AN ABSORPTION BAND NEAR 800 mμ ASSOCIATED WITH P870 IN PHOTOSYNTHETIC BACTERIA*

Abstract— Purple photosynthetic bacteria contain a component, absorbing near 805 mμ, distinct from the major light harvesting bacteriochlorophyll component. The minor component, designated P800, resembles P870 in that it resists oxidative treatments that destroy the light harvesting bacteriochlorophyll. Light induces a reversible blue-shift of P800 together with the reversible bleaching of P870. The ratio of P800 to P870 in Rhodopseudomonas spheroides is constant. Both pigments are absent in phenotypes that cannot grow photosynthetically; they reappear together in revertants to photosynthetic competence. Action spectra for light-induced bleaching of P870 and for bacteriochlorophyll fluorescence show that P800 transfers energy more efficiently to P870 than to the bulk bacteriochlorophyll. It is concluded provisionally that P800 is a specialized bacteriochlorophyll molecule in close proximity to the reaction center component P870.     

LIGHT-INDUCED ABSORBANCY CHANGES IN EIMHJELLEN''S RHODOPSEUDOMONAS*

Abstract— Light induced absorbancy changes in Rhodopseudomonas sp. NHTC 133 are analogous to those observed in other photosynthetic bacteria, but all of the features in the light-induced difference spectrum (except those reflecting cytochrome oxidation) are shifted to greater wavelengths. This general shift is consistent with the fact that the bacteriochlorophyll of this organism (BChl b ) differs from the usual BChl in that its absorption bands are shifted to greater wavelengths.
The singlet excited state of P985 (the counterpart of the P870 of Rhodopseudomonas spheroides ) is not an energy sink relative to the major component of BChl b in vivo . The absorption maximum of P985 is at 985 mp, whereas that of CBhl b in vivo is at 1012 mμ.     

ENERGY TRANSFER AND CYTOCHROME FUNCTION IN A NEW TYPE OF PHOTOSYNTHETIC BACTERIUM*

Abstract— The excitation spectrum for bacteriochlorophyll b fluorescence at 1027 mμ in Rhodopseudomonas sp. NHTC 133 indicates that the efficiency of energy transfer from caro-tenoid to bacteriochlorophyll b is between 27 and 28 %.
Light-induced absorbancy changes in anaerobic whole cells indicated the oxidation of three c -type cytochromes (C-550. 5, C-553, C-558) and one b -type cytochrome or cytochromoid C (C-560). At low light intensities C-553 is the main cytochrome oxidized, while at high light intensities mainly C-558 is oxidized in addition to C-553. The light responses of the heme proteins appear to be similar to those observed previously in purple and green photo-synthetic bacteria. No light-induced shifts in carotenoid absorption bands were detected.
In bacterial extracts C-553 and C-558 are bound to the chromatophores, while C-550. 5 is soluble.     

FLUORESCENCE FROM MAJOR AND MINOR BACTERIOCHLOROPHYLL COMPONENTS IN VIVO*

Abstract— In the photosynthetic bacteria Chromatium, Rhoahpirillum rubrum, and Rhodopseudomonus spheroides the fluorescence of bacteriochlorophyll is probably free of contamination by a “fast” component of delayed emission, judging from the characteristics of the delayed light measured 3 msec after excitation. In Rps. spheroides the pigment P870, associated with photochemical reaction centers, is non-fluorescent in its photochemically active state. Fluorescence of P870 can be induced by either of two agencies that suppress its photochemical activity: exposure to Na₂S₂O₄ and (in a dry chromatophore film) dessication. The yield of fluorescence from the major (light harvesting) component of bacteriochlorophyll in vivo is brought to a common maximum value by conditions that suppress the photochemical activity of P870. In addition to dessication and exposure to Na₂S₂O₄ these conditions include saturating illumination and exposure to K₃Fe(CN)₆. Of these four treatments only the last two bleach the long wave absorption band of P870. These experiments support the following assertions: (1) P870 traps singlet excitation energy absorbed by the light harvesting BChl; the trapping function of P870 depends on its ability to initiate and participate in photochemistry. (2) Both dessication and exposure to Na₂S₂O₄ suppress the photochemical activity of P870 by blocking an event that proceeds directly from the excited singlet state in P870. (3) The fluoresecence of BChl in vivo is emitted almost entirely by a major (light harvesting) component.     

SENSITIZATION OF PHOTOREACTIONS IN EIMHJELLEN'S RHODOPSEUDOMONAS BY A PIGMENT ABSORBING AT 830 mμ*

Abstract— The excitation spectrum for bacteriochlorophyll b fluorescence, the action spectrum for cytochrome-553 oxidation, and the action spectrum for P-985 bleaching are compared to the absorption (1-T) spectrum of a Rhodopseudomonas sp. NHTC 133 extract over the range 770 to 930 mμ. These spectra show that a minor pigment P-830 is more effective in sensitizing cytochrome oxidation and P-985 bleaching than in exciting fluorescence of Bchl b. These results are consistent with the proposal that P-830 is a form of Bchl b in special relation to the reaction center pigment P-985.     

AN IDENTIFICATION OF THE RADICAL GIVING RISE TO THE LIGHT-INDUCED ELECTRON SPIN RESONANCE SIGNAL IN PHOTOSYNTHETIC BACTERIA

Abstract— A reaction-center fraction isolated from Rhodopseudomonas spheroides chromatophores exhibits light-induced changes in its optical and electron spin-resonance (ESR) spectra. In particular, a bleaching at 870nm (P870) has been found to be closely correlated with the appearance of an ESR signal with a g factor of 2.0025 and a peak-to-peak line width of 10 G. The ESR signal is indistinguishable from light-induced signals found in chromatophores or whole cells.
A careful measurement of the spin concentration showed that the ratio of the light-induced spins to bleached P870 molecules is 1.1 ± 0.1. In addition the formation and decay kinetics are identical within experimental error under a variety of experimental conditions.
Previous work has shown that P870 is a bacteriochlorophyll molecule in a specialized environment and that the bleaching signifies oxidation. The present work provides strong evidence that the photo-bleaching of P870 produces the radical cation of bacteriochlorophyll, P870⁺, and that this radical is the source of the ESR signal in whole cells.
The quantum yield for the bleaching of P870 in reaction centers has been measured, using actinic light of wavelengths 880, 800, 760 and 680 nm. For light absorbed at 880 or 800 nm the efficiency is close to 100 per cent. In a coupled reaction, the oxidation of mammalian cytochrome c by P870⁺ proceeds with nearly the same efficiency.
The above results place definite limits on the possibilities for the identity of the primary acceptor. These possibilities are discussed.     

ACTION SPECTRA FOR THE ABSORBANCE CHANGE AT 880 nm AND FOR P870 FLUORESCENCE FROM A PHOTOSYNTHETIC REACTION CENTER*

Abstract— Photosynthetic reaction centers isolated from blue-green mutant strain R-26 of Rhodopseudomonas spheroides were used to study the action spectra for the light-induced absorption changes at 880 nm, and for the P870 fluorescence. These spectra indicated that a pigment, possibly bacteriopheophytin, with an absorption maximum at 757 nm is an intrinsic component of the reaction center and transfers energy to P870 with relatively high efficiency.     

The absolute yield of bacteriochlorophyll fluorescence in vivo

Abstract— –The method of Weber and Teale for determining absolute fluorescence quantum yield of dyes in solution was modified for determination of the yield of bacteriochlorophyll fluorescence from chromatophores and whole cells of photosynthetic bacteria. Measured yields ranged from about 1–6 per cent. The yield depended on intensity and wavelength of the exciting light. The higher yield at higher light intensity was interpreted as due to saturation of photosynthesis. The lower yield in some strains when excited at 810 nm was attributed to preferential excitation of the reaction center pigment P800. From this study and the lifetime measurements of others, the relation τ=Q.τ₀ was substantiated for the fluorescence of bacteriochlorophyll in vivo, τ being the actual lifetime, τ₀ the intrinsic lifetime as estimated from the absorption band area, and Q the quantum yield of fluorescence.     

EFFECTS OF HIGH PRESSURE ON PHOTOCHEMICAL REACTION CENTERS FROM RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Photochemical reaction centers from Rhodopseudomonas spheroides were subjected to pressures ranging from 1 to 6000 atm. Optical absorption, fluorescence and photochemical activities were studied under these conditions. Absorption spectra showed bathychromic shifts of the long-wave bands attributed to bacteriopheophytin and bacteriochlorophyll (the latter as P800 and P870). The quantum efficiency of photochemical oxidation of P870 was diminished at high pressure. The quantum yield of P870 fluorescence showed a parallel decline, as if high pressure introduced a quenching process that competed with both photochemistry and fluorescence. The original efficiencies were largely restored when the pressure was returned to 1 atm. The efficiency of oxidation of mammalian cytochrome c, coupled to the photochemical oxidation of P870 in reaction centers, was lowered by high pressure. This effect was more pronounced than the effect on P870 oxidation, and was irreversible. The kinetics of recovery of P870 following its photochemical oxidation showed effects of high pressure. The main effect was the appearance, at high pressure, of slow recovery suggesting the trapping of electrons. This effect was partly irreversible.     

SPECTROSCOPIC ANALYSIS OF BACTERIOCHLOROPHYLLS IN VITRO AND IN VIVO*

Abstract— Chromatophores from Rhodopseudonionas spheroides were treated with potassium iridic chloride so as to destroy the major complement of bacteriochlorophyll (BChl) without harming the photochemically active P870. A band at 802 mμ, attributed to a pigment P800, survived this treatment along with P870. Extraction of such chromatophores with a mixture of acetone and methanol removed the absorption bands of P800 and P870; a corresponding amount of BChl was found in the extract. The yield of BChl was too great to have been derived from either P800 or P870 alone; analysis of extinction cofficients and band areas of these pigments indicates that they are both specialized fornis of BChl, in a molecular ratio of 2P800:1P870. Bleaching of P870, without attenuation of the absorption band of P800, could be effected by adding potassium ferricyanide to the iridic chloride-treated chromatophores. Extraction of chromatophores in this condition gave a reduced yield of BChl, consistent with a 2:1 ratio of P800 to P870 under the assumption that the BChl in the extract was derived in this case from P800 alone. An absorption band at 600 mμ in iridic chloride-treated chromatophores, characteristic of BChl and ascribed to P800 and P870, is partly bleached and shifted to shoiter wavelengths upon illumination. This reversible effect, and a similar one near 375 mμ (corresponding to the Soret band maximum of BChl), has the combined attributes of the blue-shift of P800 and the bleaching of P870 seen in a spectrally resolved form near 800 and 865 mμ respectively. The 600 mμ band is bleached by about 30 per cent, again consistent with a ratio of 2P800:1P870. These data, in conjunction with information published elsewhere, support the view that two molecules of P800 and one of P870 are associated jointly with a photosynthetic reaction center. It was observed that the long wave absorption bands of BChl in vivo are sometimes narrower than the narrowest bands that have been observed for BChl in dilute organic solutions. Sharpness of these bands is most conspicuous in some forms absorbing near 800 mμ.     

CAROTENOID-TO-BACTERIOCHLOROPHYLL SINGLET ENERGY TRANSFER IN CAROTENOID-INCORPORATED B850 LIGHT-HARVESTING COMPLEXES OF Rhodobacter sphaeroides R-26.1

Four carotenoids, 3,4,7,8-tetrahydrospheroidene, 3,4,5,6-tetrahydrospheroidene, 3,4-dihydrospheroidene and spheroidene, have been incorporated into the B850 light-harvesting complex of the carotenoidless mutant, photosynthetic bacterium, Rhodobacter sphaeroides R-26.1. The extent of π-electron conjugation in these molecules increases from 7 to 10 carbon-carbon double bonds. Carotenoid-to-bacteriochlorophyll singlet state energy transfer efficiencies were measured using steady-state fluorescence excitation spectroscopy to be 54 ± 2%, 66 ± 4%, 71 ± 6% and 56 ± 3% for the carotenoid series. These results are discussed with respect to the position of the energy levels and the magnitude of spectral overlap between the S, (2′AJ state emission from the isolated carotenoids and the bacteriochlorophyll absorption of the native complex. These studies provide a systematic approach to exploring the effect of excited state energies, spectral overlap and excited state lifetimes on the efficiencies of carotenoid-to-bacteriochlorophyll singlet energy transfer in photosynthetic systems.     

IS ZEAXANTHIN CAPABLE OF ENERGY TRANSFER TO CHLOROPHYLL a IN PARTIALLY GREENED LEAVES? A STUDY OF FLUORESCENCE EXCITATION SPECTRA DURING VIOLAXANTHIN DEEPOXIDATION

Absorbance spectra and excitation spectra of chlorophyll a fluoresence were recorded during the light-induced deepoxidation of violaxauthin to zeaxanthin in bean leaves (Phaseolus coccineus) greened under intermittent light. Light minus dark fluorescence excitation difference spectra showed distinct minima at 440, 465, and 500 nm corresponding to maxima in the absorbance difference spectra. Both difference spectra were prevented by the deepoxidase inhibitor dithiothreitol and were inverted when zeaxanthin was epoxidized. The fluorescence excitation difference spectra were successfully modeled by considering the absorbance differences between violaxanthin and zeaxanthin, assuming no energy transfer from the two pigments to chlorophyll a, and accounting for light-induced scattering changes. The pigment stoichiometry and the scattering changes of the simulation were in accordance with experimental data. The results indicate that, in the early stage of leaf development, light absorbed by the cycle pigments violaxanthin and zeaxanthin is not transferred to chlorophyll.     

THE EFFECT OF PHOSPHORYLATION UNCOUPLERS AND ELECTRON TRANSPORT INHIBITORS UPON SPECTRAL SHIFTS AND DELAYED LIGHT EMISSION OF PHOTOSYNTHETIC BACTERIA

Abstract— Delayed light emission (measured 4 msec after excitation) and the light-induced red shifts of the bacteriochlorophyll and carotenoid absorption bands of chromatophores of Rhodopseudomonas spheroides were inhibited by a variety of reagents. These included anti-mycin A, NQNO, CCCP, desaspidin, quinacrine, chlorpromazine, 2,4-dinitrophenol, gramicidin D, Triton X-100 and valinomycin in the presence of potassium, cesium or ammonium ions. Delayed light emission was enhanced by orthophenanthroline, ethanol, succinate and glutathione.
Delayed light emission from chromatophores of Rhodospirillum rubrum was attenuated during photophosphorylation but restored approximately to its initial value in the presence of oligomycin. Since the delayed light and band shifts are inhibited under conditions which tend to deplete or block the formation of high energy phosphorylation intermediate, it is suggested that the presence of a high energy intermediate is a prerequisite for the appearance of each of the three phenomena.     

Artificial Light-Harvesting Antennae: Singlet Excitation Energy Transfer from Zinc Chlorin Aggregate to Bacteriochlorin in Homogeneous Hexane Solution

Abstract— Zinc chlorins possessing 3¹-hydroxyl and 13¹-carbonyl groups self-assemble in nonpolar solvents, such as hexane, in a manner similar to bacteriochlorophyll c in the chlorosomes of green photosynthetic bacteria. Visible absorption and steady-state fluorescence measurements of zinc chlorin aggregates containing a small amount of the bacteriochlorin-zinc chlorin dyad molecules showed that singlet excitation energy transfer from the zinc chlorin aggregate to the bacteriochlorin moiety of the coaggre-gated dyad occurs in the homogeneous solution. In the coaggregated dyad, the bacteriochlorin moiety plays the role of an efficient energy trap and the chlorin moiety the role of an anchor to the donor aggregate. The artificial assembly thus mimics the structure and function of natural chlorosomes and can be considered as the first in vitro supramolecular light-harvesting antenna.     

SINGLET AND TRIPLET ENERGY TRANSFER IN α-DIKETONE DERIVATIVES OF URACIL and THYMINE

Irradiation of 1-(3,4-dioxopentyl)uracil (UPD) and 1-(3.4-dioxopentyl)thymine (TPD) in acetonitrile solution at 25°C, at the wavelength (280 nm) where only the pyrimidine absorbs the light, sensitizes both fluorescence and phosphorescence of the diketone chromophore in the sidechain. From comparison of the intensity in the corrected excitation spectra with the absorption spectra in acetonitrile solution, it was estimated that the yield of singlet energy transfer in UPD was 0.17 and in TPD was 0.44. It was also observed that the ratio of phosphorescence to fluorescence was greater in the sensitized emission than in that from direct excitation of the diketone chromophore. The yield of triplet energy transfer thus measured corresponds to minimum values for the yields of intersystem crossing from singlet excited state to triplet excited state of 0.075 in the uracil chromophore of UPD and of 0.14 in the thymine chromophore of TPD. These are in agreement with other recent values for these quantities. The value of this type of system as an intramolecular triplet counter is discussed.     

Electronic energy transfer involving carotenoid pigments in chlorosomes of two green bacteria: Chlorobium tepidum and Cholroflexus aurantiacus

Electronic energy transfer processes in chlorosomes isolated from the green sulphur bacterium Chlorobium tepidum and from the green filamentous bacterium Chloroflexus aurantiacus have been investigated. Steady-state fluorescence excitation spectra and time-resolved triplet-minus-singlet (TmS) spectra, recorded at ambient temperature and under non-reducing or reducing conditions, are reported. The carotenoid (Car) pigments in both species transfer their singlet excitation to bacteriochlorophyll c (BChlc) with an efficiency which is high (between 0.5 and 0.8) but smaller than unity; BChlc and bacteriochlorophyll a (BChla) transfer their triplet excitation to the Car's with nearly 100% efficiency. The lifetime of the Car triplet states is approximately 3 micros, appreciably shorter than that of the Car triplets in the light-harvesting complex II (LHCII) in green plants and in other antenna systems. In both types of chlorosomes the yield of BChlc triplets (as judged from the yield of the Car triplets) remains insensitive to the redox conditions. In notable contrast the yield of BChlc singlet emission falls, upon a change from reducing to non-reducing conditions, by factors of 4 and 35 in Cfx. aurantiacus and Cb. tepidum, respectively. It is possible to account for these observations if one postulates that the bulk of the BChlc triplets originate either from a large BChlc pool which is essentially non-fluorescent and non-responsive to changes in the redox conditions, or as a result of a process which quenches BChlc singlet excitation and becomes more efficient under non-reducing conditions. In chlorosomes from Cfx. aurantiacus whose Car content is lowered, by hexane extraction, to 10% of the original value, nearly one-third of the photogenerated BChlc triplets still end up on the residual Car pigments, which is taken as evidence of BChlc-to-BChlc migration of triplet excitation; the BChlc triplets which escape rapid static quenching contribute a depletion signal at the long-wavelength edge of the Qy absorption band, indicating the existence of at least two pools of BChlc.     

Lipid binding to the carotenoid binding site in photosynthetic reaction centers

Lipid binding to the carotenoid binding site near the inactive bacteriochlorophyll monomer was probed in the reaction centers of carotenoid-less mutant, R-26 from Rhodobacter sphaeroides. Recently, a marked light-induced change of the local dielectric constant in the vicinity of the inactive bacteriochlorophyll monomer was reported in wild type that was attributed to structural changes that ultimately lengthened the lifetime of the charge-separated state by 3 orders of magnitude (Deshmukh, S. S.; Williams, J. C.; Allen, J. P.; Kalman, L. Biochemistry 2011, 50, 340). Here in the R-26 reaction centers, the combination of light-induced structural changes and lipid binding resulted in a 5 orders of magnitude increase in the lifetime of the charge-separated state involving the oxidized dimer and the reduced primary quinone in proteoliposomes. Only saturated phospholipids with fatty acid chains of 12 and 14 carbon atoms long were bound successfully at 8 °C by cooling the reaction center protein slowly from room temperature. In addition to reporting a dramatic increase of the lifetime of the charge-separated state at physiologically relevant temperatures, this study reveals a novel lipid binding site in photosynthetic reaction center. These results shed light on a new potential application of the reaction center in energy storage as a light-driven biocapacitor since the charges separated by ～30 ? in a low-dielectric medium can be prevented from recombination for hours.     

POLARIZATION OF FLUORESCENCE FROM BACTERIOCHLOROPHYLL IN CASTOR OIL, IN CHROMATOPHORES AND AS P870 IN PHOTOSYNTHETIC REACTION CENTERS

Abstract— The polarization of fluorescence from isolated photosynthetic reaction centers and from light harvesting chlorophyll in photosynthetic units was measured over a wide range of exciting wavelengths. In addition, the fluorescence polarization of bacteriochlorophyll was measured. The simplest interpretation of the data is that in the bacterial reaction center, the three chlorophyll molecules closely associated with photochemical oxidation do not have their transition moments parallel to one another. Highly polarized fluorescence was also observed from the intact photosynthetic unit.     

Multichannel flash spectroscopy of the reaction centers of wild-type and mutant Rhodobacter sphaeroides: bacteriochlorophyllB-mediated interaction between the carotenoid triplet and the special pair

Multichannel flash spectroscopy (with microsecond time resolution) has been applied to carotenoid (Car)-containing and Car-less reaction centers (RC) of Rhodobacter sphaeroides with a view to investigate the interaction between the Car and its neighboring pigments at room temperature. Under neutral redox potential conditions, where the primary quinone acceptor (QA) is oxidized, the light-induced spectral changes in the 350-1000 nm region are attributed to the photochemical oxidation of the special pair (denoted here as P870), the generation of P870(+)QA(-), and the attendant electrochromism of adjacent chromophores. A bathochromic shift of <1 nm in the visible absorption region of Car reveals the sensitivity of Car to the P870 photooxidation. Under low redox potential conditions, where QA is reduced, P870 triplets (P870(+)) are formed. The time-resolved triplet-minus-singlet (TmS) spectrum of Car-less RC shows a deep bleaching at 870 nm, which belongs to P870(+), and additional (but smaller) bleaching at 800 nm; the entire spectrum decays at the same rate (with a lifetime of about 50 micros). The bleaching at 800 nm arises from the pigment interaction between P870(+) and the accessory bacteriochlorophylls on A and B branches (BA,B). In Car-containing RC, the TmS spectra of Car are accompanied by two smaller, negative signals--a sharp peak at 809 +/- 2 nm and a broad band at 870 nm--which decay at the same rate as the TmS spectrum of Car (ca 10 micros). The former is ascribed to the perturbation, by Car(+), of the absorption spectrum of BB; the latter, to the TmS spectrum of P870(+), a species that appears to be in approximate thermal equilibrium with Car(+). These assignments are consistent with the absorption-detected magnetic resonance spectra obtained by other workers at low temperatures.     

HL-LH2中色素分子间的单重激发态能量传递

采用飞秒时间分辨吸收光谱手段观测了在500和800 nm激发下高光培养的紫色光合细菌Rhodopseu-domonas(Rps). palustris外周捕光天线LH2(HL-LH2)中不同共轭链长类胡萝卜素(Carotenoid, 简称Car)和细菌叶绿素a(Bacteriachlorophyll a, 简称BChl a)的特征吸收光谱. 光谱动力学分析结果表明, HL-LH2中不同Car分子间可能存在复杂的单重激发态能量平衡过程, Car分子同时向BChl a分子发生多途径的单重激发态能量传递, B800主要接受来自Car的S2和S1态能量; B850则主要接受来自长共轭链Car(共轭双键数目n=13)的S1态和B800的激发态能量, 整个能量传递过程在3~5 ps内完成.