In bacterial reaction centers rapid delivery of the second proton to QB can be achieved in the absence of L212Glu

In the reaction center (RC) of Rhodobacter capsulatus, residue L212Glu is a component of the pathway for proton transfer to the reduced secondary quinone, QB. We isolated phenotypic revertants of the photosynthetically incompetent (PS-) L212Glu-->Gln mutant; all of them retain the L212Glu-->Gln substitution and carry a second-site mutation: L227Leu-->Phe, L228Gly-->Asp, L231Arg-->Cys, or M231Arg-->Cys. We also characterized the L212Ala strain, which is a phenotypic revertant of the PS- L212Glu-L213Asp-->Ala-Ala mutant. The activities of the RCs of these strains--all of which lack L212Glu--were studied by flash-induced absorption spectroscopy. At pH 7.5, the rate of second electron transfer in the L212Q mutant is comparable to the wild-type rate. However, this mutant shows a marked decrease in the rate of cytochrome oxidation under strong continuous illumination and a very slow phase (0.66 s-1) of the proton transfer kinetics following the second flash, indicating that transfer of the second proton to QB is slowed more than 1000-fold. The levels of recovery of the functional capabilities in the revertant RCs vary widely; their rates of cytochrome oxidation were intermediate between those of the wild-type and the L212Q mutant. The kinetics of proton transfer following the second flash show a significant recovery in the L212Q + M231C and L212A RCs (330-540 s-1), but the L212Q + L227F RCs recover this function only partially. Compensation for the lack of L212Glu in revertant RCs is discussed in terms of (i) conformational changes that could allow water molecules to approach closer to QB and/or (ii) the increase in the negative electrostatic environment and the resultant rise in the free energy level of QB- that is induced by the mutations. The stoichiometries of H+/QB- proton uptake below pH 7.5 in the L212Q mutant, the L212Q + M231C revertant, and the wild-type strains are essentially equivalent, suggesting that L212Glu is protonated at neutral pH in wild-type RCs. This is also supported by the P+QB- charge recombination data. Comparison of H+/QB- proton uptake data with those obtained previously for the stoichiometries of H+/QA- proton uptake [Miksovska, J., Maróti, P., Tandori, J., Schiffer, M., Hanson, D. K., Sebban, P. (1996) Biochemistry 35, 15411-15417] suggests that L212Glu is the key to the electrostatic and perhaps structural interaction between the two quinone sites.     

Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron-proton transfer

High resolution x-ray diffraction data from crystals of the Rhodobacter sphaeroides photosynthetic reaction center (RC) have been collected at cryogenic temperature in the dark and under illumination, and the structures were refined at 2.2 and 2.6 angstrom resolution, respectively. In the charge-separated D+QAQB- state (where D is the primary electron donor (a bacteriochlorophyll dimer), and QA and QB are the primary and secondary quinone acceptors, respectively), QB- is located approximately 5 angstroms from the QB position in the charge-neutral (DQAQB) state, and has undergone a 180 degrees propeller twist around the isoprene chain. A model based on the difference between the two structures is proposed to explain the observed kinetics of electron transfer from QA-QB to QAQB- and the relative binding affinities of the different ubiquinone species in the QB pocket. In addition, several water channels (putative proton pathways) leading from the QB pocket to the surface of the RC were delineated, one of which leads directly to the membrane surface.     

Loss of inhibition by formate in newly constructed photosystem II D1 mutants, D1-R257E and D1-R257M, of Chlamydomonas reinhardtii

Formate is known to cause significant inhibition in the electron and proton transfers in photosystem II (PSII); this inhibition is uniquely reversed by bicarbonate. It has been suggested that bicarbonate functions by providing ligands to the non-heme iron and by facilitating protonation of the secondary plastoquinone QB. Numerous lines of evidence indicate an intimate relationship of bicarbonate and formate binding of PSII. To investigate the potential amino acid binding environment of bicarbonate/formate in the QB niche, arginine 257 of the PSII D1 polypeptide in the unicellular green alga Chlamydomonas reinhardtii was mutated into a glutamate (D1-R257E) and a methionine (DQ-R257M). The two mutants share the following characteristics. (1) Both have a drastically reduced sensitivity to formate. (2) A larger fraction of QA- persists after flash illumination, which indicates an altered equilibrium constant of the reaction QA-QB<-->QA QB-, in the direction of [QA-], or a larger fraction of non-QB centers. However, there appears to be no significant difference in the rate of electron transfer from QA- to QB. (3) The overall rate of oxygen evolution is significantly reduced, most likely due to changes in the equilibrium constant on the electron acceptor side of PSII or due to a larger fraction in non-QB centers. Additional effects on the donor side cannot yet be excluded. (4) The binding affinity for the herbicide DCMU is unaltered. (5) The mutants grow photosynthetically, but at a decreased (approximately 70% of the wild type) level. (6) The Fo level was elevated (approximately 40-50%) which could be due to a decrease in the excitation energy transfer from the antenna to the PSII reaction center, and/or to an increased level of [QA-] in the dark. (7) A decreased (approximately 10%) ratio of F685 (mainly from CP43) and F695 (mainly from CP47) to F715 (mainly from PSI) emission bands at 77 K suggests a change in the antenna complex. Taken together these results lead to the conclusion that D1-R257 with the positively charged side chain is important for the fully normal functioning of PSII and of growth, and is specially critical for the in vivo binding of formate. Several alternatives are discussed to explain the almost normal functioning of the D1-R257E and D1-R257M mutants.     

Room-temperature vibrational difference spectrum for S2QB-/S1QB of photosystem II determined by time-resolved Fourier transform infrared spectroscopy

Time-resolved FTIR spectroscopy has been used to kinetically characterize the vibrational properties of intact photosystem II-enriched membrane samples undergoing the S1QB-to-S2QB- transition at room temperature. To optimize the experimental conditions for the FTIR measurements, oxygen polarographic and variable chlorophyll a fluorescence measurements were used to define the decay of S2 and QA-, respectively. The flash-induced S2QB-/S1QB difference spectra were measured at a temporal resolution of 4.44 s and a spectral resolution of 4 cm-1. An intense positive band is observed at 1480 cm-1 in the difference spectrum and shows a slow decay with a half time of approximately 13 s. Based on its decay kinetics and analogy to the infrared absorption of QA- of photosystem II and QB- in bacterial reaction centers, we conclude that the 1480 cm-1 band arises from QB- of PSII and tentatively assign it to the upsilon(CO) mode of the semiquinone anion QB-. The infrared spectral features attributed to the S1-to-S2 transition of the Mn cluster at room temperature show striking similarity to the S2/S1 difference spectrum measured at cryogenic temperatures (Noguchi, T., Ono, T.-A., and Inoue, Y. (1995) Biochim. Biophys. Acta 1228, 189-200).     

Mutations in the environment of the primary quinone facilitate proton delivery to the secondary quinone in bacterial photosynthetic reaction centers

In Rhodobacter capsulatus, we constructed a quadruple mutant that reversed a structural asymmetry that contributes to the functional asymmetry of the two quinone sites. In the photosynthetically incompetent quadruple mutant RQ, two acidic residues near QB, L212Glu and L213Asp, have been mutated to Ala; conversely, in the QA pocket, the symmetry-related residues M246Ala and M247Ala have been mutated to Glu and Asp. We have selected photocompetent phenotypic revertants (designated RQrev3 and RQrev4) that carry compensatory mutations in both the QA and QB pockets. Near QA, the M246Ala --> Glu mutation remains in both revertants, but M247Asp is replaced by Tyr in RQrev3 and by Ala in RQrev4. The engineered L212Ala and L213Ala substitutions remain in the QB site of both revertants but are accompanied by an additional electrostatic-type mutation. To probe the respective influences of the mutations occurring near the QA and QB sites on electron and proton transfer, we have constructed two additional types of strains. First, "half" revertants were constructed that couple the QB site of the revertants with a wild-type QA site. Second, the QA sites of the two revertants were linked with the L212Glu-L213Asp --> Ala-Ala mutations of the QB site. We have studied the electron and proton-transfer kinetics on the first and second flashes in reaction centers from these strains by flash-induced absorption spectroscopy. Our data demonstrate that substantial improvements of the proton-transfer capabilities occur in the strains carrying the M246Ala --> Glu + M247Ala --> Tyr mutations near QA. Interestingly, this is not observed when only the M246Ala --> Glu mutation is present in the QA pocket. We suggest that the M247Ala --> Tyr mutation in the QA pocket, or possibly the coupled M246Ala --> Glu + M247Ala --> Tyr mutations, accelerates the uptake and delivery of protons to the QB anions. The M247Tyr substitution may enable additional pathways for proton transfer that are located near QA.     

Pathway of proton transfer in bacterial reaction centers: role of aspartate-L213 in proton transfers associated with reduction of quinoneto dihydroquinone

The role of Asp-L213 in proton transfer to reduced quinone QB in the reaction center (RC) from Rhodobacter sphaeroides was studied by site-directed replacement of Asp with residues having different proton donor properties. Reaction centers (RCs) with Asn, Leu, Thr, and Ser at L213 had greatly reduced (approximately 6000-fold) proton-coupled electron transfer [kAB(2)] and proton uptake rates associated with the second electron reduction of QB (QA- QB- + 2H(+)-->QAQBH2) compared to native RCs. RCs containing Glu at L213 showed faster (approximately 90-fold) electron and proton transfer rates than the other mutant RCs but were still reduced (approximately 70-fold) compared with native RCs. These results show that kAB(2) is larger when a carboxylic acid occupies the L213 site, consistent with the proposal that Asp-L213 is a component of a proton transfer chain. The reduced kAB(2) observed with Glu versus Asp at L213 suggests that Asp at L213 is important for proton transfer for some other reason in addition to its proton transfer capabilities. Glu-L213 is estimated to have a higher apparent pKa (pKa > or = 7) than Asp-L213 (pKa < or = 4), as indicated by the slower rate of charge recombination (D+QAQB(-)-->DQAQB) in the mutant RCs. The importance of the pKa and charge of the residue at L213 for proton transfer are discussed. Based on these studies, a model for proton transfer is proposed in which Asp-L213 contributes to proton transfer in native RCs in two ways: (1) it is a component of a proton transfer chain connecting the buried QB molecule with the solvent and/or (2) it provides a negative charge that stabilizes a proton on or near QB.     

Coupled activation of the donor and the acceptor side of photosystem II during photoactivation of the oxygen evolving cluster

Photoactivation of photosystem II has been studied in the FUD 39 mutant of Chlamydomonas reinhardtii that lacks the 23 kDa extrinsic subunit of photosystem II. We have taken advantage of the slow photoactivation rate of FUD 39, earlier demonstrated in Rova, E. M., et al. [(1996) J. Biol. Chem. 271, 28918-28924], to study events in photosystem II during intermediate stages of the process. By measuring the EPR multiline signal, the decay of the variable fluorescence after single flashes, and electron transfer from water to the QB site, we found a good correlation between the building of a tetrameric Mn cluster, longer recombination times between QA- and the donor side of photosystem II, and the achievement of water splitting ability. An increased rate of electron transfer from QA- to the QB site on the acceptor side of photosystem II, mainly due to enhanced efficiency of binding of QB to its site, was found to precede the building of the Mn cluster. We also showed that TyrD was oxidized simultaneously with this increase in electron-transfer rate. Thus, it appears that photoactivation is sequential, with an increased rate of electron transfer on the acceptor side occurring together with the oxidation of TyrD in the first step, followed by the assembly of the Mn cluster. We suggest that a conformational change of photosystem II is induced early in the photoactivation process facilitating electron transfer from the primary donor to the acceptor side. As a consequence, TyrD, an auxiliary electron donor to P680+/TyrZ*, is oxidized. That this occurs before the Mn cluster is fully functional serves to protect photosystem II against donor side induced photodamage.     

Conformational gating of the electron transfer reaction QA-.QB --> QAQB-. in bacterial reaction centers of Rhodobacter sphaeroides determined by a driving force assay

The mechanism of the electron transfer reaction, QA-.QB --> QAQB-., was studied in isolated reaction centers from the photosynthetic bacterium Rhodobacter sphaeroides by replacing the native Q10 in the QA binding site with quinones having different redox potentials. These substitutions are expected to change the intrinsic electron transfer rate by changing the redox free energy (i.e., driving force) for electron transfer without affecting other events that may be associated with the electron transfer (e.g., protein dynamics or protonation). The electron transfer from QA-. to QB was measured by three independent methods: a functional assay involving cytochrome c2 to measure the rate of QA-. oxidation, optical kinetic spectroscopy to measure changes in semiquinone absorption, and kinetic near-IR spectroscopy to measure electrochromic shifts that occur in response to electron transfer. The results show that the rate of the observed electron transfer from QA-. to QB does not change as the redox free energy for electron transfer is varied over a range of 150 meV. The strong temperature dependence of the observed rate rules out the possibility that the reaction is activationless. We conclude, therefore, that the independence of the observed rate on the driving force for electron transfer is due to conformational gating, that is, the rate limiting step is a conformational change required before electron transfer. This change is proposed to be the movement, controlled kinetically either by protein dynamics or intermolecular interactions, of QB by approximately 5 A as observed in the x-ray studies of Stowell et al. [Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E. & Feher, G. (1997) Science 276, 812-816].     

Proton uptake by carboxylic acid groups upon photoreduction of the secondary quinone (QB) in bacterial reaction centers from Rhodobacter sphaeroides: FTIR studies on the effects of replacing Glu H173

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, Glu H173, located approximately 7 A from the center of the secondary quinone acceptor QB, is expected to contribute to proton uptake upon QB- formation in response to the movement of an electron in its vicinity. Steady-state FTIR difference spectroscopy provides a method to monitor proton uptake by carboxylic acids upon photochemical changes. The FTIR spectra corresponding to the photoreduction of QB were obtained at pH 7 for RCs containing Glu (native), Gln (EQ H173), or Asp (ED H173) at the H173 site. No new bands were observed in the carboxylic acid region (1770-1700 cm-1) in any of the mutant RCs compared to native RCs. In addition, the positive band at 1728 cm-1, previously assigned to Glu L212 [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., M?ntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732], remained present in all of the mutant RCs. This result shows that Glu H173 is not a major contributor to proton uptake upon QB- formation and further strengthens the assignment of the 1728 cm-1 band to Glu L212. An increase in the 1728 cm-1 band was observed in the EQ H173 RCs compared to that of either the ED H173 or native RCs. These changes are consistent with Glu and Asp at H173 remaining ionized in the QB and QB- states. Changes in the absorption regions of the semiquinone and amide or side chain groups in the spectra of the mutant RCs suggest slight changes in the protein structure compared to those of native RCs, which could contribute to the altered kinetics observed in the mutant RCs.     

Comparison of the functional properties of the monomeric and dimeric forms of the isolated CP47-reaction center complex

Chlorophyll fluorescence, thermoluminescence, and EPR spectroscopy have been used to investigate the functional properties of the monomeric and dimeric forms of the photosystem II CP47-reaction center (CP47-RC) subcore complex that was isolated (Zheleva, D., Sharma, J., Panico, M., Morris, H. R., and Barber, J. (1998) J. Biol. Chem. 273, 16122-16127). Chlorophyll fluorescence yield changes induced either by the initiation of continuous actinic light or by repetitive light flashes indicated that the dimeric, but not the monomeric, form of the CP47-RC complex showed secondary electron transport properties indicative of QA reduction. Thermoluminescence measurements also clearly distinguished the monomer from the dimer in that the latter showed a ZV band, which appeared at -55 degreesC, following illumination at -80 degreesC. This band has been determined to be an indicator of the photoaccumulation of QA-. The ability of the dimeric CP47-RC to show secondary electron transport properties was clearly demonstrated by EPR studies. The dimer was characterized by organic radical signals at about g = 2 induced either by illumination or by the addition of dithionite. The dithionite-induced signal was attributed to QA-, but there was no indication of any interaction with non-heme iron. The signal induced by light was more complex, being composed not only of the QA- radical but also of radicals generated on the donor side. Difference analyses indicated that one of these radicals is likely to be due to a D1 tyrosine 161 or D2 tyrosine 161. In contrast, the monomeric CP47-RC complex did not show similar EPR-detectable radicals and instead was dominated by a high yield of the spin-polarized triplet signal generated by recombination reactions between the oxidized primary reductant, pheophytin, and the primary donor, P680. It is also concluded from EPR analyses that both the monomeric and dimeric forms of the CP47-RC subcore complex contain one cytochrome b559 per reaction center. Overall the results suggest that photosystem II normally functions as a dimer complex and that monomerization at the level of the CP47-RC subcore complex leads to destabilization of the bound plastoquinone, which functions as QA.     

The binding sites of quinones in photosynthetic bacterial reaction centers investigated by light-induced FTIR difference spectroscopy: assignment of the QA vibrations in Rhodobacter sphaeroides using 18O- or 13C-labeled ubiquinone and vitamin K1

Light-induced FTIR difference spectra of the photoreduction of the primary quinone acceptor QA have been obtained for Rhodobacter sphaeroides RCs reconstituted with a series of isotopically labeled quinones in order to separate the contributions of the quinone from those of the protein. The isotopic shifts observed in the QA-/QA spectra of RCs reconstituted with ubiquinones (Q1, Q6) or vitamin K1 18O-labeled on their carbonyl oxygens and with fully 13C-labeled Q8 lead to a clear identification of the quinone bands from both the neutral and anion forms. Double-difference spectra from pairs of QA-/QA spectra obtained from 18O/16O Q6, 18O/16O Q1, 13C/12C Q8, 13C18O/12C16O Q8, and 18O/16O vitamin K1 allow the C = O modes of QA in vivo to be identified unambiguously for the first time. For all the investigated unlabeled quinones, two carbonyl bands are demasked, at 1660 and 1628 cm-1 for neutral ubiquinones and at 1651 and 1640 cm-1 for vitamin K1, while C = C bands are found at 1608 and 1588 cm-1 for vitamin K1 and at 1601 cm-1 for ubiquinones. Compared with the spectra of the isolated quinones, the generally smaller width observed for the C = O and C = C bands in vivo suggests precise interactions between the quinone and the contours of the protein at a single, well-defined QA site. The different frequency downshifts of the two C = O bands upon binding to the QA site underscore the inequivalence of the two carbonyls in providing asymmetrical bonding interactions with the protein. The comparison of the isotopic shifts observed for the various quinone C = O and C = C bands in vitro and in vivo demonstrates that the admixture of C = O and C = C characters in these modes is strongly affected by the binding of QA to its anchoring site. In particular, the bands at 1628 and 1601 cm-1 of Q6 in vivo exhibit highly mixed C = O and C = C characters. In contrast, the methoxy groups of the ubiquinones do not appear to suffer large strain upon binding. The closeness of the QA-/QA spectra for Q1 and Q6 indicates that a possible role of the chain in providing the proper positioning of the quinone ring in the site for both the oxidized and reduced states of QA cannot extend significantly beyond the first isoprene unit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic phases in the electron transfer from P+QA-QB to P+QAQB- and the associated processes in Rhodobacter sphaeroides R-26 reaction centers

Electron transfer from P+QA-QB to form P+QAQB- was measured in Rhodobacter sphaeroides R-26 reaction centers (RCs) where the native primary quinone, ubiquinone-10 (UQA), was replaced by 2-methyl-3-phytyl-1,4-naphthoquinone (MQA). The native secondary quinone, UQ-10, was retained as UQB. The difference spectrum of the semiquinone MQA- minus UQB- absorption is very similar to that of MQ- minus UQ- in solution (398-480 nm). Thus, the absorption change provides a direct monitor of the electron transfer from MQA- to UQB. In contrast, when both QA and QB are UQ-10 the spectral difference between UQA- and UQB- arises from electrochromic responses of RC chromophores. Three kinetic processes are seen in the near UV (390-480 nm) and near-IR (740-820 nm). Analysis of the time-correlated spectra support the conclusion that the changes at tau1 approximately 3 micros are mostly due to electron transfer, electron transfer and charge compensation are mixed in tau2 approximately 80 micros, while little or no electron transfer occurs at 200-600 micros (tau3) in MQAUQB RCs. The 80-micros rate has been previously observed, while the fast component has not. The fast phase represents 60% of the electron-transfer reaction (398 nm). The activation energy for electron transfer is DeltaG approximately 3.5 kcal/mol for both tau1 and tau2 between 0 and 30 degrees C. In isolated RCs with UQA, if there is any fast component, it appears to be faster and less important than in the MQA reconstituted RCs.     

A new metal-binding site in photosynthetic bacterial reaction centers that modulates QA to QB electron transfer

Isolated reaction centers (RCs) from Rhodobacter sphaeroides were found to bind Zn(II) stoichiometrically and reversibly in addition to the 1 equiv of non-heme Fe(II). Metal and EPR analyses confirm that Zn(II) is ligated to a binding site that is distinct from the Fe site. When Zn(II) is bound to this site, electron transfer between the quinones QA and QB (QA-QB --> QAQB-) is slowed and the room-temperature kinetics become distributed across the microsecond to millisecond time domain. This effect of metal binding on the kinetics is similar to the more global effect of cooling RCs to 2 degreesC in the absence of Zn(II). This suggests that Zn(II) binding alters localized protein motions that are necessary for rapid QA-QB --> QAQB- electron transfer. Inspection of the RC crystal structure suggests a cluster of histidine ligands located beneath the QB binding pocket as a potential binding site.     

Correlation between protein flexibility and electron transfer from QA-* to QB in PSII membrane fragments from spinach

To analyze a possible correlation between the extent of QA-* reoxidation and protein dynamics, fluorometric and M?ssbauer spectroscopic measurements were performed in photosystem II membrane fragments from spinach. Numerical evaluation of the flash-induced change of the normalized fluorescence quantum yield revealed that the extent of reoxidation starts to decrease below 275 K and is almost completely suppressed at 230 K. Detailed analyses of M?ssbauer spectra measured at different temperatures in 57Fe-enriched material indicate that the onset of fluctuations between conformational substates of the protein matrix occurs also at around 230 K. Based on this correspondence, protein flexibility is inferred to play a key role for QA-* reoxidation in photosystem II. Taking into account the striking similarities with purple bacteria and the latest structural information on these reaction centers [Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Science 276, 812-816], it appears most plausible that also the headgroup of plastoquinone-9 bound to the QB-site in PSII requires a structural reorientation for its reduction to the semiquinone.     

A relative energy failure is associated with low-Mg2+ but not with 4-aminopyridine induced seizure-like events in entorhinal cortex

Interactions of the primary quinone acceptor QA of photosystem II (PS II) with surrounding amino acid residues were studied by analysis of FTIR difference spectra of QA upon its photoreduction (QA-/QA). Structural coupling with a His side chain was revealed by identifying the imidazole bands in the QA-/QA spectrum using the PS II core complexes from Synechocystis PCC 6803 in which both of the two imodazole nitrogens of His side chains were specifically labeled with 15N. Strong hydrogen bonding of the imidazole NH was shown by (i) the presence of several peaks at 2600-3000 cm-1, which arise from Fermi resonance of harmonics or combinations of imidazole ring modes with the hydrogen bonding NH stretching vibration, and (ii) the 1179 cm-1 band, which can be assigned to the mode including NH deformation, is at a frequency significantly higher than the corresponding 1151 cm-1 band of model compounds 4- and 5-methylimidazole in aqueous solution. Also, the presence of the bands specific to the Npi-protonated state at 1109/1102/1090 and 1359 cm-1 suggests that the QA-coupled His is protonated at the Npi site. These results are in good agreement with the model of QA interaction in which His215 (D2), which coordinates to the non-heme iron at Ntau, is hydrogen bonded to the QA carbonyl through the Npi-H bond. In contrast, no bands of Trp side chains were detected in the QA-/QA spectrum upon labeling of the indole ring of Trp residues with indole-d5. This result indicates that Trp254 (D2), which corresponds to Trp252 (M) of the bacterial reaction center that is located in van der Waals contact with QA, is not strongly coupled with QA in PS II. Probably, the predicted pi-pi interaction is not strong enough to influence the vibrations of the indole ring of Trp upon QA reduction, or Trp254 (D2) is located rather far from QA in PS II.     

Measurement of cofactor distances between P700.+ and A1.- in native and quinone-substituted photosystem I using pulsed electron paramagnetic resonance spectroscopy

The radical pair P700.+Q.- (P700 = primary electron donor, Q = quinone acceptor) in native photosystem I and in preparations in which the native acceptor (vitamin K1) is replaced by different quinones is investigated by pulsed EPR spectroscopy. In a two-pulse experiment, the light-induced radical pair causes an out-of-phase electron spin echo, showing an envelope modulation. From the modulation frequency, the dipolar coupling, and therefore the distance between the two cofactors, can be derived. The observation of nearly identical distances of about 25.4 A between P700.+ and Q.- in all preparations investigated here leads to the conclusion that the reconstituted quinones are bound to the native A1 binding pocket. Since the orientation of the reconstituted naphthoquinone relative to the axis joining P700.+ and Q*- differs drastically from that of the native vitamin K1, it cannot be bonded to the protein in the same way as the native acceptor. This implies that the function of A1 as an electron acceptor does not depend on the orientation or hydrogen bonding of the quinone.     

A one-tube quantitative HIV-1 RNA NASBA nucleic acid amplification assay using electrochemiluminescent (ECL) labelled probes

Quantification of HIV-1 viral RNA based on co-amplification of an internal standard Q-RNA dilution series requires a number of NASBA nucleic acid amplification reactions. For each internal standard Q-RNA concentration a separate NASBA amplification has to be performed. The development of an electrochemiluminescent (ECL) detection system with a dynamic signal detection range over 5 orders of magnitude enabled simplification of the Q-NASBA protocol. Three distinguishable Q-RNAs (QA, QB and QC) are mixed with the wild-type sample at different amounts (i.e. 10(4) QA, 10(3) QB and 10(2) QC molecules) and co-amplified with the wild-type RNA in one tube. Using ECL-labelled oligonucleotides the wild-type, QA, QB and QC amplificates are separately detected with a semi-automated ECL detection instrument and the ratio of the signals determined. The amount of initial wild-type RNA can be calculated from the ratio of wild-type signal to QA, QB and QC signals. This one-tube Q-NASBA protocol was compared to the earlier described protocol with six amplifications per quantification using model systems and HIV-1 RNA isolated from plasma of HIV-1-infected individuals. In all cases the quantification results of HIV-1 RNA were comparable between the two methods tested. Due to the use of only one amplification per quantification in the one-tube Q-NASBA protocol the QA, QB and QC internal standard RNA molecules can be added to the sample before nucleic acid isolation. The ratio of QA:QB:QC:WT RNAs, from which the initial input of WT-RNA is calculated, will remain constant independent of any loss that might occur during the nucleic acid isolation.     

Modification of the water oxidizing complex in leaves of the dgd1 mutant of Arabidopsis thaliana deficient in the galactolipid digalactosyldiacylglycerol

The primary biochemical defect in the genetically well characterized dgd1 mutant of Arabidopsis thaliana causes a 90% reduction in the relative amount of the galactolipid digalactosyldiacylglycerol (DGDG). To study the effect of this DGDG deficiency on photosystem II (PS II), time-resolved transients of laser-flash-induced changes of the relative fluorescence quantum yield Fvar,rel(t) were measured in whole leaves from wild-type and the dgd1 mutant. The results obtained reveal (i) in untreated leaves the decay kinetics of Fvar, rel(t) reflecting QA.- reoxidation by endogenous plastoquinone are very similar in wild-type and the dgd1 mutant at room temperature, (ii) the Arrhenius plot of the temperature dependence of electron transfer from QA.- to QB exhibits a break point at about 19 degrees C in wild-type and about 12 degrees C in the dgd1 mutant, (iii) in leaves treated with DCMU the slow reoxidation of QA.- by the PS II donor side is blocked to a much higher extent in the dgd1 mutant (about 50%) compared to wild-type (about 10%), and iv) the normalized amplitude of Fvar,rel(t = 1 micros) reflecting the percentage of fast P680.+ reduction by YZ exhibits a characteristic period four oscillation in wild-type while this feature is strongly damped in the dgd1 mutant. Presumably, the severe DGDG deficiency is causing the thermal down shift of a lipid phase transition that affects the QA.- reoxidation by QB. Most strikingly, the properties of the WOC are modified as a result of reduced DGDG content. Thus, the lipid DGDG appears to be of structural relevance for the WOC.     

Absorbance changes due to the charge-accumulating species in system 2 of photosynthesis

Absorbance changes are reported associated with Photosystem II and showing a periodicity of two and four as a function of flash number. The absorbance changes showing a periodicity of two were found to occur in the presence of artificial electron donors as well and are presumably caused by the secondary electron acceptor R of Photosystem II. The absorbance difference spectra suggest that R is a plastoquinone molecule, which is reduced to its semiquinone anion after an uneven number of flashes. After an even number of flashes, the semiquinone is reoxidized. The absorbance changes showing a periodicity of four are tentatively ascribed to the charge accumulating donor complex of Photosystem II.