Fluorescence properties of the allenic carotenoid fucoxanthin: Implication for energy transfer in photosynthetic pigment systems

The fluorescence spectrum of an allenic carotenoid, all-trans-fucoxanthin isolated from a brown alga, has been reported for the first time. This carotenoid is known to function efficiently as a primary photosynthetic antenna pigment in marine algae. The emission bands were located around 630, 685 and 750 nm in CS₂ at 20°C, absorption bands being located at 448, 476 and 505 nm. The energy difference between the 0-0 bands of absorption and emission spectra was about 3900 cm^-1 and location of the emission maximum was less sensitive to the polarizability of solvents than that of the absorption maximum. These clearly indicate that the emission originates from the optically forbidden singlet state (2A_g). This is in contrast to other carotenoids whose emission is assigned to 1B_u state, probably due to the symmetric structure of the conjugated double bond responsible for the absorption in the visible region. A rapid internal conversion from 1B_u to 2A_g state might be facilitated by distorted structure of the conjugated double bond of fucoxanthin. The energy level responsible for the emission is almost identical to the Q_y level of the acceptor molecule (Chl a), thus we propose an energy transfer pathway from the optically forbidden 2A_g state of the carotenoid to the Q_y transition of Chl a in algal pigment systems.     

Primary electron transfer in reaction centers of YM210L and YM210L/HL168L mutants of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The role of tyrosine M210 in charge separation and stabilization of separated charges was studied by analyzing of the femtosecond oscillations in the kinetics of decay of stimulated emission from P* and of a population of the primary charge separated state P⁺B_A⁻ in YM210L and YM210L/HL168L mutant reaction centers (RCs) of Rhodobacter sphaeroides in comparison with those in native Rba. sphaeroides RCs. In the mutant RCs, TyrM210 was replaced by Leu. The HL168L mutation placed the redox potential of the P⁺/P pair 123 mV below that of native RCs, thus creating a theoretical possibility of P⁺B_A⁻ stabilization. Kinetics of P* decay at 940 nm of both mutants show a significant slowing of the primary charge separation reaction in comparison with native RCs. Distinct damped oscillations in these kinetics with main frequency bands in the range of 90–150 cm⁻¹ reflect mostly nuclear motions inside the dimer P. Formation of a very small absorption band of B_A⁻ at 1020 nm is registered in RCs of both mutants. The formation of the B_A⁻ band is accompanied by damped oscillations with main frequencies from ∼10 to ∼150 cm⁻¹. Only a partial stabilization of the P⁺B_A⁻ state is seen in the YM210L/HL168L mutant in the form of a small non-oscillating background of the 1020-nm kinetics. A similar charge stabilization is absent in the YM210L mutant. A model of oscillatory reorientation of the OH-group of TyrM210 in the electric fields of P⁺ and B_A⁻ is proposed to explain rapid stabilization of the P⁺B_A⁻ state in native RCs. Small oscillatory components at ∼330–380 cm⁻¹ in the 1020-nm kinetics of native RCs are assumed to reflect this reorientation. We conclude that the absence of TyrM210 probably cannot be compensated by lowering of the P⁺B_A⁻ free energy that is expected for the double YM210L/HL168L mutant. An oscillatory motion of the HOH55 water molecule under the influence of P⁺ and B_A⁻ is assumed to be another potential contributor to the mechanism of P⁺B_A⁻ stabilization.     

Femtosecond stage of electron transfer in reaction centers of the triple mutant SL178K/GM203D/LM214H of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Coherent processes in an initial phase of charge transfer in reaction centers (RCs) of the triple mutant S(L178)K/G(M203)D/L(M214)H of Rhodobacter sphaeroides were investigated by difference (light — dark) absorption spectroscopy with 18 fsec time resolution. Electron transfer in the B cofactor branch is activated in this mutant, while the A-branch electron transfer is slowed in comparison with native RCs of Rba. sphaeroides. A bulk of absorption difference spectra was analyzed in the 940–1060 nm range (stimulated emission of excited bacteriochlorophyll dimer P* and absorption of bacteriochlorophyll anions B_A⁻ and β⁻, where β is a bacteriochlorophyll substituting the native bacteriopheophytin H_A) and in the 735–775 nm range (bleaching of the absorption band of the bacteriopheophytin H_B in the B-branch) in the −0.1 to 4 psec range of delays with respect to the moment of photoexcitation of P at 870 nm. Spectra were measured at 293 and 90 K. The kinetics of P* stimulated emission at 940 nm shows its decay with a time constant of ∼14 psec at 90 K and ∼18 psec at 293 K, which is accompanied by oscillations with a frequency of ∼150 cm⁻¹. A weak absorption band is found at 1018 nm that is formed ∼100 fsec after excitation of P and reflects the electron transfer from P* to β and/or B_A with accumulation of the P⁺β⁻ and/or P⁺B_A⁻ states. The kinetics of ΔA at 1018 nm contains the oscillations at ∼150 cm⁻¹ and distinct low-frequency oscillations at 20–100 cm⁻¹; also, the amplitude of the oscillations at 150 cm⁻¹ is much smaller at 293 than at 90 K. The oscillations in the kinetics of the 1018 nm band do not contain a 32 cm⁻¹ mode that is characteristic for native Rba. sphaeroides RCs having water molecule HOH55 in their structure. The ΔA kinetics at 751 nm reflects the electron transfer to HB with formation of the P⁺H_B⁻ state. The oscillatory part of this kinetics has the form of a single peak with a maximum at ∼50 fsec completely decaying at ∼200 fsec, which might reflect a reversible electron transfer to the B-branch. The results are analyzed in terms of coherent nuclear wave packet motion induced in the P* excited state by femtosecond light pulses, of an influence of the incorporated mutations on the mutual position of the energy levels of charge separated states, and of the role of water HOH55 in the dynamics of the initial electron transfer.     

Primary processes of charge separation in reaction centers of YM210L/FM197Y and YM210L mutants of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Difference femtosecond absorption spectroscopy with 20-fsec temporal resolution was applied to study a primary stage of charge separation and transfer processes in reaction centers of YM210L and YM210L/FM197Y site-directed mutants of the purple bacterium Rhodobacter sphaeroides at 90 K. Photoexcitation was tuned to the absorption band of the primary electron donor P at 880 nm. Coherent oscillations in the kinetics of stimulated emission of P* excited state at 940 nm and of anion absorption of monomeric bacteriochlorophyll B_A⁻ at 1020 nm were monitored. The absence of tyrosine YM210 in RCs of both mutants leads to strong slowing of the primary reaction P* → P⁺B_A⁻ and to the absence of stabilization of separated charges in the state P⁺B_A⁻. Mutation FM197Y increases effective mass of an acetyl group of pyrrole ring I in the bacteriochlorophyll molecule P_B of the double mutant YM210L/FM197Y by a hydrogen bond with OH-TyrM197 group that leads to a decrease in the frequency of coherent nuclear motions from 150 cm⁻¹ in the single mutant YM210L to ∼100 cm⁻¹ in the double mutant. Oscillations with 100–150 cm⁻¹ frequencies in the dynamics of the P* stimulated emission and in the kinetics of the reversible formation of P⁺B_A⁻ state of both mutants reflect a motion of the P_B molecule relatively to P_A in the area of mutual overlapping of their pyrrole rings I. In the double mutant YM210L/FM197Y the oscillations in the P* emission band and the B_A⁻ absorption band are conserved within a shorter time ∼0.5 psec (1.5 psec in the YM210L mutant), which may be a consequence of an increase in the number of nuclei forming a wave packet by adding a supplementary mass to the dimer P.     

Kinetic processes initiated by a nanosecond high-current discharge in hot air

Results from numerical investigations of kinetic processes initiated by a pulsed nanosecond discharge in hot (T ₀ ≥ 1000 K) air at atmospheric pressure are presented. The calculated results on the dynamics of the electron density, the population of the N₂(B³Π _g ) and N₂(C³Π _u ) states, and the atomic oxygen density in the axial discharge region agree with experiment. The method for determining the gas temperature by measuring the rotational structure of the transitions N₂(C³Π _u , ν) → N₂(B³Π _g , ν′) of the 2⁺ nitrogen system is analyzed. It is shown that, in relatively weak reduced electric fields typical of secondary discharge pulses, the electron impact excitation of the N₂(C³Π _u ) state from the ground state N₂(X¹Σ _g ⁺) can be accompanied by its additional step population from the N₂(B³Π _g ), N₂(a′Σ _u ⁻), and other electronic states. This effect substantially influences the rotational distribution of nitrogen molecules in the N₂(C³Π _u , ν) state; moreover, the temperature determined from this distribution can be substantially higher than the true gas temperature.     

Towards elucidating the energy of the first excited singlet state of xanthophyll cycle pigments by X-ray absorption spectroscopy

The first excited singlet state (S₁) of carotenoids (also termed 2A_g⁻) plays a key role in photosynthetic excitation energy transfer due to its close proximity to the S₁ (Q_y) level of chlorophylls. The determination of carotenoid 2A_g⁻ energies by optical techniques is difficult; transitions from the ground state (S₀, 1A_g⁻) to the 2A_g⁻ state are forbidden (“optically dark”) due to parity (g ← //→ g) as well as pseudo-parity selection rules (− ← //→ −). Of particular interest are S₁ energies of the so-called xanthophyll-cycle pigments (violaxanthin, antheraxanthin and zeaxanthin) due to their involvement in photoprotection in plants. Previous determinations of S₁ energies of violaxanthin and zeaxanthin by different spectroscopic techniques vary considerably. Here we present an alternative approach towards elucidation of the optically dark states of xanthophylls by near-edge X-ray absorption fine structure spectroscopy (NEXAFS). The indication of at least one π* energy level (about 0.5 eV below the lowest 1B_u⁺ vibronic sublevel) has been found for zeaxanthin. Present limitations and future improvements of NEXAFS to study optically dark states of carotenoids are discussed. NEXAFS combined with simultaneous optical pumping will further aid the investigation of these otherwise hardly accessible states.     

B-branch electron transfer in reaction centers of Rhodobacter sphaeroides assessed with site-directed mutagenesis

Mutants of Rhodobacter (Rba.) sphaeroides are described which were designed to study electron transfer along the so-called B-branch of reaction center (RC) cofactors. Combining the mutation L(M214)H, which results in the incorporation of a bacteriochlorophyll, β, for H_A [Kirmaier et al. (1991) Science 251: 922–927] with two mutations, G(M203)D and Y(M210)W, near B_A, we have created a double and a triple mutant with long lifetimes of the excited state P^* of the primary donor P, viz. 80 and 160 ps at room temperature, respectively. The yield of P⁺Q_A ⁻ formation in these mutants is reduced to 50 and 30%, respectively, of that in wildtype RCs. For both mutants, the quantum yield of P⁺H_B ⁻ formation was less than 10%, in contrast to the 15% B-branch electron transfer demonstrated in RCs of a similar mutant of Rba. capsulatus with a P^* lifetime of 15 ps [Heller et al. (1995) Science 269: 940–945]. We conclude that the lifetime of P^* is not a governing factor in switching to B-branch electron transfer. The direct photoreduction of the secondary quinone, Q_B, was studied with a triple mutant combining the G(M203)D, L(M214)H and A(M260)W mutations. In this triple mutant Q_A does not bind to the reaction center [Ridge et al. (1999) Photosynth Res 59: 9–26]. It is shown that B-branch electron transfer leading to P⁺Q_B ⁻ formation occurs to a minor extent at both room temperature and at cryogenic temperatures (about 3% following a saturating laser flash at 20 K). In contrast, in wildtype RCs P⁺Q_B ⁻ formation involves the A-branch and does not occur at all at cryogenic temperatures. Attempts to accumulate the P⁺Q_B ⁻ state under continuous illumination were not successful. Charge recombination of P⁺Q_B ⁻ formed by B-branch electron transfer in the new mutant is much faster (seconds) than has been previously reported for charge recombination of P⁺Q_B ⁻ trapped in wildtype RCs (10⁵ s) [Kleinfeld et al. (1984b) Biochemistry 23: 5780–5786]. This difference is discussed in light of the different binding sites for Q_B and Q_B ⁻ that recently have been found by X-ray crystallography at cryogenic temperatures [Stowell et al. (1997) Science 276: 812–816]. We present the first low-temperature absorption difference spectrum due to P⁺Q_B ⁻. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Nature of the Intramolecular Charge Transfer State in Peridinin

Experimental and theoretical evidence is presented that supports the theory that the intramolecular charge transfer (ICT) state of peridinin is an evolved state formed via excited-state bond-order reversal and solvent reorganization in polar media. The ICT state evolves in <100 fs and is characterized by a large dipole moment (∼35 D). The charge transfer character involves a shift of electron density within the polyene chain, and it does not involve participation of molecular orbitals localized in either of the β-rings. Charge is moved from the allenic side of the polyene into the furanic ring region and is accompanied by bond-order reversal in the central portion of the polyene chain. The electronic properties of the ICT state are generated via mixing of the “1¹B_u⁺” ionic state and the lowest-lying “2¹A_g^–” covalent state. The resulting ICT state is primarily ¹B_u⁺-like in character and exhibits not only a large oscillator strength but an unusually large doubly excited character. In most solvents, two populations exist in equilibrium, one with a lowest-lying ICT ionic state and a second with a lowest-lying “2¹A_g^–” covalent state. The two populations are separated by a small barrier associated with solvent relaxation and cavity formation.     

Paramagnetic 1H NMR spectroscopy of the reduced, unbound Photosystem I subunit PsaC: sequence-specific assignment of contact-shifted resonances and identification of mixed-and equal-valence Fe-Fe pairs in [4Fe-4S] centers FA − and FB −

A and F_B. The g-tensor orientation of F_A ⁻ and F_B ⁻ is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]⁺ cluster. The preferential position of the mixed-valence and equal-valence pairs, in turn, can be inferred from the study of the temperature dependence of contact-shifted resonances by ¹H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The ¹H NMR spectrum of reduced, unbound PsaC from Synechococcus sp. PCC 7002 at 280.4 K in 99% D₂O solution shows 18 hyperfine-shifted resonances. The non-solvent-exchangeable, hyperfine-shifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters F_A ⁻ and F_B ⁻ by their downfield chemical shifts, by their temperature dependencies, and by their short T ₁ relaxation times. The usual fast method of assigning the ¹H NMR spectra of reduced [4Fe-4S] proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC. Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2[4Fe-4S] cluster ferredoxin at 0.94 Å resolution as a model. The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other [4Fe-4S]-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in F_A ⁻ and F_B ⁻ have the same preferential positions relative to the protein. The analysis reveals that the magnetic properties of the two [4Fe-4S] clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex. Received: 1 February 2000 / Accepted: 20 March 2000     

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: Photooxidation of the primary electron donor

Photochemical oxidation of the primary electron donor P in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus (C.) aurantiacus was examined by light-induced Fourier transform infrared (FTIR) difference spectroscopy at 95 K in the spectral range of 4000–1200 cm⁻¹. The light-induced P⁺Q_A⁻/PQ_A IR spectrum of C. aurantiacus RCs is compared to the well-characterized FTIR difference spectrum of P photooxidation in the purple bacterium Rhodobacter (R.) sphaeroides R-26 RCs. The presence in the P⁺Q_A⁻/PQ_A FTIR spectrum of C. aurantiacus RCs of specific low-energy electronic transitions at ∼2650 and ∼2200 cm⁻¹, as well as of associated vibrational (phase-phonon) bands at 1567, 1481, and 1294–1285 cm⁻¹, indicates that the radical cation P⁺ in these RCs has dimeric structure, with the positive charge distributed between the two coupled bacteriochlorophyll a molecules. The intensity of the P⁺ absorbance band at ∼1250 nm (upon chemical oxidation of P at room temperature) in C. aurantiacus RCs is approximately 1.5 times lower than that in R. sphaeroides R-26 RCs. This fact, together with the decreased intensity of the absorbance band at ∼2650 cm⁻¹, is interpreted in terms of the weaker coupling of bacteriochlorophylls in the P⁺ dimer in C. aurantiacus compared to R. sphaeroides R-26. In accordance with the previous (pre)resonance Raman data, FTIR measurements in the carbonyl stretching region show that in C. aurantiacus RCs (i) the 13¹-keto C=O groups of P_A and P_B molecules constituting the P dimer are not involved in hydrogen bonding in either neutral or photooxidized state of P and (ii) the 3¹-acetyl C=O group of P_B forms a hydrogen bond (probably with tyrosine M187) absorbing at 1635 cm⁻¹. Differential signals at 1757(+)/1749(−) and 1741(+)/1733(−) cm⁻¹ in the FTIR spectrum of C. aurantiacus RCs are attributed to the 13³-ester C=O groups of P in different environments.     

New insights into the photochemistry of carotenoid spheroidenone in light-harvesting complex 2 from the purple bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Light-harvesting complex 2 (LH2) from the semi-aerobically grown purple phototrophic bacterium Rhodobacter sphaeroides was studied using optical (static and time-resolved) and resonance Raman spectroscopies. This antenna complex comprises bacteriochlorophyll (BChl) a and the carotenoid spheroidenone, a ketolated derivative of spheroidene. The results indicate that the spheroidenone-LH2 complex contains two spectral forms of the carotenoid: (1) a minor, “blue” form with an S₂ (1¹B _u ⁺ ) spectral origin band at 522 nm, shifted from the position in organic media simply by the high polarizability of the binding site, and (2) the major, “red” form with the origin band at 562 nm that is associated with a pool of pigments that more strongly interact with protein residues, most likely via hydrogen bonding. Application of targeted modeling of excited-state decay pathways after carotenoid excitation suggests that the high (92%) carotenoid-to-BChl energy transfer efficiency in this LH2 system, relative to LH2 complexes binding carotenoids with comparable double-bond conjugation lengths, derives mainly from resonance energy transfer from spheroidenone S₂ (1¹B _u ⁺ ) state to BChl a via the Q_x state of the latter, accounting for 60% of the total transfer. The elevated S₂ (1¹B _u ⁺ ) → Q_x transfer efficiency is apparently associated with substantially decreased energy gap (increased spectral overlap) between the virtual S₂ (1¹B _u ⁺ ) → S₀ (1¹A _g ^? ) carotenoid emission and Q_x absorption of BChl a. This reduced energetic gap is the ultimate consequence of strong carotenoid–protein interactions, including the inferred hydrogen bonding.     

Toxic effects of antimony on photosystem II of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. as probed by in vivo chlorophyll fluorescence

It has been demonstrated that antimony (Sb) at concentrations ranging from 1.0 to 10.0 mg L⁻¹ inhibits O₂ evolution. Deeper insight into the influence of Sb on PSII was obtained with measurements of in vivo chlorophyll fluorescence. The donor and the acceptor sides of PSII were shown to be the target of Sb. Sb treatment induces inhibition of electron transport from Q_A⁻ to Q_B/Q_B⁻ and accumulation of P₆₈₀⁺. S₂(Q_AQ_B)⁻ charge recombination and oxidation by PQ9 molecules became more important in Q_A⁻ reoxidation as the electron transfer in PSII was inhibited. Sb exposure caused a steady increase in the proportion of PSII_X and PSII_β. These changes resulted in increased fluxes of dissipated energy and decreased index of photosynthesis performance, of maximum quantum yield, and of the overall photosynthetic driving force of PSII.     

Adaptation of the photosynthetic apparatus of Nitellopsis obtusa to changing light intensity at the molecular level: different pathways of a singlet excitation quenching

The effect of prolonged illumination (60 min) with photosynthetically active monochromatic radiation of low intensity (3 μmol m⁻² s⁻¹) and high intensity (60 μmol m⁻² s⁻¹), corresponding to the physiological conditions and light stress conditions, respectively, was studied in the algae Nitellopsis obtusa. Illumination of Nitellopsis obtusa cells with strong light was associated with activation of the xanthophyll cycle, manifested by the deepoxidation of violaxanthin and accumulation of antheraxanthin and zeaxanthin. At the same time, the efficient singlet excitation quenching in the photosynthetic apparatus was activated, as demonstrated by the decrease in the intensity of the chlorophyll a fluorescence emission by ca 50 %. The difference of the fluorescence excitation spectra recorded before and after the light treatment match the difference absorption spectrum of the xanthophyll cycle pigments. The illumination with low light intensity resulted also in the chlorophyll a fluorescence quenching but the effect was very small (less than 10 %). The fluorescence quenching is interpreted in terms of the energy transfer between the Q_y energy level of chlorophyll a and the 2¹ A_g ⁻ energy level of zeaxanthin. The singlet energy levels of carotenoids, corresponding to the green spectral region, are also taken into consideration in the interpretation of the excitation energy exchange between the carotenoids and chlorophylls. Possible molecular mechanisms involved in the activation of the strong and the weak excitation quenching, including violaxanthin isomerization, and possible physiological functions of such pathways of energy transfer are discussed.     

Cationic state distribution over the P700 chlorophyll pair in photosystem I

The primary electron donor P700 in photosystem I is composed of two chlorophylls, P_A and P_B. P700 forms the cationic [P_A/P_B]^•+ state as a result of light-induced electron transfer. We obtained a P_A^•+/P_B^•+ ratio of 28:72 and a spin distribution of 22:78 for the entire PSI protein-pigment complex. By considering the influence of the protein components on the redox potential for one-electron oxidation of P_A/P_B monomers, we found that the following three factors significantly contributed to a large P_B^•+ population relative to P_A^•+: 1), Thr-A743 forming a H-bond with P_A; 2), P_A as a chlorophyll a epimer; and 3), a conserved PsaA/PsaB pair, the Arg-A750/Ser-B734 residue. In addition, 4), the methyl-ester groups of the accessory chlorophylls A_−1A/A_−1B significantly stabilized the cationic [P_A/P_B]^•+ state and 5), the methyl-ester group orientations were completely different in A_−1A and A_−1B as seen in the crystal structure. When the methyl-ester group was rotated, the spin-density distribution over P_A/P_B ranged from 22:78 to 15:85.     

Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins and zearalenone in the Rio de Janeiro State, Brazil

The intake of mycotoxin-contaminated feeds can lead to nutrient losses and may have adverse effects on animal health and on productivity. The aims of this study were (1) to determine the mycobiota present in poultry feed samples, and (2) to evaluate the natural occurrence of aflatoxin B₁, fumonisin B₁ and zearalenone. Fungal counts were similar between all culture media tested (10³ CFU g⁻¹). The most frequent genus isolated was Penicillium spp. (41.26%) followed by Aspergillus spp. (33.33%) and Fusarium spp. (20.63%). High precision liquid chromatography was applied to quantify aflatoxin B₁ and fumonisin B₁. Thin layer chromatography was used to determine zearalenone levels. Aflatoxin B₁ values ranged between 1.2 and 17.5 μg kg⁻¹. Fumonisin B₁ levels ranged between 1.5 and 5.5 μg g⁻¹. Zearalenone levels ranged between 0.1 and 7 μg g⁻¹. The present study shows the simultaneous occurrence of two carcinogenic mycotoxins, aflatoxin B₁ and fumonisin B₁, together with another Fusarium mycotoxin (zearalenone) in␣feed intended for poultry consumption. Many samples contained AFB₁ levels near the permissible maximum and it could affect young animals. A synergistic toxic response is possible in animals under simultaneous exposure.     

Comparative biotransformation of pentachlorophenol in soils by solid substrate cultures of Lentinula edodes

Sterilised and non-sterilised soils contaminated with pentachlorophenol (PCP) were inoculated with solid substrate cultures of Lentinula edodes LE2 (“shiitake” mushroom) to simulate monoculture bioremediation treatments and treatments in which the fungus competes with natural microflora. With monocultures of L. edodes, rates of PCP depletion were rapid for the initial 4 weeks and, although thereafter the rate decreased, 99% biotransformation was obtained in 10 weeks. In mixed culture, PCP biotransformation by L.␣edodes was markedly slower and only 42% of the PCP was depleted after 10 weeks. Maximal rates of PCP transformation, biomass (ergosterol) accumulation and oxidative enzymes (phenol oxidase and manganese-peroxidase) production were observed after 2 weeks of incubation. In monocultures, phenol oxidase activity was 195.5 U g⁻¹ and Mn-peroxidase 138.4 U g⁻¹. In mixed cultures, fungal enzyme activities were markedly lower: 70.33 U g⁻¹ for phenol oxidase and 85.0 g⁻¹ for Mn-peroxidase. Analyses of soil metabolites after 10 weeks revealed that monocultures of L.edodes had eliminated both PCP and pentachloroanisole. Pentachloroanisole, however, was detected in soils with the mixed microflora. Both dechlorination and mineralisation of the xenobiotic compound were effected by L. edodes LE2. Received: 7 April 1997 / Accepted: 14 June 1997     

Femtosecond charge separation in dry films of reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> and <Emphasis Type="Italic">Chloroflexus aurantiacus</Emphasis>

In this work, the influence of the crystallographic water on electron transfer between primary donor P and acceptor B_A was studied in reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides and the green bacterium Chloroflexus aurantiacus. For this purpose, time constants and oscillations of charge separation kinetics are compared between dry film RCs and RCs in glycerol-water buffer at 90 K. A common result of the drying of Rba. sphaeroides and Cfx. aurantiacus RCs is slowing of the charge separation process, decrease in amplitude of the oscillatory components of the kinetics, and the depletion of its spectrum. Thus, the major time constant of stimulated emission decay of P* bacteriochlorophyll dimer at 940 nm is increased from 1.1 psec for water-containing Rba. sphaeroides RCs to 1.9 psec for dry films of Rba. sphaeroides RCs. An analogous increase from 3.5 to 4.2 psec takes place in Cfx. aurantiacus RCs. In dry films of Rba. sphaeroides RCs, the amplitude of coherent oscillations of the absorption band of monomeric bacteriochlorophyll B_A⁻ at 1020 nm is 1.8 times less for the 130-cm⁻¹ component and 2.3 times less for the 32-cm⁻¹ component than the analogous amplitudes for water-containing RCs. Measurements in the analogous band of Cfx. aurantiacus RCs show that strong decrease (∼5-10 times) of the B_A⁻ absorption band and strong slowing (from ∼0.8 to ∼3 psec) of B_A⁻ accumulation together with ∼3-fold decrease in oscillation amplitude occurs on drying of these RCs. The overtones of the 32-cm⁻¹ component disappeared from the oscillations of the kinetics at 940 and 1020–1028 nm after drying of the Rba. sphaeroides and Cfx. aurantiacus RCs. The results are in agreement with the results for GM203L mutant of Rba. sphaeroides, in which the HOH55 water molecule is sterically removed, and with the results for dry films of pheophytin-modified RCs of Rba. sphaeroides R-26 and for YM210W and YM210L Rba. sphaeroides mutant RCs. The data are discussed in terms of the influence (or participation) of the HOH55 water molecule on electron transfer along the chain of polar atomic groups N-Mg(P_B)-N-C-N(HisM202)-HOH55-O=(B_A) connecting P_B and B_A in Rba. sphaeroides RCs.     

Charge separation in photosynthesis via a spin exchange coupling mechanism

A new mechanism for the primary photoinduced charge separation in photosynthesis is proposed. It involves as a real intermediate between the excited special pair state P^* and the primary charge separated state P⁺ H_L ^– a trip-trip-singlet B^T B_L ^T, which consists of a triplet on the dimer P and a further triplet on the monomer B_L. Both combine to a singlet. The electron transfer is caused by spin exchange couplings. The transient spectrum of the short lived intermediate, formerly taken as evidence for the charge transfer state P⁺ B_L ^–, is reinterpreted as a transient excitation of this trip-trip singlet. Received: 2 June 1997 / Accepted: 18 July 1997     

Colonization of contaminated soil by an introduced bacterium: effects of initial pentachlorophenol levels on the survival of Sphingomonas chlorophenolica strain RA2

The survival of a Sphingomonas species that was introduced into pentachlorophenol (PCP)-contaminated soil was monitored with two complementary methods, a respiration-based assay and a most probable number (MPN) technique. Sphingomonas chlorophenolicastrain RA2 is a PCP-mineralizing bacterium that was introduced into soil contaminated with a range of PCP concentrations (0–300 μg PCP g⁻¹ soil). The population of introduced microorganisms was followed for 170 days using a substrate-induced growth-response method and a MPN assay that specifically targets PCP-mineralizing bacteria. Varying the initial PCP concentration resulted in the emergence of three distinct patterns of survival. In soil contaminated with 300 μg PCP g⁻¹ the population of S. chlorophenolica strain RA2 immediately declined following introduction, increased by 200-fold and leveled off by the end of the 170-day incubation. In contrast, populations of S. chlorophenolica strain RA2 declined to levels below detection limits in uncontaminated soil by the end of the experiment. Intermediate PCP concentrations (10–100 μg PCP g⁻¹ soil) resulted in the establishment of S. chlorophenolica strain RA2 that slowly declined in numbers. These results indicate that Sphingomonas chlorophenolica strain RA2 is an effective colonizer of PCP-contaminated soil but will not persist in the absence of PCP. Received 14 April 1999/ Accepted in revised form 24 June 1999