Time-resolved infrared spectroscopy of electron transfer in bacterial photosynthetic reaction centers: dynamics of binding and interaction upon QA and QB reduction.

Light-induced forward electron transfer in the bacterial photosynthetic reaction center from Rhodobacter sphaeroides was investigated by time-resolved infrared spectroscopy. Using a highly sensitive kinetic photometer based on a tunable IR diode laser source [M?ntele, W., Hienerwadel, R., Lenz, F., Riedel, W. J., Grisar, R., & Tacke, M. (1990a) Spectrosc. Int. 2, 29-35], molecular processes concomitant with electron-transfer reactions were studied in the microsecond-to-second time scale. Infrared (IR) signals in the 1780-1430-cm-1 spectral region, appearing within the instrument time resolution of about 0.5 microseconds, could be assigned to molecular changes of the primary electron donor upon formation of a radical cation and to modes of the primary quinone electron acceptor QA and its environment upon formation of QA-. These IR signals are consistent with steady-state FTIR difference spectra of the P+Q- formation [M?ntele, W., Nabedryk, E., Tavitian, B. A., Kreutz, W., & Breton, J. (1985) FEBS Lett. 187, 227-232; M?ntele, W., Wollenweber, A., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472; Nabedryk, E., Bagley, K. A., Thibodeau, D. L., Bauscher, M., M?ntele, W., & Breton, J. (1990) FEBS Lett. 266, 59-62] and with time-resolved FTIR studies [Thibodeau, D. L., Nabedryk, E., Hienerwadel, R., Lenz, F., M?ntele, W., & Breton, J. (1990) Biochim. Biophys. Acta 1020, 253-259]. At given wavenumbers, kinetic components with a half-time of approximately 120 microseconds were observed and attributed to QA----QB electron transfer. The time-resolved IR signals, in contrast to steady-state experiments where full protein relaxation after electron transfer can occur, allow us to follow directly the modes of QA and QB and their protein environment under conditions of forward electron transfer. Apart from signals attributed to the primary electron donor, signals are proposed to arise not only from the C = O and C = C vibrational modes of the neutral quinones and from the C-O and C-C vibrations of their semiquinone anion form but also from amino acid groups forming their binding sites. Some of the signals appearing with the instrument rise time as well as the transient 120-microseconds signals are interpreted in terms of binding and interaction of the primary and secondary quinone electron acceptor in the Rb. sphaeroides reaction center and of the conformational changes in their binding site.(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)     

Probing the primary quinone environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy

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-.     

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.     

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.     

Is bicarbonate in Photosystem II the equivalent of the glutamate ligand to the iron atom in bacterial reaction centers?

Photosystem II of oxygen-evolving organisms exhibits a bicarbonate-reversible formate effect on electron transfer between the primary and secondary acceptor quinones, QA and QB. This effect is absent in the otherwise similar electron acceptor complex of purple bacteria, e.g., Rhodobacter sphaeroides. This distinction has led to the suggestion that the iron atom of the acceptor quinone complex in PS II might lack the fifth and sixth ligands provided in the bacterial reaction center (RC) by a glutamate residue at position 234 of the M-subunit in Rb. sphaeroides RCs (M232 in Rps. viridis). By site-directed mutagenesis we have altered GluM234 in RCs from Rb. sphaeroides, replacing it with valine, glutamine and glycine to form mutants M234EV, M234EQ and M234EG, respectively. These mutants grew competently under phototrophic conditions and were tested for the formate-bicarbonate effect. In chromatophores there were no detectable differences between wild type (Wt) and mutant M234EV with respect to cytochrome b-561 reduction following a flash, and no effect of bicarbonate depletion (by incubation with formate). In isolated RCs, several electron transfer activities were essentially unchanged in Wt and M234EV, M234EQ and M234EG mutants, and no formate-bicarbonate effect was observed on: (a) the fast or slow phases of recovery of the oxidized primary donor (P+) in the absence of exogenous donor, i.e., the recombination of P+Q-A or P+Q-B, respectively; (b) the kinetics of electron transfer from Q-A to QB; or (c) the flash dependent oscillations of semiquinone formation in the presence of donor to P+ (QB turnover). The absence of a formate-bicarbonate effect in these mutants suggests that GluM234 is not responsible for the absence of the formate-bicarbonate effect in Wt bacterial RCs, or at least that other factors must be taken into account. The mutant RCs were also examined for the fast primary electron transfer along the active (A-)branch of the pigment chain, leading to reduction of QA. The kinetics were resolved to reveal the reduction of the monomer bacteriochlorophyll (tau = 3.5 ps), followed by reduction of the bacteriopheophytin (tau = 0.9 ps). Both steps were essentially unaltered from the wild type. However, the rate of reduction of QA was slowed by a factor of 2 (tau = 410 +/- 30 and 47 +/- 30 ps for M234EQ and M234EV, respectively, compared to 220 ps in the wild type).(ABSTRACT TRUNCATED AT 400 WORDS)     

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

The titration of amino acids and the energetics of electron transfer from the primary electron acceptor (QA) to the secondary electron acceptor (QB) in the photosynthetic reaction center of Rhodobacter sphaeroides are calculated using a continuum electrostatic model. Strong electrostatic interactions between titrating sites give rise to complex titration curves. Glu L212 is calculated to have an anomalously broad titration curve, which explains the seemingly contradictory experimental results concerning its pKa. The electrostatic field following electron transfer shifts the average protonation of amino acids near the quinones. The pH dependence of the free energy between Q-AQB and QAQ-B calculated from these shifts is in good agreement with experiment. However, the calculated absolute free energy difference is in severe disagreement (by approximately 230 meV) with the observed experimental value, i.e., electron transfer from Q-A to QB is calculated to be unfavorable. The large stabilization energy of the Q-A state arises from the predominantly positively charged residues in the vicinity of QA in contrast to the predominantly negatively charged residues near QB. The discrepancy between calculated and experimental values for delta G(Q-AQB-->QAQ-B) points to limitations of the continuum electrostatic model. Inclusion of other contributions to the energetics (e.g., protein motion following quinone reduction) that may improve the agreement between theory and experiment are discussed.     

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 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.     

Exploring the primary electron acceptor (QA)-site of the bacterial reaction center from Rhodobacter sphaeroides. Binding mode of vitamin K derivatives.

The functional replacement of the primary ubiquinone (QA) in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides with synthetic vitamin K derivatives has provided a powerful tool to investigate the electron transfer mechanism. To investigate the binding mode of these quinones to the QA binding site we have determined the binding free energy and charge recombination rate from QA(-) to D+ (kAD) of 29 different 1,4-naphthoquinone derivatives with systematically altered structures. The most striking result was that none of the eight tested compounds carrying methyl groups in both positions 5 and 8 of the aromatic ring exhibited functional binding. To understand the binding properties of these quinones on a molecular level, the structures of the reaction center-naphthoquinone complexes were predicted with ligand docking calculations. All protein--ligand structures show hydrogen bonds between the carbonyl oxygens of the quinone and AlaM260 and HisM219 as found for the native ubiquinone-10 in the X-ray structure. The center-to-center distance between the naphthoquinones at QA and the native ubiquinone-10 at QB (the secondary electron acceptor) is essentially the same, compared to the native structure. A detailed analysis of the docking calculations reveals that 5,8-disubstitution prohibits binding due to steric clashes of the 5-methyl group with the backbone atoms of AlaM260 and AlaM249. The experimentally determined binding free energies were reproduced with an rmsd of approximately 4 kJ x mol(-1) in most cases providing a valuable tool for the design of new artificial electron acceptors and inhibitors.     

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.     

Investigations on the influence of headgroup substitution and isoprene side-chain length in the function of primary and secondary quinones of bacterial reaction centers

The contributions of headgroup and side-chain in the binding and function of the primary (QA) and secondary (QB) quinones of isolated reaction centers (RCs) from Rhodobacter sphaeroides were investigated. Various ubiquinones and structurally similar quinones were reconstituted into RCs depleted of one (1Q-RCs) or both (0Q-RCs) quinones. The influence of partition coefficients on the apparent binding affinities was minimized by expressing dissociation constants in terms of the mole fraction of quinone partitioned into the detergent. It was then apparent that the size of the isoprenyl side-chain was of little consequence in determining the binding affinity or the functional competence of either QA or QB, although an alkyl chain of equivalent size was a poor substitute. The degree of substitution of the headgroup, however, was a sensitive determinant of binding. For both quinone sites, the trisubstituted plastoquinones bond more weakly than the fully substituted ubiquinones. Similarly, for binding to the QA site, duroquinone (tetramethylbenzoquinone) bound much more strongly than trimethylbenzoquinone. The affinity of the QA site for ubiquinones was about 20-times stronger than the QB site, but the QB site is probably not more specific than the QA site. However, QB function depends on a suitable redox free-energy drop from QA as well as binding, and of all the quinones tested only the ubiquinones simultaneously supported full QA and QB activity. Even plastoquinone-A, which fills both roles in Photosystem II, was unable to do so in bacterial RCs, although it did bind. The unique ability of ubiquinones to both bind and provide the appropriate redox span is discussed. The temperature dependence of binding of the isoprenyl ubiquinones at the QA site changed markedly with chain length. For Q-10-Q-7, the binding enthalpy was positive and net binding was entirely driven by entropic factors. For the shorter-chain ubiquinones, Q-6-Q-1, both entropy and enthalpy of binding were favorable. This strong entropy-enthalpy compensation is suggested to arise from antagonistic interactions (anticooperativity) between headgroup and tail binding. For QB function by hydrophobic quinones, the temperature dependence of the micelle properties prevented easy access to thermodynamic parameters. However, for water-soluble Q-0, binding to the QB site was determined to be enthalpically driven.(ABSTRACT TRUNCATED AT 400 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.     

Site-directed mutations at D1-His198 and D1-Thr179 of photosystem II in Synechocystis sp. PCC 6803: deciphering the spectral properties of the PSII reaction centre

Site-directed mutations were constructed in photosystem II of Synechocystis sp. PCC6803 in which the axial ligand, D1-His198, of special pair chlorophyll PD1 was replaced with Gln and where D1-Thr179, which overlies monomeric chlorophyll ChlD1, was replaced with His. The D1-His198Gln mutation produces a 3nm displacement to the blue of the bleaching minimum in the Soret and in the Qy region of the (P+QA--PQA) absorbance difference spectrum. To a first approximation, the bleaching can be assigned to the low-energy exciton transition of the special pair chlorophylls PD1/PD2. The D1-Thr179His mutation produces a 2nm displacement to the red of the bleaching minimum in the Qy region of the (3P-1P) absorbance difference spectrum. Analysis of the flash-induced (P+QA--PQA) and (3P-1P) absorbance difference spectra of both mutants compared with wild-type at 80K indicate that the cation of the oxidized donor P+ is predominantly localized on the chlorophyll PD1 of the special pair and that the reaction centre triplet state, produced upon charge recombination from 3[P+Pheo-], when the primary quinone electron acceptor QA is doubly reduced, is primarily localized on ChlD1.     

Does different orientation of the methoxy groups of ubiquinone-10 in the reaction centre of Rhodobacter sphaeroides cause different binding at QA and QB?

The different roles of ubiquinone-10 (UQ10) at the primary and secondary quinone (QA and QB) binding sites of Rhodobacter sphaeroides R26 reaction centres are governed by the protein microenvironment. The 4C=O carbonyl group of QA is unusually strongly hydrogen-bonded, in contrast to QB. This asymmetric binding seems to determine their different functions. The asymmetric hydrogen-bonding at QA can be caused intrinsically by distortion of the methoxy groups or extrinsically by binding to specific amino-acid side groups. Different X-ray-based structural models show contradictory orientations of the methoxy groups and do not provide a clear picture. To elucidate if distortion of the methoxy groups induces this hydrogen-bonding, their (ring-)C-O vibrations were assigned by use of site-specifically labelled [5-13C]UQ10 and [6-13C]UQ10 reconstituted at either the QA or the QB binding site. Two infrared bands at 1288 cm(-1) and 1264 cm(-1) were assigned to the methoxy vibrations. They did not shift in frequency at either the QA or QB binding sites, as compared with unbound UQ10. As the frequencies of these vibrations and their coupling are sensitive to the conformations of the methoxy groups, different conformations of the C(5) and C(6) methoxy groups at the QA and QB binding sites can now be excluded. Both methoxy groups are oriented out of plane at QA and QB. Therefore, hydrogen-bonding to His M219 combined with electrostatic interactions with the Fe2+ ion seems to determine the strong asymmetric binding of QA.     

Asymmetric binding of the 1- and 4-C=O groups of QA in Rhodobacter sphaeroides R26 reaction centres monitored by Fourier transform infra-red spectroscopy using site-specific isotopically labelled ubiquinone-10.

Using 1-, 2-, 3- and 4-13C site-specifically labelled ubiquinone-10, reconstituted at the QA site of Rhodobacter sphaeroides R26 reaction centres, the infra-red bands dominated by the 1- and 4-C = O vibration of QA are assigned in the QA(-)-QA difference spectra. The mode dominated by the 4-C = O vibration is drastically downshifted in the reaction centres as compared with its absorption frequency in free ubiquinone-10. In contrast, the mode dominated by the 1-C = O vibration absorbs at similar frequencies in the free and the bound forms. The frequency shift of the 4-C = O vibration is due to a large decrease in bond order and indicates a strong interaction with the protein microenvironment in the ground state. In the charge-separated state the mode dominated by the semiquinone 4-C = O vibration is characteristic of strong hydrogen bonding to the microenvironment, whereas the mode dominated by the 1-C = O vibration indicates a weaker interaction. The asymmetric binding of the 1- and 4-C = O groups to the protein might contribute to the factors governing different redox reactions of ubiquinone-10 at the QA site as compared with its reactions at the QB site.     

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.     

Effect of hydrogen bonds on the energetics of electron transfer

For a model system consisting of a special pair of bacteriochlorophyll molecules (P) and a primary quinone with the nearest environment (QA) (which are acceptor and donor in the recombination reaction in Rhodobacter sphaeroides reaction center, respectively), energies of P+QA(-) and PQA states were calculated. Calculations were performed using several stable QA conformations differing by the positions of hydrogen bond protons. Essential influence of proton positions on the energy of vertical transition P+QA(-) --> PQA was shown.     

Electron-transfer kinetics in photosynthetic reaction centers cooled to cryogenic temperatures in the charge-separated state: evidence for light-induced structural changes

We have compared the electron-transfer kinetics in reaction centers (RCs) cooled in the dark with those cooled under illumination (i.e., in the charge-separated state). Large differences between the two cases were observed. We interpreted these findings in terms of light-induced structural changes. The kinetics of charge recombination D+QA-----DQA in RCs containing one quinone were modeled in terms of a distribution of donor-acceptor electron-transfer distances. For RCs cooled under illumination the distribution broadened and shifted to larger distances compared to the distribution for RCs cooled in the dark. The model accounts for the nonexponential decay observed at low temperatures [McElroy, J. D., Mauzerall, D. C., & Feher, G. (1974) Biochim. Biophys. Acta 333, 261-277; Morrison, L.E., & Loach, P.A. (1978) Photochem. Photobiol. 27, 751-757]. A possible physiological role of the structural changes is an enhanced charge stabilization. For RCs with two quinones, the recombination kinetics D+QAQB-----DQAQB were found to be strongly temperature dependent. This was interpreted in terms of temperature-dependent transitions between structural states [Agmon, N., & Hopfield, J.J. (1983) J. Chem. Phys. 78, 6947-6959]. This interpretation requires that these transitions occur at cryogenic temperatures on a time scale t greater than or approximately 10(3) s. The electron transfer from QA- to QB was found to not take place in RCs cooled in the dark (tau ABdark greater than 10(-1) s). In RCs cooled under illumination, we found tau ABlight less than 10(-3) s. We suggest the possibility that the drastic decrease in tau AB observed in RCs cooled under illumination is due to the trapping of a proton near QB-.     

Calculated coupling of electron and proton transfer in the photosynthetic reaction center of Rhodopseudomonas viridis.

Based on new Rhodopseudomonas (Rp.) viridis reaction center (RC) coordinates with a reliable structure of the secondary acceptor quinone (QB) site, a continuum dielectric model and finite difference technique have been used to identify clusters of electrostatically interacting ionizable residues. Twenty-three residues within a distance of 25 A from QB (QB cluster) have been shown to be strongly electrostatically coupled to QB, either directly or indirectly. An analogous cluster of 24 residues is found to interact with QA (QA cluster). Both clusters extend to the cytoplasmic surface in at least two directions. However, the QB cluster differs from the QA cluster in that it has a surplus of acidic residues, more strong electrostatic interactions, is less solvated, and experiences a strong positive electrostatic field arising from the polypeptide backbone. Consequently, upon reduction of QA or QB, it is the QB cluster, and not the QA cluster, which is responsible for substoichiometric proton uptake at neutral pH. The bulk of the changes in the QB cluster are calculated to be due to the protonation of a tightly coupled cluster of the three Glu residues (L212, H177, and M234) within the QB cluster. If the lifetime of the doubly reduced state QB2- is long enough, Asp M43 and Ser L223 are predicted to also become protonated. The calculated complex titration behavior of the strongly interacting residues of the QB cluster and the resulting electrostatic response to electron transfer may be a common feature in proton-transferring membrane protein complexes.