RELATIONS BETWEEN PHOTOCHEMISTRY AND FLUORESCENCE IN CELLS AND EXTRACTS OF PHOTOSYNTHETIC BACTERIA*

Abstract— Light-induced changes in the yield of bacteriochlorophyll fluorescence have been measured in cells and chromatophores of photosynthetic bacteria, and coordinated with light-induced absorbancy changes. Comparisons were drawn during transitions between dark and light steady states and also between steady states established at different light intensities. Aerobic cell suspensions of Rhodospirillum rubrum, Rhodopseudomonas spheroides, Chromatium and Rhodopseudomonas sp. NHTC 133 showed a strict correspondence between changes in the fluorescence yield and the bleaching of P870 (P985 in Rps. sp. NHTC 133), as reported by Vredenberg and Duysens for R. rubrum cells. The relationship shows that singlet excitation energy in bacteriochlorophyll is quenched by P870 at a rate proportional to the concentration of unbleached P870. This implies that the photosynthetic units are not independent with respect to energy transfer. In anaerobic cell suspensions the change in fluorescence did not follow the bleaching of P870 in the manner described by Vredenberg and Duysens. Here a change in fluorescence may have resulted from the reduction of a primary photochemical electron acceptor as well as from the oxidation (bleaching) of P870. In chromatophore preparations there were further deviations from the Vredenberg and Duysens relationship which could be attributed to changes in the rate constants for quenching of singlet excitation energy. Finally there was a light-induced increase in the fluorescence yield which was related to a band shift of bacteriochlorophyll and not to the bleaching of P870. Aerobic cell suspensions presented a limiting case in which these complications were absent. No change in the fluorescence was associated uniquely with the oxidation of cytochrome or band shifts of carotenoid pigments. These results, when coordinated with earlier findings about the fluorescence of bacteriochlorophyll and P870, indicate that the singlet excitation quantum is the only energy carrier linking the absorption of light with the initiation of photochemistry in bacterial photosynthesis.     

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.     

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.     

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.     

Fluorescence studies on photosynthetic pigment development in Rhodopseudomonas spheroides

Abstract— When bleached, aerobically grown cells of Rhodopseudomonas spheroides are transferred to semi-aerobic conditions to induce bacteriochlorophyll synthesis, a new fluorescence band, with a maximum at 790 nm, is observed in addition to the 885 nm emission maximum normally seen in pigmented cells. The 790 nm fluorescence may be due to bacterio-chlorophyll which has not been bound into the chromatophore membrane. The quantum yield of the 885 nm fluorescence is at first relatively high and then, about 1 hour after transfer, drops to the level found in pigmented photosynthetic cells. The coupling to the rest of the photo-synthetic apparatus, as indicated by the effect of dithionite on the fluorescence, also seems to occur during the first hour of pigment development, which suggests that the onset of fluorescence quenching is due at least in part to the synthesis of photochemical reaction centers. Continuation of these studies should provide new information on the formation, structure and molecular interactions of the pigments and the photosynthetic membranes.     

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.     

Effect of intramolecular charge transfer on fluorescence and singlet oxygen production of phthalocyanine analogues

Intramolecular charge transfer (ICT) was studied on a series of magnesium, metal-free and zinc complexes of unsymmetrical tetrapyrazinoporphyrazines and tribenzopyrazinoporphyrazines bearing two dialkylamino substituents (donors) and six alkylsulfanyl or aryloxy substituents (non-donors). The dialkylamino substituents were responsible for ICT that deactivated excited states and led to considerable decrease of fluorescence and singlet oxygen quantum yields. Photophysical and photochemical properties were compared to corresponding macrocycles that do not bear any donor centers. The data showed high feasibility of ICT in the tetrapyrazinoporphyrazine macrocycle and significantly lower efficiency of this deactivation process in the tribenzopyrazinoporphyrazine type molecules. Considerable effect of non-donor peripheral substituents on ICT was also described. The results imply that tetrapyrazinoporphyrazines may be more suitable for development of new molecules investigated in applications based on ICT.     

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.     

Photoactivated Fluorescence Enhancement in F,N‐Doped Carbon Dots with Piezochromic Behavior

Photoactivation in CdSe/ZnS quantum dots (QDs) on UV/Vis light exposure improves photoluminescence (PL) and photostability. However, it was not observed in fluorescent carbon quantum dots (CDs). Now, photoactivated fluorescence enhancement in fluorine and nitrogen co‐doped carbon dots (F,N‐doped CDs) is presented. At 1.0 atm, the fluorescence intensity of F,N‐doped CDs increases with UV light irradiation (5 s–30 min), accompanied with a blue‐shift of the fluorescence emission from 586 nm to 550 nm. F,N‐doped CDs exhibit photoactivated fluorescence enhancement when exposed to UV under high pressure (0.1 GPa). F,N‐doped CDs show reversible piezochromic behavior while applying increasing pressure (1.0 atm to 9.98 GPa), showing a pressure‐triggered aggregation‐induced emission in the range 1.0 atm–0.65 GPa. The photoactivated CDs with piezochromic fluorescence enhancement broadens the versatility of CDs from ambient to high‐pressure conditions and enhances their anti‐photobleaching.     

Photoactivated Fluorescence Enhancement in F,N-Doped Carbon Dots with Piezochromic Behavior

Photoactivation in CdSe/ZnS quantum dots (QDs) on UV/Vis light exposure improves photoluminescence (PL) and photostability. However, it was not observed in fluorescent carbon quantum dots (CDs). Now, photoactivated fluorescence enhancement in fluorine and nitrogen co-doped carbon dots (F,N-doped CDs) is presented. At 1.0 atm, the fluorescence intensity of F,N-doped CDs increases with UV light irradiation (5 s–30 min), accompanied with a blue-shift of the fluorescence emission from 586 nm to 550 nm. F,N-doped CDs exhibit photoactivated fluorescence enhancement when exposed to UV under high pressure (0.1 GPa). F,N-doped CDs show reversible piezochromic behavior while applying increasing pressure (1.0 atm to 9.98 GPa), showing a pressure-triggered aggregation-induced emission in the range 1.0 atm–0.65 GPa. The photoactivated CDs with piezochromic fluorescence enhancement broadens the versatility of CDs from ambient to high-pressure conditions and enhances their anti-photobleaching.     

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.     

ISOLATION OF PHOTOCHEMICAL REACTION CENTERS FROM A CAROTENOIDLESS MUTANT OF RHODOSPIRILLUM RUBRUM*

Lauryl dimethyl amine oxide was used to isolate reaction centers from a carotenoid-less mutant, strain G-9, of Rhodospirillum rubrum. These reaction centers have absorption spectra and light or chemically induced difference spectra very similar to those obtained from Rhodopseudomonas spheroides, strain R-26. But, unlike those from Rps. spheroides, they are more labile to higher detergent concentrations and to ammonium sulfate. The cytochrome content was estimated to be less than one per 10 P870.     

THE FUNCTION OF P700 AND CYTOCHROME f IN THE PHOTOSYNTHETIC REACTION CENTER OF SYSTEM 1 IN RED ALGAE

Abstract— Kinetics and quantum yields of light-induced oxidation of P700 and the f -type cytochrome were measured in marine red algae from absorbance changes in the region 420, 435 and 705 nm. The quantum yield for cytochrome oxidation in Iridaea splendens and Schizymenia pacifica was 0.5-0.65 in far-red light, at 20 and at 0C.
Oxidation rates of P700 measured when varying amounts of cytochrome were in the oxidized state indicated that a reaction center of system 1 in Iridaea contains 1 P700 and 4 cytochrome molecules. Oxidized P700 only accumulates when all 4 cytochromes are oxidized. The rate of photochemistry of system 1, measured as the sum of the rates of P700 and cytochrome oxidation, was independent of the oxidation level of cytochrome, but decreased with accumulation of oxidized P700. This decrease was less than proportional to the fraction of P700 that was in the oxidized state, which suggested transfer of excitation energy between reaction centers.
The quantum yield for cytochrome oxidation after dark periods of 1 min or more was only about 0.2. This effect was tentatively ascribed to a dark reduction of the cytochrome coupled to phosphorylation.     

CINETIQUE DES ECHANGES D'OXYGENE ET DE LA FLUORESCENCE DES CHLOROPLASTES ISOLES

Abstract Oxygen evolution and fluorescence have been studied with isolated chloroplasts illuminated, in the absence of Hill reagents, by flashes or continuous light. As in whole cells, at least two substances are involved in the primary process leading to the oxygen evolution. The first, called E, probably is the photochemical “complex” of System II. After a long period of darkness, E is not active. It is activated in two steps. Step one is a photochemical reaction, induced by a quantum of light absorbed by pigment-system II, which results in the production of E in a reduced state. Step two is a dark oxidation of the reduced E by the second substance, A. The oxidized E can then enter the normal photochemical cycle of system II. Reduced E might alternatively be oxidized by oxygen, this reaction being responsible for a very rapid and brief light-induced oxygen uptake. Substance A is measured by the oxygen burst and is present in the chloroplasts at the approximate ratio of 1 molecule for 70 molecules of total chlorophyll while E is at the ratio of about 1/2800. This gives a E over A value of 1/35 which is much smaller than the one found in whoe cells (ca. 1/10). This independent behavior of E and A suggests that chloroplast extraction destroys some photochemical centers without having a direct impact on A, which might diffuse from one center to another. Besides the brief light-induced oxygen uptake above mentioned, there is another one which is related to System I functioning. The kinetics of the oxygen evolution and of the fluorescence have been compared. During the activation process of the oxygen evolving ability, rate of oxygen evolution and fluorescence yield increase in a parallel way. After the maximum velocity of the oxygen burst is reached (i.e. after activation), the fluorescence yield keeps growing up until the steady-state is attained (with an intermediary plateau), whereas the rate of oxygen emission slows down. The time-course curves of fluorescence obtained with inactivated or activated chloroplasts are essentially different in that the initial yield is higher in the latter case.     

Atrazine and Methyl Viologen Effects on Chlorophyll‐a Fluorescence Revisited—Implications in Photosystems Emission and Ecotoxicity Assessment

In this work, we use the effect of herbicides that affect the photosynthetic chain at defined sites in the photosynthetic reaction steps to derive information about the fluorescence emission of photosystems. The interpretation of spectral data from treated and control plants, after correction for light reabsorption processes, allowed us to elucidate current controversies in the subject. Results were compatible with the fact that a nonnegligible Photosystem I contribution to chlorophyll fluorescence in plants at room temperature does exist. In another aspect, variable and nonvariable chlorophyll fluorescence were comparatively tested as bioindicators for detection of both herbicides in aquatic environment. Both methodologies were appropriate tools for this purpose. However, they showed better sensitivity for pollutants disconnecting Photosystem II–Photosystem I by blocking the electron transport between them as Atrazine. Specifically, changes in the (experimental and corrected by light reabsorption) red to far red fluorescence ratio, in the maximum photochemical quantum yield and in the quantum efficiency of Photosytem II for increasing concentrations of herbicides have been measured and compared. The most sensitive bioindicator for both herbicides was the quantum efficiency of Photosystem II.     

Elevated atmospheric CO₂ mitigated photoinhibition in a tropical tree species, Gmelina arborea

Effects of elevated CO? on photosynthetic CO? assimilation, PSII photochemistry and photoinhibition were investigated in the leaves of a fast growing tropical tree species, Gmelina arborea (Verbenaceae) during summer days of peak growth season under natural light. Elevated CO? had a significant effect on CO? assimilation rates and maximal efficiency of PSII photochemistry. Chlorophyll a fluorescence induction kinetics were measured to determine the influence of elevated CO? on PSII efficiency. During midday, elevated CO?-grown Gmelina showed significantly higher net photosynthesis (p<0.001) and greater F(V)/F(M) (p<0.001) than those grown under ambient CO?. The impact of elevated CO? on photosynthetic rates and Chl a fluorescence were more pronounced during midday depression where the impact of high irradiance decreased in plants grown under elevated CO? compared to ambient CO?-grown plants. Our results clearly demonstrate that decreased susceptibility to photoinhibition in elevated CO? grown plants was associated with increased accumulation of active PSII reaction centers and efficient photochemical quenching. We conclude that elevated CO? treatment resulted in easy diminution of midday photosynthetic depression.     

FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE STUDIES OF THE INTERACTION OF PHENAZINE METHOSULFATE WITH REACTION-CENTER PREPARATIONS FROM THE R-26 MUTANT OF RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Using the technique of flash photolysis-electron spin resonance, we have shown, by means of a kinetic analysis, that phenazine methosulfate (PMS) interacts with reaction-center preparations from the blue-green mutant R26 of Rhodopseudomonas spheroides. At intermediate concentrations of PMS, biphasic decay kinetics of the P870⁺ ESR signal are observed demonstrating that the PMS radical interacts with reaction centers by a specific binding mechanism. With PMS bound to reaction centers, the P870⁺ ESR signal decays in ˜ 1 ms; whereas, in unbound reaction centers the decay is ˜ 120 ms. A model is proposed involving the interaction of PMS on the donor side of P870.     

Kinetic modeling of propane oxidation and pyrolysis

Propane oxidation in jet-stirred reactor was modeled using a comprehensive kinetic reaction mechanism including the most recent findings concerning the kinetics of the reactions involved in the oxidation of C₁? C₄ hydrocarbons. The present detailed mechanism is able to reproduce experimental species concentration profiles obtained in our high-pressure jet-stirred reactor (900 ? T/K ? 1200; 1 ? P/atm ? 10; 0.15 ? ? ? 4) and in a turbulent flow reactor at 1 atm; ignition delay times measured in shock tube (1200 ? T/K ? 1700; 2 ? P/atm ? 15; 0.125 ? ? ? 2); H-atoms concentrations measured in shock tube during the pyrolysis of propane and burning velocities of freely propagating premixed propane-air laminar flames. The computed results are discussed in terms of pressure and equivalence ratio (?) effects on propane oxidation. The same detailed kinetic reaction mechanism can also be used to model the oxidation of methane, ethylene, ethane, and propene in similar conditions. © John Wiley & Sons, Inc.     

Direct electrosynthesis and characterization of a new soluble polyfluorene derivative containing carboxyl group in boron trifluoride diethyl etherate

High-quality poly(fluorene-9-acetic acid) (PFAA), a new soluble polyfluorene derivative, was synthesized electrochemically by direct anodic oxidation of fluorene-9-acetic acid (FAA) in boron trifluoride diethyl etherate (BFEE) containing a certain amount of trifluoroacetic acid (TFA). This electrolyte enables facile anodic oxidation of FAA monomer at lower potential (1.05 V vs. SCE). PFAA films with conductivity of 0.53 S cm⁻¹ obtained from this medium showed better redox activity and thermal stability in relation to unsoluble poly(fluorene-9-carboxylic acid). Fluorescent spectral studies indicate that PFAA film with high fluorescence quantum yields and photochemical stability is a good blue-light emitter. The structure and morphology of the polymer were studied by UV–vis, FT-IR, ¹H NMR spectra and scanning electron microscopy, respectively.     

Optomechanical Control of Quantum Yield in Trans–Cis Ultrafast Photoisomerization of a Retinal Chromophore Model

The quantum yield of a photochemical reaction is one of the most fundamental quantities in photochemistry, as it measures the efficiency of the transduction of light energy into chemical energy. Nature has evolved photoreceptors in which the reactivity of a chromophore is enhanced by its molecular environment to achieve high quantum yields. The retinal chromophore sterically constrained inside rhodopsin proteins represents an outstanding example of such a control. In a more general framework, mechanical forces acting on a molecular system can strongly modify its reactivity. Herein, we show that the exertion of tensile forces on a simplified retinal chromophore model provokes a substantial and regular increase in the trans ‐to‐cis photoisomerization quantum yield in a counterintuitive way, as these extension forces facilitate the formation of the more compressed cis photoisomer. A rationale for the mechanochemical effect on this photoisomerization mechanism is also proposed.