flow, from the quinone QA to QB and beyond, in the photosynthetic bacterium Rhodobacter sphaeroides. No measurable effect of bicarbonate depletion, obtained by competition with formate, was observed on cytochrome b-561 reduction in chromatophores; on the flash-dependent oscillation of semiquinone formation in reaction centers; on electron transfer from QA- to QB; or on either the fast or slow recovery of the oxidized primary donor (P+) which reflects the P+QA- ----PQA or the P+QB- ----PQB reaction. The lack of an observed effect in Rhodobacter sphaeroides in contrast to the effect seen in Photosystem II is suggested to be due to the amino-acid sequence differences between the reaction centers of the two systems.     

Kinetic properties of the acceptor quinone complex in Rhodopseudomonas viridis

The kinetics of electron transfer from the primary (QA) to the secondary (QB) quinone acceptor in whole cells and chromatophores of Rhodopseudomonas viridis was studied as a function of the redox state of QB and of pH by using a photovoltage technique. Under conditions where QB was oxidized, the reoxidation of QA- was found to be essentially monophasic and independent of pH with a half-time of about 20 microseconds. When QB was reduced to the semiquinone form by a preflash, the reoxidation of QA- was slowed down showing a half-time between 40 and 80 microseconds at pH less than or equal to 9. Above pH 9, the rate of the second electron transfer decreased nearly one order of magnitude per pH unit. After a further preflash, the fast and pH-independent kinetics of QA- reoxidation was essentially restored. The concentration of QA still reduced 100 microseconds after its complete reduction by a flash showed distinct binary oscillations as a function of the number of preflashes, confirming the interpretation that the electron-transfer rate depends on the redox state of QB. After addition of o-phenanthroline, the reoxidation of QA- is slowed down to the time range of seconds as expected for a back-reaction with oxidized cytochrome. Under conditions where inhibitors of the electron transfer between the quinones fail to block this reaction in a fraction of the reaction centers due to the presence of the extremely stable and strongly bound semiquinone, QB-, these reaction centers show a slow electron transfer on the first flash and a fast one on the second, i.e., an out-of-phase oscillation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monitoring the pH dependence of IR carboxylic acid signals upon Q(B)- formation in the Glu-L212 --> Asp/Asp-L213 --> Glu swap mutant reaction center from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter sphaeroides, proton-coupled electron-transfer reactions occur at the secondary quinone (QB) site. Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations in the 1770-1700 cm-1 spectral range that are sensitive to 1H/2H isotopic exchange. With respect to the native RC, a novel protonation pattern for carboxylic acids upon QB photoreduction has been identified in the Glu-L212 --> Asp/Asp-L213 --> Glu mutant RC using light-induced FTIR difference spectroscopy (Nabedryk, E., Breton, J., Okamura, M. Y., and Paddock, M. L. (2004) Biochemistry 43, 7236-7243). These carboxylic acids are structurally close and have been implicated in proton transfer to reduced QB. In this work, we extend previous studies by measuring the pH dependence of the QB-/QB FTIR difference spectra of the mutant in 1H2O and 2H2O. Large pH dependent changes were observed in the 1770-1700 cm-1 spectral range between pH 8 and pH 4. The IR fingerprints of the protonating carboxylic acids upon QB- formation were obtained from the calculated double-difference spectra 1H2O minus 2H2O. These IR fingerprints are specific for each pH, indicative of the contribution of different titrating groups. In particular, the 1752 cm-1 signal indicates that Glu-L213 protonates upon QB- formation at pH >or= 5, whereas the 1746 cm-1 signal indicates protonation of Asp-L212 even at pH 4. An unidentified carboxylic acid absorbing at approximately 1765 cm-1 could be the proton donor between pH 8 and 5. The observation that in the swap mutant there are several uniquely behaving carboxylic acids shows that electrostatic interactions occurring between them are sufficiently modified from the native RC to reveal their IR signatures.     

A Mutation in the D-de Loop of D1 Modifies the Stability of the S2QA- and S2QB- States in Photosystem II

Photosystem II electron transfer, charge stabilization, and photoinhibition were studied in three site-specific mutants of the D1 polypeptide of Synechocystis PCC 6803: E243K, E229D, and CA1 (deletion of three glutamates 242-244 and a substitution, glutamine-241 to histidine). The phenotypes of the E229D and E243K mutants were similar to that of the control strain (AR) in all of the studied aspects. The characteristics of CA1 were very different. Formate, which inhibits the QA- to QB- reaction, was severalfold less effective in CA1 than in AR. The S2QA- and S2QB- states were stabilized in CA1. It was previously shown that the electron transfer between QA- and QB was modified in CA1 (P Maenpaa, T. Kallio, P. Mulo, G. Salih, E.-M. Aro, E. Tyystjarvi, C. Jansson [1993] Plant Mol Biol 22: 1-12). A change in the redox potential of the QA/QA- couple, which renders the reoxidation of QA- by back or forward reactions more difficult, could explain the phenotype of CA1. Although the rates of photoinhibition measured as inhibition of oxygen evolution, Chl fluorescence quenching, and decrease of thermoluminescence B and Q bands were similar in AR and CA1, the CA1 strain more quickly reached a state from which the cells were unable to recover their activity. The results described in this paper suggest that a modification in the structure of the D-de loop of D1 could influence the properties of the couple QA/QA- in D2 and the mechanism of recovery from photoinhibition.     

Primary donor recovery kinetics in reaction centers from Rhodopseudomonas viridis. The influence of ferricyanide as a rapid oxidant of the acceptor quinones

In reaction centers from Rhodopseudomonas viridis that contain a single quinone, the decay of the photo-oxidized primary donor, P+, was found to be biphasic when the bound, donor cytochromes were chemically oxidized by ferricyanide. The ratio of the two phases was dependent on pH with an apparent pK of 7.6. A fast phase, which dominated at high pH (t1/2 = 1 ms at pH 9.5), corresponded to the expected charge recombination of P+ and the primary acceptor QA-. A much slower phase dominated at low pH and was shown to arise from a slow reduction of P+ by ferrocyanide in reaction centers where QA- has been rapidly oxidized by ferricyanide. The rate of QA- oxidation was linear with respect to ferricyanide activity and was strongly pH-dependent. The second-order rate constant, corrected for the activity coefficient of ferricyanide, approached a maximum of 2 X 10(8) M-1 X s-1 at low pH, but decreased steadily as the pH was raised above a pK of 5.8, indicating that a protonated state of the reaction center was involved. The slow reduction of P+ by ferrocyanide was also second-order, with a maximum rate constant at low pH of 8 X 10(5) M-1 X s-1 corrected for the activity coefficient of ferrocyanide. This rate also decreased at higher pH, with a pK of 7.4, indicating that ferrocyanide also was most reactive with a protonated form of the reaction center. The oxidation of QA- by ferricyanide was unaffected by the presence of o-phenanthroline, implying that access to QA- was not via the QB-binding site. In reaction centers supplemented with ubiquinone, oxidation of reduced secondary quinone, QB-, by ferricyanide was observed but was substantially slower than that for QA-. It is suggested that Q-B may be oxidized via QA so that the rate is modulated by the equilibrium constant for QA-QB in equilibrium with QAQB-.     

Study of QB- stabilization in herbicide-resistant mutants from the purple bacterium Rhodopseudomonas viridis

The pH dependences of the rate constants of P+QB- (kBP) and P+QA- (kAP) charge recombination decays have been studied by flash-induced absorbance change technique, in chromatophores of three herbicide-resistant mutants from Rhodopseudomonas (Rps.) viridis, and compared to the wild type. P, QA, and QB are the primary electron donor and the primary and the secondary quinone acceptors, respectively. The triazine resistant mutants T1 (Arg L217----His and Ser L223----Ala), T3 (Phe L216----Ser and Val M263----Phe), and T4 (Tyr L222----Phe), all mutated in the QB binding pocket of the reaction center, have previously been characterized (Sinning, I., Michel, H., Mathis, P., & Rutherford, A. W. (1989) Biochemistry 28, 5544-5553). The pH dependence curves of kBP in T4 and the wild type are very close. This confirms that the sensitivity toward DCMU of T4 is mainly due to a structural rearrangement in the QB pocket rather than to a change in the charge distribution in this part of the protein. In T3, a 6-fold increase of kAP is observed (kAP = 4200 +/- 300 s-1 at pH 8) compared to that of the wild type (kAP = 720 +/- 50 s-1 at pH 8). We propose that the Val M263----Phe mutation induces a free energy decrease between P+QA- and P+I- (delta G zero IA) (I is the primary electron acceptor) of about 49 meV. The very different pH dependence of kAP in T3 suggests a substantial change in the QA pocket. The 2.5 times increase of kAP above pH 9.5 in the wild type is no longer detected in T3.(ABSTRACT TRUNCATED AT 250 WORDS)