Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides. Evidence for two absorption bands of the dimeric primary electron donor.

Chromatophores from Rhodopseudomonas sphaeroides were oriented by allowing aqueous suspensions to dry on glass plates. Orientation of reaction center pigments was investigated by studying the linear dichroism of chromatophores in which the absorption by antenna bacteriochlorophyll had been attenuated through selective oxidation. Alternatively the light-induced absorbance changes, in the ranges 550-650 and 700-950nm, were studied in untreated chromatophores. The long wave transition moment of reaction center bacteriochlorophyll (P-870) was found to be nearly parallel to the plane of the membrane, whereas the long wave transition moments of bacteriopheophytin are polarized out of this plane. For light-induced changes the linear dichroic ratios, defined as deltaav/deltaah, are nearly the same for untreated and for oxidized chromatophores. Typical values are 1.60 at 870 nm, 0.80 at 810nm, 1.20 at 790 nm, 0.70 at 765 nm, 0.30 at 745 nm , and 0.50 at 600 nm. The different values for the absorbance decrease at 810 nm (0.80) and the increase at 790 nm (1.20) are incompatible with the hypothesis that these changes are due to the blue-shift of a single band. We propose that the decreases at 870 and 810 nm reflect bleaching of the two components of a bacteriochlorophyll dimer, the "special pair" that shares in the photochemical donation of a single electron. The increase at 790 nm then represents the appearance of a monomer band in place of the dimer spectrum, as a result of electron donation. This hypothesis is consistent with available data on circular dichroism. It is confirmed by the presence of a shoulder at 810 nm in the absorption spectrum of reaction centers at low temperature; this band disappears upon photooxidation of the reaction centers. For the changes near 760 nm, associated with bacteriopheophytin, the polarization and the shape of the "light-dark" difference spectrum (identical to the first derivative of the absorption spectrum) show that the 760 nm band undergoes a light-induced shift to greater wavelengths.     

Analysis of absorption spectra of purple bacterial reaction centers in the near infrared region by higher order derivative spectroscopy

Reaction centers (RCs) of purple bacteria are uniquely suited objects to study the mechanisms of the photosynthetic conversion of light energy into chemical energy. A recently introduced method of higher order derivative spectroscopy [I.K. Mikhailyuk, H. Lokstein, A.P. Razjivin, A method of spectral subband decomposition by simultaneous fitting the initial spectrum and a set of its derivatives, J. Biochem. Biophys. Methods 63 (2005) 10-23] was used to analyze the NIR absorption spectra of RC preparations from Rhodobacter (R.) sphaeroides strain 2R and Blastochloris (B.) viridis strain KH, containing bacteriochlorophyll (BChl) a and b, respectively. Q(y) bands of individual RC porphyrin components (BChls and bacteriopheophytins, BPheo) were identified. The results indicate that the upper exciton level P(y+) of the photo-active BChl dimer in RCs of R. sphaeroides has an absorption maximum of 810nm. The blue shift of a complex integral band at approximately 800nm upon oxidation of the RC is caused primarily by bleaching of P(y+), rather than by an electrochromic shift of the absorption band(s) of the monomeric BChls. Likewise, the disappearance of a band peaking at 842nm upon oxidation of RCs from B. viridis indicates that this band has to be assigned to P(y+). A blue shift of an absorption band at approximately 830nm upon oxidation of RCs of B. viridis is also essentially caused by the disappearance of P(y+), rather than by an electrochromic shift of the absorption bands of monomeric BChls. Absorption maxima of the monomeric BChls, B(B) and B(A) are at 802 and 797nm, respectively, in RCs of R. sphaeroides at room temperature. BPheo co-factors H(B) and H(A) peak at 748 and 758nm, respectively, at room temperature. For B. viridis RCs the spectral positions of H(B) and H(A) were found to be 796 and 816nm, respectively, at room temperature.     

Orientation of chromophores in reaction centers of Rhodopseudomonas sphaeroides. Evidence for two absorption bands of the dimeric primary electron donor

Chromatophores from Rhodopseudomonas sphaeroides were oriented by allowing aqueous suspensions to dry on glass plates. Orientation of reaction center pigments was investigated by studying the linear dichroism of chromatophores in which the absorption by antenna bacteriochlorophyll had been attenuated through selective oxidation. Alternatively the light-induced absorbance changes, in the ranges 550–650 and 700–950 nm, were studied in untreated chromatophores. The long wave transition moment of reaction center bacteriochlorophyll (P-870) was found to be nearly parallel to the plane of the membrane, whereas the long wave transition moments of bacteriopheophytin are polarized out of this plane. For light-induced changes the linear dichroic ratios, defined as Δa_v/Δa_h, are nearly the same for untreated and for oxidized chromatophores. Typical values are 1.60 at 870 nm, 0.80 at 810 nm, 1.20 at 790 nm, 0.70 at 765 nm, 0.30 at 745 nm, and 0.50 at 600 nm. The different values for the absorbance decrease at 810 nm (0.80) and the increase at 790 nm (1.20) are incompatible with the hypothesis that these changes are due to the blue-shift of a single band. We propose that the decreases at 870 and 810 nm reflect bleaching of the two components of a bacteriochlorophyll dimer, the “special pair” that shares in the photochemical donation of a single electron. The increase at 790 nm then represents the appearance of a monomer band in place of the dimer spectrum, as a result of electron donation. This hypothesis is consistent with available data on circular dichroism. It is confirmed by the presence of a shoulder at 810 nm in the absorption spectrum of reaction centers at low temperature; this band disappears upon photooxidation of the reaction centers. For the changes near 760 nm, associated with bacteriopheophytin, the polarization and the shape of the “light-dark” difference spectrum (identical to the first derivative of the absorption spectrum) show that the 760 nm band undergoes a light-induced shift to greater wavelengths.     

Photoinduced transient absorbance spectra of P840/P840(+) and the FMO protein in reaction centers of Chlorobium vibrioforme.

The kinetics of photoinduced absorbance changes in the 400-ns to 100-ms time range were studied between 770 and 1025 nm in reaction center core (RCC) complexes isolated from the green sulfur bacterium Chlorobium vibrioforme. A global, multiple stretched-exponential analysis shows the presence of two distinct but strongly overlapping spectra. The spectrum of the 70-micros component consists of a broad bleaching with two minima at 810 and 825 nm and a broad positive band at wavelengths greater than 865 nm and is assigned to the decay of (3)Bchl a of the Fenna-Matthews-Olson (FMO) protein. The contribution of the 70-micros component correlates with the amount of FMO protein in the isolated RCC complex. The spectrum of the 1.6-micros component has a sharp bleaching at 835 nm, a maximum at 805 nm, a broad positive band at wavelengths higher than 865 nm, and a broad negative band at wavelengths higher than 960 nm. When the RCC is incubated with inorganic iron and sulfur, the 1.6-micros component is replaced by a component with a lifetime of approximately 40 micros, consistent with the reconstruction of the F(X) cluster. We propose that the 1.6-micros component results from charge recombination between P840(+) and an intermediate electron acceptor operating between A(0) and F(X). Our studies in Chlorobium RCCs show that approaches that employ a single wavelength in the measurement of absorption changes have inherent limitations and that a global kinetic analysis at multiple wavelengths in the near-infrared is required to reliably separate absorption changes due to P840/P840(+) from the decay of (3)Bchl a in the FMO protein.     

Nuclear wavepacket motion between P and P(+)B(A)(-) potential surfaces with subsequent electron transfer to H(A) in bacterial reaction centers. 1. Room temperature

Formation and coherent propagation of nuclear wavepackets on potential energy surfaces of the excited state of the primary electron donor P and of the charge transfer states P(+)B(A)(-) and P(+)H(A)(-) were studied in native and pheophytin-modified Rhodobacter sphaeroides R-26 reaction centers (RCs) induced by 25 fs excitation (where B(A) and H(A) are the primary and secondary electron acceptors, respectively). The processes were monitored by measuring coherent oscillations in kinetics of the time evolution of the stimulated emission band of P at 935 nm, of the absorption band of B(A)(-) at 1020 nm, and of the bleaching band of H(A) at 760 nm. It was found that the nuclear wavepacket motion on the 130-140 cm(-1) surface of P is directly induced by light absorption in P. When the wavepacket approaches the intersection between P and P(+)B(A)(-) surfaces at 120 and 380 fs delays, the formation of intermediate mixed-state emitting light at 935 nm (P) and absorbing light at 1020 nm (P(+)B(A)(-)) takes place. At the latter time, the wavepacket is transferred to the 32 cm(-1) mode which can belong to the P hypersurface effectively transferring the wavepacket to the P(+)B(A)(-) surface or can represent a diabatic surface which is formed by the states P and P(+)B(A)(-). The wavepacket motion on the P(+)B(A)(-) surface or on the P(+)B(A)(-) part of the mixing surface is accompanied by irreversible electron transfer to H(A). This process is monitored by the kinetics of 1020 nm band development and 760 nm band bleaching (delayed with respect to 1020 nm band development) which both have the enhanced 32 cm(-1) mode in Fourier transform (FT) spectra. The mechanism of wavepacket transfer from the 130-140 cm(-1) to the 32 cm(-1) mode is discussed.     

Absorption and structure of an LM unit isolated with sodium dodecyl sulfate from reaction centers of Rhodopseudomonas sphaeroides

Low-temperature absorption, circular dichroism and resonance Raman spectra of the LM units isolated with sodium dodecyl sulfate from wild-type Rhodopseudomonas sphaeroides reaction centers (Agalidis, I. and Reiss-Husson, F. (1983) Biochim. Biophys. Acta 724, 340–351) are described in comparison with those of intact reaction centers. In LM unit, the Q_y absorption band of P-870 at 77 K shifted from 890 nm (in reaction center) to 870 nm and was broadened by about 30%. In contrast, the 800 nm bacteriochlorophyll absorption band including the 810 species remained unmodified. It was concluded that the 810 nm transition is not the higher excitonic component of P-870. The Q_x band of P-870 shifted from 602 nm (in reaction center) to 598 nm in LM, whereas the Q_x band of the other bacteriochlorophylls was the same in reaction center and LM and had two components at about 605 and 598 nm. The Q_xII band of bacteriopheophytin was upshifted to 538 nm and a slight blue shift of the Q_y band of bacteriopheophytin was observed. Resonance Raman spectra of spheroidene in LM showed that its native cis-conformation was preserved. Resonance Raman spectroscopy also demonstrated that in LM the molecular interactions assumed by the conjugated carbonyls of bacteriochlorophyll molecules were altered, but not those assumed by the bacteriopheophytins carbonyls. In particular at least one Keto group of bacteriochlorophyll free in reaction center, becomes intermolecularly bounded in LM (possibly with extraneous water). This group may belong to the primary donor molecules.     

The carotenoid band shift in reaction centers from from Rhodopseudomonas sphaeroides.

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the "special pair" of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.     

Energy transfer and bacteriochlorophyll fluorescence in purple bacteria at low temperature

Emission spectra of bacteriochlorophyll a fluorescence and absorption spectra of various purple bacteria were measured at temperatures between 295 and 4 K. For Rhodospirillum rubrum the relative yield of photochemistry was measured in the same temperature region. In agreement with earlier results, sharpening and shifts of absorption bands were observed upon cooling to 77 K. Below 77 K further sharpening occurred. In all species an absorption band was observed at 751-757 nm. The position of this band and its amplitude relative to the concentration of reaction centers indicate that this band is due to reaction center bacteriopheophytin. The main infrared absorption band of Rhodopseudomonas sphaeroides strain R26 is resolved in two bands at low temperature, which may suggest that there are two pigment-protein complexes in this species. Emission bands, like the absorption bands, shifted and sharpened upon cooling. The fluorescence yield remained constant or even decreased in some species between room temperature and 120 K, but showed an increased below 120 K. This increase was most pronounced in species, such as R. rubrum, which showed single banded emission spectra. In Chromatium vinosum three (835, 893 and 934 nm) and in Rps. sphaeroides two (888 and 909 nm) emission bands were observed at low temperature. The temperature dependence of the amplitudes of the short wavelength bands indicated the absence of a thermal equilibrium for the excitation energy distribution in C. vinosum and Rps. sphaeroides. In all species the increased in the yield was larger when all reaction centers were photochemically active than when the reaction centers were closed. In R. rubrum the increase in the fluorescence yield was accompanied by a decrease of the quantum yield of charge separation upon excitation of the antenna but not of the reaction center chlorophyll. Calculation of the F?rster resonance integral at various temperatures indicated that the increase in fluorescence yield and the decrease in the yield of photochemistry may be due to a decrease in the rate of energy transfer between antenna bacteriochlorophyll molecules. The energy transfer from carotenoids to bacteriochlorophyll was independent of the temperature in all species examined. The results are discussed in terms of existing models for energy transfer in the antenna pigment system.     

Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa

Reaction centers were isolated from a carotenoidless mutant of Rhodopseudomonas gelatinosa by hydroxyapatite chromatography of purified chromatophores treated with lauryl dimethyl amine oxide. Absorption spectra and spectra of light-induced absorbance changes are similar to those of reaction centers from Rhodopseudomonas sphaeroides. The ratio of absorbance at 280 nm to that at 799 nm was 1.8 in the purest preparations. The extinction coefficient at the 799 nm absorption maximum was estimated to be 305 +/- 20 mM--1 . CM--1. The molecular weight based on protein and chromophore assays was found to be 1.5 . 10(5); the reaction center protein accounted for 6% of the total membrane protein. These reaction centers contained no cytochrome and showed just two components of apparent molecular weights 33 000 and 25 000 in polyacrylamide gel electrophoresis. The chromatophores contained 42 molecules of antenna bacteriochlorophyll for each reaction center.     

Site-directed mutations at D1-Thr179 of photosystem II in Synechocystis sp. PCC 6803 modify the spectroscopic properties of the accessory chlorophyll in the D1-branch of the reaction center

D1-Thr179, which overlies the reaction center chlorophyll Chl D1 of Photosystem II was replaced with His and Glu through site-directed mutation in Synechocystis sp. PCC 6803. Spectroscopic characterization of the mutants indicates that, compared to wild type, the main bleaching in the triplet-minus-singlet absorbance difference spectrum and the electrochromic band shift in the (P680 (+)Q A (-)-P680Q A) absorbance difference spectrum are displaced to the red by approximately 2 nm in the D1-Thr179His mutant and to the blue by approximately 1 nm in the D1-Thr179Glu mutant. These difference spectra are compared with the absorbance difference spectra, measured on the same states in the D1-His198Gln mutant in which the axial ligand D1-His198 of the special pair chlorophyll, P D1, was replaced by glutamine. Together, these results give direct evidence that (a) the reaction center triplet state, produced upon charge recombination from (3)[P (+)Pheo (-)], is primarily localized on Chl D1; (b) the cation of the oxidized donor P (+) is predominantly localized on chlorophyll P D1 of the special pair; and (c) the Q Y band of the accessory chlorophyll Chl D1 is electrochromically shifted in response to charges on P (+) and Q A (-). Light-induced absorbance difference spectra (between 650 and 710 nm), associated with the oxidation of secondary donors and the reduction of Q A, exhibit a bleaching attributed to the oxidation of a Chl Z and strong electrochromic band shifts. On the basis of mutation-induced spectroscopic changes and of structure-based calculations, we conclude that the experimental spectra are best explained by a blue-shift of the Q Y band of the accessory chlorophyll Chl D1, arising from charges on Car D2 (+) and Chl ZD2 (+) and on reduced Q A.     

Primary light-energy conversion in tetrameric chlorophyll structure of photosystem II and bacterial reaction centers: II. Femto- and picosecond charge separation in PSII D1/D2/Cyt b559 complex

In Part I of the article, a review of recent data on electron-transfer reactions in photosystem II (PSII) and bacterial reaction center (RC) has been presented. In Part II, transient absorption difference spectroscopy with 20-fs resolution was applied to study the primary charge separation in PSII RC (DI/DII/Cyt b 559 complex) excited at 700 nm at 278 K. It was shown that the initial electron-transfer reaction occurs within 0.9 ps with the formation of the charge-separated state P680(+)Chl(D1)(-), which relaxed within 14 ps as indicated by reversible bleaching of 670-nm band that was tentatively assigned to the Chl(D1) absorption. The subsequent electron transfer from Chl(D1)(-) within 14 ps was accompanied by a development of the radical anion band of Pheo(D1) at 445 nm, attributable to the formation of the secondary radical pair P680(+)Pheo(D1)(-). The key point of this model is that the most blue Q(y) transition of Chl(D1) in RC is allowing an effective stabilization of separated charges. Although an alternative mechanism of charge separation with Chl(D1)* as a primary electron donor and Pheo(D1) as a primary acceptor can not be ruled out, it is less consistent with the kinetics and spectra of absorbance changes induced in the PSII RC preparation by femtosecond excitation at 700 nm.     

Properties of reaction centers of Rhodopseudomonas sphaeroides in dried gelatin films.: Linear dichroism and low temperature spectra

Methods of preparing dried gelatin films containing purified reaction centers of Rhodopseudomonas sphaeroides are described. The spectral properties of reaction centers in solution are essentially maintained in dried gelatin films. These films are uniform and have excellent optical properties, showing little particulate scattering at temperatures down to about 4K. Film contraction on cooling to 90K is less than 1% in linear dimension. Linear dichroism spectra are reported for films at room and low temperature. Reaction centers show a moderate amount of linear dichroism in unstretched gelatin films; the magnitude of the linear dichroism becomes much greater when the films are stretched. In stretched films, linear dichroic ratios ($\text{A}{}_{\mathrm{\parallel }}\text{A}{}_{\mathrm{\perp }}$; absorbance measured with electric vector parallel and perpendicular to stretching direction) between 1.7 and 2.2 were obtained for the 860 nm absorption band of the bacteriochlorophyll component that undergoes primary photooxidation. The relative polarizations of light-induced absorption changes of reaction centers in stretched films are similar to those reported by Vermeglio and Clayton ((1976) Biochim. Biophys. Acta 449, 500–515) and support their hypothesis that absorbance decreases, maximal near 860 and 810 nm, and an increase near 790 nm are associated with the respective disappearance and appearance of discrete bands characteristic of the reduced and oxidized bacteriochlorophyll dimer. This interpretation is also supported by the polarization of the absolute absorption spectrum near 810 and 860 nm. An absorption band near 540 nm, ascribed to the Q_x transitions of two molecules of bacteriopheophytin in the reaction center, is split at low temperatures into two bands having similar polarizations. This splitting is probably not due to exciton coupling of the two molecules, since excition theory predicts different polarizations.     

Bacteriochlorophyll a-types in chromatophore and subchromatophore preparations from Rhodopseudomonas sphaeroides.

Comparison of absorption and circular dichroism (CD) spectra in the near infrared region was made with chromatophore and subchromatophore preparations obtained from Rhodopseudomonas sphaeroides. The 850 nm absorption band had a positive correlation with the 850 nm and 870 nm CD bands. The 800 nm and 870 nm absorption bands seemed not to correlate with any CD bands. Lipid contents in chromatophores and subchromatophores were measured. Lipids in membranes seemed to contribute to the appearance of the 870 nm absorption band, but not to that of the 800 nm and 850 nm absorption bands. The time courses of absorbance changes were compared at 800, 850, and 870 nm in detergent-treated chromatophores. Relative changes of absorbances differed from one another. The present results suggest that the three absorption bands are due to three different bacteriochlorophyll a-types and the 850 nm absorption band originates from exciton-coupling of bacteriochlorophyll a.     

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.     

Photochemical activities of reaction centers from Rhodopseudomonas sphaeroides at low temperature and in the presence of chaotropic agents.

Light-induced absorbance changes were measured at low temperatures in reaction center preparations from Rhodopseudomonas sphaeroides. Absorbance difference spectra measured at 100 degrees K show that ubiquinone is photoreduced at this temperature, both by continuous light and by a short actinic flash. The reduction occurred with relatively high efficiency. These results give support to the idea that ubiquinone is involved in the primary photochemical reaction in Rhodopseudomonas sphaeroides. Reduction of ubiquinone was accompanied by a shift of the infrared absorption band of bacteriopheophytin. The rate of decay of the primary photoproducts (P+870 and ubisemiquinone) appeared to be approximately independent of temperature below 180 degrees K and above 270 degrees K; in the region between 180 and 270 degrees K it increased with decreasing temperature. The rate of decay was not affected by 0-phenanthroline. Secondary reactions were inhibited by lowering the temperature. The light-induced absorbance changes were inhibited by chaotropic agents, like thiocyanate and perchlorate. It was concluded that these agents lower the efficiency of the primary photoconversion. The kinetics indicated that the degree of inhibition was not the same for all reaction centers. The absorption spectrum of the photoconverted reaction centers appeared to be somewhat modified by thiocyanate.     

Site-directed mutations at D1-His198 and D2-His197 of photosystem II in Synechocystis PCC 6803: sites of primary charge separation and cation and triplet stabilization

Site-directed mutations were introduced to replace D1-His198 and D2-His197 of the D1 and D2 polypeptides, respectively, of the photosystem II (PSII) reaction center of Synechocystis PCC 6803. These residues coordinate chlorophylls P(A) and P(B) which are homologous to the special pair Bchlorophylls of the bacterial reaction centers that are coordinated respectively by histidines L-173 and M-200 (202). P(A) and P(B) together serve as the primary electron donor, P, in purple bacterial reaction centers. In PS II, the site-directed mutations at D1 His198 affect the P(+)--P-absorbance difference spectrum. The bleaching maximum in the Soret region (in WT at 433 nm) is blue-shifted by as much as 3 nm. In the D1 His198Gln mutant, a similar displacement to the blue is observed for the bleaching maximum in the Q(y) region (672.5 nm in WT at 80 K), whereas features attributed to a band shift centered at 681 nm are not altered. In the Y(Z*)--Y(Z)-difference spectrum, the band shift of a reaction center chlorophyll centered in WT at 433--434 nm is shifted by 2--3 nm to the blue in the D1-His198Gln mutant. The D1-His198Gln mutation has little effect on the optical difference spectrum, (3)P--(1)P, of the reaction center triplet formed by P(+)Pheo(-) charge recombination (bleaching at 681--684 nm), measured at 5--80 K, but becomes visible as a pronounced shoulder at 669 nm at temperatures > or =150 K. Measurements of the kinetics of oxidized donor--Q(A)(-) charge recombination and of the reduction of P(+) by redox active tyrosine, Y(Z), indicate that the reduction potential of the redox couple P(+)/P can be appreciably modulated both positively and negatively by ligand replacement at D1-198 but somewhat less so at D2-197. On the basis of these observations and others in the literature, we propose that the monomeric accessory chlorophyll, B(A), is a long-wavelength trap located at 684 nm at 5 K. B(A)* initiates primary charge separation at low temperature, a function that is increasingly shared with P(A)* in an activated process as the temperature rises. Charge separation from B(A)* would be potentially very fast and form P(A)(+)B(A)(-) and/or B(A)(+)Pheo(-) as observed in bacterial reaction centers upon direct excitation of B(A) (van Brederode, M. E., et al. (1999) Proc. Natl. Acad Sci. 96, 2054--2059). The cation, generated upon primary charge separation in PSII, is stabilized at all temperatures primarily on P(A), the absorbance spectrum of which is displaced to the blue by the mutations. In WT, the cation is proposed to be shared to a minor extent (approximately 20%) with P(B), the contribution of which can be modulated up or down by mutation. The band shift at 681 nm, observed in the P(+)-P difference spectrum, is attributed to an electrochromic effect of P(A)(+) on neighboring B(A). Because of its low-energy singlet and therefore triplet state, the reaction center triplet state is stabilized on B(A) at < or =80 K but can be shared with P(A) at >80 K in a thermally activated process.     

Correlation of proton release and electrochromic shifts of the optical spectrum due to oxidation of tyrosine in reaction centers from Rhodobacter sphaeroides

Reaction centers from the Y(L167) mutant of Rhodobacter sphaeroides, containing a highly oxidizing bacteriochlorophyll dimer and a tyrosine residue substituted at Phe L167, were compared to reaction centers from the Y(M) mutant, with a tyrosine at M164, and a quadruple mutant containing a highly oxidizing dimer but no nearby tyrosine residue. Distinctive features in the light-induced optical and EPR spectra showed that the oxidized bacteriochlorophyll dimer was reduced by Tyr L167 in the Y(L167) mutant, resulting in a tyrosyl radical, as has been found for Tyr M164 in the Y(M) mutant. In the Y(L167) mutant, the net proton uptake after formation of the tyrosyl radical and the reduced primary quinone ranged from +0.1 to +0.3 H(+)/reaction center between pH 6 and pH 10, with a dependence that is similar to the quadruple mutant but different than the large proton release observed in the Y(M) mutant. In the light-induced absorption spectrum in the 700-1000 nm region, the Y(L167) mutant exhibited unique changes that can be assigned as arising primarily from an approximately 30 nm blue shift of the dimer absorption band. The optical signals in the Y(L167) mutant were pH dependent, with a pK(a) value of approximately 8.7, indicating that the tyrosyl radical is stabilized at high pH. The results are modeled by assuming that the phenolic proton of Tyr L167 is trapped in the protein after oxidation of the tyrosine, resulting in electrostatic interactions with the tetrapyrroles and nearby residues.     

Titles of related papers in other sections

A method is described for isolation of the Rhodopseudomonas viridis reaction center complex free of altered, 685 nm absorbing pigment. This improved preparation contains two c-type cytochromes in the ratio P-960: cytochrome c-558: cytochrome c-553 of 1 : 2 : 2 to 3. The near infrared spectral forms of the reduced preparation are located at 790, 832, 846 and 987 nm at 77 K; the oxidized complex absorbs at 790, 808, 829 and approx. 1310 nm. The 790 nm band is attributed to bacteriophaeophytin b and the other absorbances to bacteriochlorophyll b. The visible absorption bands may be assigned to these pigments and to the cytochromes present and, probably, to a carotenoid. The presence of two bacteriochlorophyll b spectral forms in the P⁺-830 band suggests that exciton interactions occur among pigments in the oxidized, as well as the reduced, reaction center. Changes in the 790 and 544 nm bands upon illumination of the reaction center preparation at low redox potential may be indicative of a role for bacteriophaeophytin b in primary photochemical events.     

Dependence of tyrosine oxidation in highly oxidizing bacterial reaction centers on pH and free-energy difference

The pH and temperature dependences of tyrosine oxidation were measured in reaction centers from mutants of Rhodobacter sphaeroides containing a tyrosine residue near a highly oxidizing bacteriochlorophyll dimer. Under continuous illumination, a rapid increase in the absorption change at 420 nm was observed because of the formation of a charge-separated state involving the oxidized dimer and reduced primary quinone, followed by a slow absorption decrease attributed to tyrosine oxidation. Both the amplitude and rate of the slow absorption change showed a pH dependency, indicating that, at low pH, the rate of tyrosine oxidation is limited by the transfer of the phenolic proton to a nearby base. Below 17 degrees C, the rate of the slow absorption change had a strong exponential dependence on the temperature, indicating a high activation energy. At higher pH and temperature, the overall rate of tyrosyl formation appears to be limited by a proposed conformational change in the reaction center that is also observed in reaction centers that do not undergo tyrosine oxidation. The yield of tyrosyl formation measured using electron paramagnetic resonance spectroscopy decreased significantly at 4 degrees C compared to 20 degrees C and was lower at both temperatures in mutants expected to have a slightly smaller driving force for tyrosyl formation.     

High-resolution optical absorption-difference spectra of the triplet state of the primary donor in isolated reaction centers of the photosynthetic bacteria Rhodopseudomonas sphaeroides R-26 and Rhodopseudomonas viridis measured with optically detected magnetic resonance at 1.2 K

We have recorded triplet optical absorption-difference spectra of the reaction center triplet state of isolated reaction centers from Rhodopseudomonas sphaeroides R-26 and Rps. viridis with optical absorption-detected electron spin resonance in zero magnetic field (ADMR) at 1.2 K. This technique is one to two orders of magnitude more sensitive than conventional flash absorption spectroscopy, and consequently allows a much higher spectral resolution. Besides the relatively broad bleachings and appearances found previously (see, e.g., Shuvalov V.A. and Parson W.W. (1981) Biochim. Biophys. Acta 638, 50–59) we have found strong, sharp oscillations in the wavelength regions 790–830 nm (Rps. sphaeroides) and 810–890 nm (Rps. viridis). For Rps. viridis these features are resolved into two band shifts (a blue shift at about 830 nm and a red shift at about 855 nm) and a strong, narrow absorption band at 838 nm. For Rps. sphaeroides R-26 the features are resolved into a red shift at about 810 nm and a strong absorption band at 807 nm. We conclude that the appearance of the absorption bands at 807 and 838 nm, respectively, is due to monomeric bacteriochlorophyll. Apparently, the exciton interaction between the pigments constituting the primary donor is much weaker in the triplet state than in the singlet state, and at low temperature the triplet is localized on one of the bacteriochlorophylls on an optical time scale. The fact that for Rps. sphaeroides the strong band shift and the monomeric band found at 1.2 K are absent at 293 K and very weak at 77 K indicates that these features are strongly temperature dependent. It seems, therefore, premature to ascribe the temperature dependence between 293 and 77 K of the intensity of the triplet absorption-difference spectrum at 810 nm (solely) to a delocalization of the triplet state on one of the accessory bacteriochlorophyll pigments.