P+QA- and P+QB- charge recombinations in Rhodopseudomonas viridis chromatophores and in reaction centers reconstituted in phosphatidylcholine liposomes. Existence of two conformational states of the reaction centers and effects of pH and o-phenanthroline

The P+QA- and P+QB- charge recombination decay kinetics were studied in reaction centers from Rhodopseudomonas viridis reconstituted in phosphatidylcholine bilayer vesicles (proteoliposomes) and in chromatophores. P represents the primary electron donor, a dimer of bacteriochlorophyll; QA and QB are the primary and secondary stable quinone electron acceptors, respectively. In agreement with recent findings for reaction centers isolated in detergent [Sebban, P., & Wraight, C.A. (1989) Biochim. Biophys. Acta 974, 54-65] the P+QA- decay kinetics were biphasic (kfast and kslow). Arrhenius plots of the kinetics were linear, in agreement with the hypothesis of a thermally activated process (probably via P+I-; I is the first electron acceptor, a bacteriopheophytin) for the P+QA- charge recombination. Similar activation free energies (delta G) for this process were found in chromatophores and in proteoliposomes. Significant pH dependences of kfast and kslow were observed in chromtophores and in proteoliposomes. In the pH range 5.5-11, the pH titration curves of kfast and kslow were interpreted in terms of the existence of three protonable groups, situated between I- and QA-, which modulate the free energy difference between P+I- and P+QA-. In proteoliposomes, a marked effect of o-phenanthroline was observed on two of the three pKs, shifting one of them by more than 2 pH units. On the basis of recent structural data, we suggest a possible interpretation for this effect, which is much smaller in Rhodobacter sphaeroides. The decay kinetics of P+QB- were also biphasic. Marked pH dependences of the rate constants and of the relative proportions of both phases were also detected for these decays. The major conclusion of this work comes from the biphasicity of the P+QB- decay kinetics. We had suggested previously that biphasicity of the P+QA- charge recombination in Rps. viridis comes from nonequilibrium between protonation states of the reaction centers due to comparable rates of the protonation events and charge recombination. This hypothesis does not hold since the P+QB- decays occur on a time scale (tau approximately 300 ms at pH 8) much longer than protonation events. This leads to the conclusion that kfast and kslow (for both P+QA- and P+QB-) are related to conformational states of the reaction centers, existing before the flash. In addition, the fast and slow decays of P+QB- are related to those measured for P+QA-, via the calculations of the QA-QB in equilibrium QAQB- apparent equilibrium constants, K2.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)     

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.     

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.     

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)     

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.     

The acceptor quinone complex of Rhodopseudomonas viridis reaction centers

The acceptor complex of isolated reaction centers from Rhodopseudomonas viridis contains both menaquinone and ubiquinone. In a series of flashes the ubiquinone was observed to undergo binary oscillations in the formation and disappearance of a semiquinone, indicative of secondary acceptor (QB) activity. The oscillating signal, Q-B, was typical of a ubisemiquinone anion with a peak at 450 nm (delta epsilon = 6 mM-1 X cm-1) and a shoulder at 430 nm. Weak electrochromic bandshifts in the infrared were also evident. The spectrum of the reduced primary acceptor (Q-A) exhibited a major peak at 412 nm (delta epsilon = 10 mM-1 X cm-1) consistent with the assignment of menaquinone as QA. The Q-A spectrum also had minor peaks at 385 and 455 nm in the blue region. The same spectrum was recorded after quantitative removal of the secondary acceptor, when only menaquinone was present in the reaction centers. Spectral features in the near-infrared due to Q-A were attributed to electrochromic effects on bacteriochlorophyll (BChl) b and bacteriopheophytin (BPh) b pigments resulting in a distinctive split peak at 810 and 830 nm (delta epsilon = 8 mM-1 X cm-1). The menaquinone was identified as 2-methyl-3-nonylisoprenyl-1,4-naphthoquinone (menaquinone-9). The native QA activity was uniquely provided by this menaquinone and ubiquinone was not involved. QB activity, on the other hand, displayed at least a 40-fold preference for ubiquinone (Q-10) as compared to menaquinone. Thus, both quinone-binding sites display remarkable specificity for their respective quinones. In the absence of donors to P+, charge recombination of the P+Q-A and P+Q-B pairs had half-times of 1.1 +/- 0.2 and 110 +/- 20 ms, respectively, at pH 9.0, indicating an electron-transfer equilibrium constant (Kapp2) of at least 100 for Q-AQB in equilibrium QAQ-B. Also observed was a slow recombination of the cytochrome c-558+ Q-A pair, with t 1/2 = 2 +/- 0.5 s at pH 6.     

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.     

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-.     

Steady-state FTIR spectra of the photoreduction of QA and QB in Rhodobacter sphaeroides reaction centers provide evidence against the presence of a proposed transient electron acceptor X between the two quinones

In the reaction center (RC) of the photosynthetic bacterium Rhodobacter sphaeroides, two ubiquinone molecules, QA and QB, play a pivotal role in the conversion of light energy into chemical free energy by coupling electron transfer to proton uptake. In native RCs, the transfer of an electron from QA to QB takes place in the time range of 5-200 micros. On the basis of time-resolved FTIR step-scan measurements in native RCs, a new and unconventional mechanism has been proposed in which QB- formation precedes QA- oxidation [Remy, A., and Gerwert, K. (2003) Nat. Struct. Biol. 10, 637-644]. The IR signature of the proposed transient intermediary electron acceptor (denoted X) operating between QA and QB has been recently measured by the rapid-scan technique in the DN(L210) mutant RCs, in which the QA to QB electron transfer is slowed 8-fold compared to that in native RCs. This IR signature has been reported as a difference spectrum involving states X+, X, QA, and QA- [Hermes, S., et al. (2006) Biochemistry 45, 13741-13749]. Here, we report the steady-state FTIR difference spectra of the photoreduction of either QA or QB measured in both native and DN(L210) mutant RCs in the presence of potassium ferrocyanide. In these spectra, the CN stretching marker modes of ferrocyanide and ferricyanide allow the extent of the redox reactions to be quantitatively compared and are used for a precise normalization of the QA-/QA and QB-/QB difference spectra. The calculated QA- QB/QA QB- double-difference spectrum in DN(L210) mutant RCs is closely equivalent to the reported QA- X+/QA X spectrum in the rapid-scan measurement. We therefore conclude that species X+ and X are spectrally indistinguishable from QB and QB-, respectively. Further comparison of the QA- QB/QA QB- double-difference spectra in native and DN(L210) RCs also allows the possibility that QB- formation precedes QA- reoxidation to be ruled out for native RCs.     

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.     

Study of wild type and genetically modified reaction centers from Rhodobacter capsulatus: structural comparison with Rhodopseudomonas viridis and Rhodobacter sphaeroides.

Reaction centers from the purple bacterium Rhodobacter (Rb.) capsulatus and from two mutants ThrL226-->Ala and IleL229-->Ser, modified in the binding protein pocket of the secondary quinone acceptor (QB), have been studied by flash-induced absorbance spectroscopy. In ThrL226-->Ala, the binding affinities for endogenous QB (ubiquinone 10) and UQ6 are found to be two to three times as high as the wild type. In contrast, in IleL229-->Ser, the binding affinity for UQ6 is decreased about three times compared to the wild type. In ThrL226-->Ala, a markedly increased sensitivity (approximately 30 times) to o-phenanthroline is observed. In Rhodopseudomonas viridis, where Ala is naturally in position L226, the sensitivity to o-phenanthroline is close to that observed in ThrL226-->Ala. We propose that the presence of Ala in position L226 is responsible for the high sensitivity to that inhibitor. The pH dependencies of the rate constants of P+QB- (kBP) charge recombination kinetics (P is a dimer of bacteriochlorophyll, and QB is the secondary quinone electron acceptor) show destabilization of QB- in ThrL226-->Ala and IleL229-->Ser, compared to the wild type. At low pH, similar apparent pK values of protonation of amino acids around QB- are measured in the wild type and the mutants. In contrast to Rb. sphaeroides, in the wild type Rb. capsulatus, kBP substantially increases in the pH range 7-10.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functioning of quinone acceptors in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus aurantiacus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be reconstituted by the addition of ubiquinone-10. The equilibrium constant of electron transfer between primary (QA) and secondary (QB) quinones was much higher than that in RC of purple bacteria. The QB binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash-induced electric responses were about 200 mV. An additional electrogenic phase due to the QB protonation was observed after the second flash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P+QA-) formation. Since the C. aurantiacus RC lacks H-subunit, this subunit was not an obligatory component for electrogenic QB protonation.     

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.     

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.     

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)     

Primary photochemistry in the facultative green photosynthetic bacterium Chloroflexus aurantiacus

The mechanism of primary photochemistry has been investigated in purified cytoplasmic membranes and isolated reaction centers of Chloroflexus aurantiacus. Redox titrations on the cytoplasmic membranes indicate that the midpoint redox potential of P870, the primary electron donor bacteriochlorophyll, is +362 mV. An early electron acceptor, presumably menaquinone has Em 8.1 = -50 mV, and a tightly bound photooxidizable cytochrome c554 has Em 8.1 = +245 mV. The isolated reaction center has a bacteriochlorophyll to bacteriopheophytin ratio of 0.94:1. A two-quinone acceptor system is present, and is inhibited by o-phenanthroline. Picosecond transient absorption and kinetic measurements indicate the bacteriopheophytin and bacteriochlorophyll form an earlier electron acceptor complex.     

Probing the secondary quinone (QB) environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy

The photoreduction of the secondary electron acceptor, QB, has been characterized by light-induced Fourier transform infrared difference spectroscopy of Rb. sphaeroides and Rp. viridis reaction centers. The reaction centers were supplemented with ubiquinone (UQ10 or UQ0). The QB- state was generated either by continuous illumination at very low intensity or by single flash in the presence of redox compounds which rapidly reduce the photooxidized primary electron donor P+. This approach yields spectra free from P and P+ contributions making possible the study of the microenvironment of QB and QB-. Assignments are proposed for the C...O vibration of QB- and tentatively for the C = O and C = C vibrations of QB.