Isolation and pigment composition of the reaction centers from purple photosynthetic bacterium Rhodopseudomonas palustris species.

The reaction centers (RCs) from several species of a purple photosynthetic bacterium, Rhodopseudomonas palustris, were first isolated by ammonium-sulfate fractionation of the isolated core complexes, and were successfully purified by anion-exchange and gel-filtration chromatography as well as sucrose-density gradient centrifugation. The RCs were characterized by spectroscopic and biochemical analyses, indicating that they were sufficiently pure and had conserved their redox activity. The pigment composition of the purified RCs was carefully analyzed by LCMS. Significant accumulation of both bacteriochlorophyll(BChl)-a and bacteriopheophytin(BPhe)-a esterified with various isoprenoid alcohols in the 17-propionate groups was shown in RCs for the first time. Moreover, a drastic decrease in BPhe-a with the most dehydrogenated and rigid geranylgeranyl(GG) ester was observed, indicating that BPhe-a in RC preferably took partially hydrogenated and flexible ester groups, i.e. dihydro-GG and tetrahydro-GG in addition to phytyl. Based on the reported X-ray crystal structures of purple bacterial RCs, the meaning of flexibility of the ester groups in BChl-a and BPhe-a as the cofactors of RCs is proposed.

Vibrational coherence in bacterial reaction centers with genetically modified B-branch pigment composition.

Femtosecond absorption difference spectroscopy was applied to study the time and spectral evolution of low-temperature (90 K) absorbance changes in isolated reaction centers (RCs) of the HM182L mutant of Rhodobacter (Rb.) sphaeroides. In this mutant, the composition of the B-branch RC cofactors is modified with respect to that of wild-type RCs by replacing the photochemically inactive BB accessory bacteriochlorophyll (BChl) by a photoreducible bacteriopheophytin molecule (referred to as PhiB). We have examined vibrational coherence within the first 400 fs after excitation of the primary electron donor P with 20-fs pulses at 870 nm by studying the kinetics of absorbance changes at 785 nm (PhiB absorption band), 940 nm (P*-stimulated emission), and 1020 nm (BA- absorption band). The results of the femtosecond measurements are compared with those recently reported for native Rb. sphaeroides R-26 RCs containing an intact BB BChl. At delay times longer than approximately 50 fs (maximum at 120 fs), the mutant RCs exhibit a pronounced BChl radical anion (BA-) absorption band at 1020 nm, which is similar to that observed for Rb. sphaeroides R-26 RCs and represents the formation of the intermediate charge-separated state P+ BA-. Femtosecond oscillations are revealed in the kinetics of the absorption development at 1020 nm and of decay of the P*-stimulated emission at 940 nm, with the oscillatory components of both kinetics displaying a generally synchronous behavior. These data are interpreted in terms of coupling of wave packet-like nuclear motions on the potential energy surface of the P* excited state to the primary electron-transfer reaction P*-->P+ BA- in the A-branch of the RC cofactors. At very early delay times (up to 80 fs), the mutant RCs exhibit a weak absorption decrease around 785 nm that is not observed for Rb. sphaeroides R-26 RCs and can be assigned to a transient bleaching of the Qy ground-state absorption band of the PhiB molecule. In the range of 740-795 nm, encompassing the Qy optical transitions of bacteriopheophytins HA, HB, and PhiB, the absorption difference spectra collected for mutant RCs at 30-50 fs resemble the difference spectrum of the P+ PhiB- charge-separated state previously detected for this mutant in the picosecond time domain (E. Katilius, Z. Katiliene, S. Lin, A.K.W. Taguchi, N.W. Woodbury, J. Phys. Chem., B 106 (2002) 1471-1475). The dynamics of bleaching at 785 nm has a non-monotonous character, showing a single peak with a maximum at 40 fs. Based on these observations, the 785-nm bleaching is speculated to reflect reduction of 1% of PhiB in the B-branch within about 40 fs, which is earlier by approximately 80 fs than the reduction process in the A-branch, both being possibly linked to nuclear wave packet motion in the P* state.

Energy dissipation in photosynthesis: does the quenching of chlorophyll fluorescence originate from antenna complexes of photosystem II or from the reaction center?

Dissipation of light energy was studied in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst., and in leaves of Spinacia oleracea L. and Arabidopsis thaliana (L.) Heynh., using chlorophyll fluorescence as an indicator reaction. Maximum chlorophyll fluorescence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated spinach leaves, as produced by saturating light and studied between and -20 degrees C, revealed an activation energy delta E of 0.11 eV. As this suggested recombination fluorescence produced by charge recombination between the oxidized primary donor of photosystem II and reduced pheophytin, a mathematical model explaining fluorescence, and based in part on known characteristics of primary electron-transport reactions, was developed. The model permitted analysis of different modes of fluorescence quenching, two localized in the reaction center of photosystem II and one in the light-harvesting system of the antenna complexes. It predicted differences in the relationship between quenching of variable fluorescence Fv and quenching of basal, so-called F0 fluorescence depending on whether quenching originated from antenna complexes or from reaction centers. Such differences were found experimentally, suggesting antenna quenching as the predominant mechanism of dissipation of light energy in the moss Rhytidiadelphus, whereas reaction-center quenching appeared to be important in spinach and Arabidopsis. Both reaction-center and antenna quenching required activation by thylakoid protonation but only antenna quenching depended on or was strongly enhanced by zeaxanthin. De-protonation permitted relaxation of this quenching with half-times below 1 min. More slowly reversible quenching, tentatively identified as so-called qI or photoinhibitory quenching, required protonation but persisted for prolonged times after de-protonation. It appeared to originate in reaction centers.

Light-harvesting chlorophyll a/b-binding protein stably inserts into etioplast membranes supplemented with Zn-pheophytin a/b.

Light-harvesting chlorophyll a/b-binding protein, LHCP, or its precursor, pLHCP, cannot be stably inserted into barley etioplast membranes in vitro. However, when these etioplast membranes are supplemented with the chlorophyll analogs Zn-pheophytin a/b, synthesized in situ from Zn-pheophorbide a/b and digeranyl pyrophosphate, pLHCP is inserted into a protease-resistant state. This proves that chlorophyll is the only component lacking in etioplast membranes that is necessary for stable LHCP insertion. Synthesis of Zn-pheophytin b alone promotes insertion of LHCP in vitro into a protease-resistant state, whereas synthesis of Zn-pheophytin a alone does not. Insertion of pLHCP into etioplast membranes can also be stimulated by adding chlorophyll a and chlorophyll b to the membranes, albeit at a significantly lower efficiency as compared with Zn-pheophytin a/b synthesized in situ. When pLHCP is inserted into chlorophyll- or Zn-pheophytin-supplemented etioplast membranes and then assayed with protease, only the protease digestion product indicative of the monomeric major light-harvesting chlorophyll a/b complex (LHCII) is found but not the one indicating trimeric complexes. In this respect, chlorophyll- or Zn-pheophytin-supplemented etioplast membranes resemble thylakoid membranes at an early greening stage: pLHCP inserted into plastid membranes from greening barley is assembled into trimeric LHCII only after more than 1 h of greening.

Electrostatic control of charge separation in bacterial photosynthesis.

Electrostatic interaction energies of the electron carriers with their surroundings in a photosynthetic bacterial reaction center are calculated. The calculations are based on the detailed crystal structure of reaction centers from Rhodopseu-domonas viridis, and use an iterative, self-consistent procedure to evaluate the effects of induced dipoles in the protein and the surrounding membrane. To obtain the free energies of radical-pair states, the calculated electrostatic interaction energies are combined with the experimentally measured midpoint redox potentials of the electron carriers and of bacteriochlorophyll (BChl) and bacteriopheophytin (BPh) in vitro. The P+HL- radical-pair, in which an electron has moved from the primary electron donor (P) to a BPh on the 'L' side of the reaction center (HL), is found to lie approx. 2.0 kcal/mol below the lowest excited singlet state (P*), when the radical-pair is formed in the static crystallographic structure. The reorganization energy for the subsequent relaxation of P+HL- is calculated to be 5.0 kcal/mol, so that the relaxed radical-pair lies about 7 kcal/mol below P*. The unrelaxed P+BL- radical-pair, in which the electron acceptor is the accessory BChl located between P and HL, appears to be essentially isoenergetic with P*.P+BM-, in which an electron moves to the BChl on the 'M' side, is calculated to lie about 5.5 kcal/mol above P*. These results have an estimated error range of +/- 2.5 kcal/mol. They are shown to be relatively insensitive to various details of the model, including the charge distribution in P+, the atomic charges used for the amino acid residues, the boundaries of the structural region that is considered microscopically and the treatments of the histidyl ligands of P and of potentially ionizable amino acids. The calculated free energies are consistent with rapid electron transfer from P* to HL by way of BL, and with a much slower electron transfer to the pigments on the M side. Tyrosine M208 appears to play a particularly important role in lowering the energy of P+BL-. Electrostatic interactions with the protein favor localization of the positive charge of P+ on PM, one of the two BChl molecules that make up the electron donor.

A protein conformational change associated with the photoreduction of the primary and secondary quinones in the bacterial reaction center.

A comparison is made between the PQA----P+QA- and PQAQB----P+QAQB-transitions in Rps. viridis and Rb. sphaeroides reaction centers (RCs) by the use of light-induced Fourier transform infrared (FTIR) difference spectroscopy. In Rb. sphaeroides RCs, we identify a signal at 1650 cm-1 which is present in the P+QA-minus-PQA spectrum and not in the P+QAQB(-)-minus-PQAQB spectrum. In contrast, this signal is present in both P+QA(-)-minus-PQA- and P+QAQB(-)-minus-PQAQB spectra of Rps. viridis RCs. These data are interpreted in terms of a conformational change of the protein backbone near QA (possible at the peptide C = O of a conserved alanine residue in the QA pocket) and of the different bonding interactions of QB with the protein in the RC of the two species.

Analysis of the electron transfer from Pheo- to QA in PS II membrane fragments from spinach by time resolved 325 nm absorption changes in the picosecond domain.

Absorption changes at 325 nm (delta A325) induced by 15 ps laser flashes (lambda = 650 nm) in PS II membrane fragments were measured with picosecond time-resolution. In samples with the reaction centers (RCs) kept in the open state (P I QA) the signals are characterized by a very fast rise (not resolvable by our equipment) followed by only small changes within our time window of 1.6 ns. In the closed state (PI QA-) of the reaction center the signal decays with an average half-life time of about 250 ps. It is shown that under our excitation conditions (E = 2 x 10(14) photons/cm2 per pulse) subtraction of the absorption changes in closed RCs (delta A closed 325) from those in open RCs (delta A open 325) leads to a difference signal which is dominated by the reduction kinetics of QA. From the rise kinetics of this signal and by comparison with data in the literature it is inferred that QA becomes reduced by direct electron transfer from Pheo- with a time constant of about 350 +/- 100 ps.

[Functions of pheophytin, plastoquinone, iron and carotenoids in plant photosystem 2 reaction centers]. / Issledovanie funktsii feofitina, plastokhinona, zheleza i karotinoidov v reaktsionnykh tsentrakh fotosistemy 2 rastenii.

Photoreduction of the intermediary electron acceptor, pheophytin (Ph), in photosystem-2 (PS-2) reaction centers of spinach chloroplasts or subchloroplast particles (TSF-II and TSF-IIa) at 220 K and Eh approximately -450 mV produces a narrow ESR signal of Ph. (g = 2.0033; delta H approximately 13 G) and a "doublet" centered at g = 2.00 with a splitting of 52 G at 7 K. The doublet (but not the narrow signal) is eliminated after extraction of lyophylized TSF-II with hexane, containing 0.1-0.2% methanol, or after extraction of Fe with LiClO4 and o-phenantroline, and the signal is restored by reconstitution with plastoquinone-A (PQ) or Fe++, respectively. The Fe removal results also in the development of a photoinduced ESR signal of PQ. (g approximately 2.0044; delta H approximately 9.2 G). The conclusion is made that the primary electron acceptor, Q, is in fact a complex PQ-Fe++ and that the exchange interaction of Ph. with PQ. -Fe++ is responsible for the ESR doublet. Photoreduction of Ph in TSF-IIa is accompanied by the 3-fold decrease in the formation of carotenoid triplet state (measured by the characteristic flash-induced absorbance changes) which is suggested to be a result of charge recombination in the pair [P680+ .PH.].