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1.
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. 相似文献
2.
Breton J 《Biochemistry》2007,46(15):4459-4465
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. 相似文献
3.
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. 相似文献
4.
Alexov E Miksovska J Baciou L Schiffer M Hanson DK Sebban P Gunner MR 《Biochemistry》2000,39(20):5940-5952
Numerical calculations of the free energy of the first electron transfer in genetically modified reaction centers from Rhodobacter (Rb.) sphaeroides and Rb. capsulatus were carried out from pH 5 to 11. The multiconformation continuum electrostatics (MCCE) method allows side chain, ligand, and water reorientation to be embedded in the calculations of the Boltzmann distribution of cofactor and amino acid ionization states. The mutation sites whose effects have been modeled are L212 and L213 (the L polypeptide) and two in the M polypeptide, M43(44) and M231(233) in Rb. capsulatus (Rb. sphaeroides). The results of the calculations were compared to the experimental data, and very good agreement was found especially at neutral pH. Each mutation removes or introduces ionizable residues, but the protein maintains a net charge close to that in native RCs through ionization changes in nearby residues. This reduces the effect of mutation and makes the changes in state free energy smaller than would be found in a rigid protein. The state energy of QA-QB and QAQB- states have contributions from interactions among the residues as well as with the quinone which is ionized. For example, removing L213Asp, located in the QB pocket, predominantly changes the free energy of the QA-QB state, where the Asp is ionized in native RCs rather than the QAQB- state, where it is neutral. Side chain, hydroxyl, and water rearrangements due to each of the mutations have also been calculated showing water occupancy changes during the QA- to QB electron transfer. 相似文献
5.
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. 相似文献
6.
Nabedryk E 《Biochimica et biophysica acta》1999,1411(1):206-213
The photoreduction of the secondary quinone acceptor QB in reaction centers (RCs) of the photosynthetic bacteria Rhodobacter (Rb.) capsulatus has been investigated by light-induced FTIR difference spectroscopy in 1H2O and 2H2O. The Q-B/QB FTIR spectra reflect reorganization of the protein upon electron transfer, changes of protonation state of carboxylic acid groups, and (semi)quinone-protein interactions. As expected from the conservation of most of the amino acids near QB in the RCs from Rb. capsulatus and Rb. sphaeroides, several protein and quinone IR bands are common to both spectra, e.g., the 1728 cm-1 band is assigned to proton uptake by a carboxylic acid residue, most probably Glu L212 as previously proposed for Rb. sphaeroides RCs. However, noticeable changes are observed at 1709 cm-1 (deprotonation of a Glu or Asp residue), 1674 and 1659 cm-1 (side chain and/or backbone), around 1540 cm-1 (amide II), and in the semiquinone absorption range. This FTIR study demonstrates that the environment of the secondary quinone in Rb. capsulatus is close but not identical to that in Rb. sphaeroides suggesting slight differences in the structural organization of side chains and/or ordered water molecules near QB. 相似文献
7.
Probing the primary quinone environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy 总被引:1,自引:0,他引:1
J Breton D L Thibodeau C Berthomieu W M?ntele A Verméglio E Nabedryk 《FEBS letters》1991,278(2):257-260
The photoreduction of the primary electron acceptor, QA, has been characterized by light-induced Fourier transform infrared difference spectroscopy for Rb. sphaeroides reaction centers and for Rsp. rubrum and Rp. viridis chromatophores. The samples were treated both with redox compounds, which rapidly reduce the photooxidized primary electron P+, and with inhibitors of electron transfer from QA- to the secondary quinone QB. This approach yields spectra free from P and P+ contributions which makes possible the study of the microenvironment of QA and QA-. 相似文献
8.
D K Hanson L Baciou D M Tiede S L Nance M Schiffer P Sebban 《Biochimica et biophysica acta》1992,1102(2):260-265
The mechanisms of proton conduction to the reduced secondary quinone in bacterial reaction centers were studied in wild-type and genetically modified reaction centers from Rhodobacter capsulatus. In the L212-213AA double mutant (L212Glu----Ala, L213Asp----Ala), reaction center function is severely altered. However, a photocompetent revertant of this strain which carries a third 'compensating' mutation, M231Arg----Leu, at about 15 A from the secondary quinone, displays the normal proton binding function of the reaction center. Furthermore, the apparent pK values of group(s) involved in the stabilization of the semiquinone anion are restored by that mutation. We conclude that L212Glu and L213Asp are not obligatory residues for proton donation to QB in Rb. capsulatus. We suggest that protons can be delivered to the QB site from the cytoplasm via a network of proton channels activated by compensatory mutations, possibly involving water molecules bound in the interior of the reaction center. 相似文献
9.
Cheap H Tandori J Derrien V Benoit M de Oliveira P Koepke J Lavergne J Maroti P Sebban P 《Biochemistry》2007,46(15):4510-4521
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. 相似文献
10.
The interaction of quinone and detergent with reaction centers of purple bacteria. I. Slow quinone exchange between reaction center micelles and pure detergent micelles.
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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. 相似文献
11.
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. 相似文献
12.
The extent of electrostatic contributions from the protein environment was assessed by the introduction of ionizable residues near the bacteriochlorophyll dimer in reaction centers from Rhodobacter sphaeroides. Two mutations at symmetry-related sites, M199 Asn to Asp and L170 Asn to Asp, resulted in a 48 and 44 mV lowering of the midpoint potential, respectively, compared to the wild type at pH 8, while a 75 mV decrease in the midpoint potential was observed for the mutation L168 His to Glu. The decrease relative to wild type was found to be approximately additive, up to 147 mV, for various combinations of the mutations. As the pH was lowered from 9.5 to 6.0, the relative decrease in the midpoint potential became smaller for each of these three mutations. Titration of the pH dependence of the change in midpoint potential of the M199 Asn to Asp mutant compared to wild type yielded a pK(a) value of 7.9 and a change in midpoint potential from low to high pH of 59 mV. The major effect of the mutation on the midpoint potential of the dimer is interpreted as stemming from a negative charge on the residue. An average dielectric constant of approximately 20 was estimated for the local protein environment, consistent with a relatively hydrophobic environment for residue M199. The rate of charge recombination between the primary quinone acceptor and the bacteriochlorophyll dimer decreased in the M199 Asn to Asp mutant at high pH, reflecting the decrease in midpoint potential. 相似文献
13.
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. 相似文献
14.
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) 相似文献
15.
Laible Philip D. Zhang Yuenian Morris Andrea L. Snyder Seth W. Ainsworth Clint Greenfield Scott R. Wasielewski Michael R. Parot Pierre Schoepp Barbara Schiffer Marianne Hanson Deborah K. Thurnauer Marion C. 《Photosynthesis research》1997,52(2):93-103
Site-specific mutations in the quinone binding sites of the photosynthetic reaction center (RC) protein complexes of Rhodobacter (R.) capsulatus caused pronounced effects on sequential electron transfer. Conserved residues that break the twofold symmetry in this region of the RC – M246Ala and M247Ala in the QA binding pocket, and L212Glu and L213Asp in the QB binding pocket – were targeted. We constructed a QB-site mutant, L212Glu-L213Asp Ala-Ala, and a QA-site mutant, M246Ala–M247Ala Glu-Asp, to partially balance the differences in charge distribution normally found between the two quinone binding sites. In addition, two photocompetent revertants were isolated from the photosynthetically-incompetent M246Glu-M247Asp mutant: M246Ala–M247Asp and M246Gly–M247Asp. Sequential electron transfer was investigated by continuous light excitation and time-resolved electron paramagnetic resonance (EPR), and time-resolved optical techniques. Several lines of EPR evidence suggested that the forward electron transfer rate to QA, kQ, was slowed in those strains containing altered QA sites. The slower rates of secondary electron transfer were confirmed by time-resolved optical results with the M246Glu-M247Asp mutations in the QA site resulting in a dramatically lowered secondary electron transfer efficiency [kQ < (2 ns)-1] in comparison with either the native R. capsulatus RC or the QB site mutant [kQ (200 ps)-1]. Secondary electron transfer in the two revertants was intermediate between that of the native RC and the QA mutant. The P+ QA- PQA charge recombination rates were also changed in the strains that carried altered QA sites. We show that local mutations in the QA site, presumably through local electrostatic changes, significantly alter binding and electron transfer properties of QA. 相似文献
16.
Onidas D Stachnik JM Brucker S Krätzig S Gerwert K 《European journal of cell biology》2010,89(12):983-989
In photosynthesis, the central step in transforming light energy into chemical energy is the coupling of light-induced electron transfer to proton uptake and release. Despite intense investigations of different photosynthetic protein complexes, including the photosystem II (PS II) in plants and the reaction center (RC) in bacteria, the molecular details of this fundamental process remain incompletely understood. In the RC of Rhodobacter (Rb.) sphaeroides, fast formation of the charge separated state, P(+)Q(A)(-), is followed by a slower electron transfer from the primary acceptor, Q(A), to the secondary acceptor, Q(B), and the uptake of a proton from the cytoplasm. The proton transfer to Q(B) takes place via a protonated water chain. Mutation of the amino acid AspL210 to Asn (L210DN mutant) near the entry of the proton pathway can disturb this water chain and consequently slow down proton uptake. Time-resolved step-scan Fourier transform infrared (FTIR) measurements revealed an intermediate X in the Q(A)(-)Q(B) to Q(A)Q(B)(-) transition. The nature of this transition remains a matter of debate. In this study, we investigated the role of the iron-histidine complex located between Q(A) and Q(B). We used time-resolved fast-scan FTIR spectroscopy to characterize the Rb. sphaeroides L210DN RC mutant marked with isotopically labeled histidine. FTIR marker bands of the intermediate X between 1120 cm(-1) and 1050 cm(-1) are assigned to histidine vibrations and indicate the protonation of a histidine, most likely HisL190, during the disappearance of the intermediate. Based on these results we propose a novel mechanism of the coupling of electron and proton transfer in photosynthesis. 相似文献
17.
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. 相似文献
18.
G Venturoli M Trotta R Feick B A Melandri D Zannoni 《European journal of biochemistry》1991,202(2):625-634
The temperature dependence of charge recombination from the P+QA- and from the P+QB- states produced by a flash was studied in reaction centers isolated from the photosynthetic thermophilic bacterium Chloroflexus aurantiacus. P designates the primary electron donor; QA and QB the primary and secondary quinone electron acceptors respectively. In QB-depleted reaction centers the rate constant (kAP) for P+QA- recombination was temperature independent between 0-50 degrees C (17.6 +/- 0.7 s-1 at pH 8 and pH 10). The same value was obtained in intact membranes in the presence of o-phenanthroline. Upon lowering the temperature from 250 K to 160 K, kAP increased by a factor of two and remained constant down to 80 K. The overall temperature dependence of kAP was consistent with an activationless process. Ubiquinone (UQ-3) and different types of menaquinone were used for QB reconstitution. In UQ-3 reconstituted reaction centers charge recombination was monoexponential (rate constant k = 0.18 +/- 0.03 s-1) and temperature independent between 5-40 degrees C. In contrast, in menaquinone-3- and menaquinone-4-reconstituted reaction centers P+ rereduction following a flash was markedly biphasic and temperature dependent. In menaquinone-6-reconstituted reaction centers a minor contribution from a third kinetic phase corresponding to P+QA- charge recombination was detected. Analysis of these kinetics and of the effects of the inhibitor o-phenanthroline at high temperature suggest that in detergent suspensions of menaquinone-reconstituted reaction centers a redox reaction removing electrons from the quinone acceptor complex competes with charge recombination. Instability of the semiquinone anions is more pronounced when QB is a short-chain menaquinone. From the temperature dependence of P+ decay the activation parameters for the P+QB- recombination and for the competing side oxidation of the reduced menaquinone acceptor have been derived. For both reactions the activation enthalpies and entropies change markedly with menaquinone chain length but counterbalance each other, resulting in activation free energies at ambient temperature independent of the menaquinone tail. When reaction centers are incorporated into phospholipid vesicles containing menaquinone-8 a temperature-dependent, monophasic, o-phenanthroline-sensitive recombination from the P+QB- state is observed, which is consistent with the formation of stable semiquinone anions. This result seems to indicate a proper QB functioning in the two-subunit reaction center isolated from Chlorflexus aurantiacus when the complex is inserted into a lipid bilayer. 相似文献
19.
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) 相似文献
20.
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. 相似文献