Juan Feng

Radical Cation Generation from Singlet and Triplet Excited States of All-trans-Lycopene in Chloroform¶

Abstract On direct photoexcitation, subpicosecond time-resolved absorption spectroscopy revealed that the 1Bu-type singlet excited state of all-trans-lycopene in chloroform was about seven times more efficient than all-trans-β-carotene in generating the radical cation. The time constant of radical cation generation from the 1Bu-type state was found to be ∼0.14 ps, a value that was comparable for the two carotenoids. On anthracene-sensitized triplet excitation, radical cation generation was found to be much less efficient for lycopene than for β-carotene. A slow rising phase (20–30 μs) in the bleaching of ground-state absorption was common for both lycopene and β-carotene in chloroform and was ascribed to an efficient secondary reaction with a solvent radical leading to the formation of carotenoid radical cations. The reverse ordering in the tendency of the excited states of different multiplicities for the two carotenoids to generate radical cations is discussed in relation to the two carotenoids as scavengers of free radicals.

Triplet excitation transfer between carotenoids in the LH2 complex from photosynthetic bacterium Rhodopseudomonas palustris

We have studied, by means of sub-microsecond time-resolved absorption spectroscopy, the triplet-excited state dynamics of carotenoids (Cars) in the intermediate-light adapted LH2 complex (ML–LH2) from Rhodopseudomonas palustris containing Cars with different numbers of conjugated double bonds. Following pulsed photo-excitation at 590 nm at room temperature, rapid spectral equilibration was observed either as a red shift of the isosbestic wavelength on a time scale of 0.6–1.0 μs, or as a fast decay in the shorter-wavelength side of the Tn←T1 absorption of Cars with a time constant of 0.5–0.8 μs. Two major spectral components assignable to Cars with 11 and 12 conjugated double bonds were identified. The equilibration was not observed in the ML–LH2 at 77 K, or in the LH2 complex from Rhodobacter sphaeroides G1C containing a single type of Car. The unique spectral equilibration was ascribed to temperature-dependent triplet excitation transfer among different Car compositions. The results suggest that Cars of 11 and 12 conjugated bonds, both in close proximity of BChls, may coexist in an α,β-subunit of the ML–LH2 complex.

Spectroscopic study on the photophysical properties of chlorine substituted tetraphenylporphyrin-histidine and its zinc (II) complexes

The photophysical properties ofortho- CI,meta-CI andpara-CI substituted tetraphenylporphyrin-histidine and their zinc (II) complexes have been studied by means of steady-state absorption and fluorescence spectroscopies, as well as time-resolved fluorescence spectroscopy. For the cases of both free-base and zinc complexes, it was found that theortho-chlorine substitution onto the phenyl rings significantly altered the fluorescence quantum yield, the fluorescence lifetime and the ratio between radiative and nonradiative deactivation rates of the porphyrin chromophore, i.e. the photophysical parameters were quite different from those ofmeta- andpara-substituted compounds. On the other hand, however, the introduction of covalently-linked histidine did not exert much effects on the photophysical behavior of the porphyrin chromophore. The results are interpreted in terms of the steric effect and the heavy-atom effect from the chlorine atoms substituted onto the phenyl rings.

Cross-linked enzyme aggregates (CLEAs) of halohydrin dehalogenase from Agrobacterium radiobacter AD1: Preparation, characterization and application as a biocatalyst

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential to produce valuable optically pure epoxides and β-substituted alcohols. However, this enzyme has been reported to be very sensitive and less stable under oxidative conditions. Enzyme immobilization represents a powerful means to overcome this limitation and provides the enzyme characteristics of a biocatalyst. In this study, the crude extract of HheC was directly subjected to enzyme immobilization using a carrier-free cross-linked enzyme aggregates (CLEAs) method. The results showed that under the optimized conditions, the obtained HheCCLEAs retained more than 90% activity of the free enzyme; preserved more than 50% of their original activity after storage at 4 °C for 2 months, even in the absence of a reducing agent; displayed a strong tolerance to organic solvents with fully active after incubation in the presence of 50% cyclohexane and n-hexane for 5 h; the presence of organic solvents could minimize the negative effect of enzyme immobilization on the enzntioselectivity of HheC. Most importantly, HheCCLEAs maintained more than 70% activity after 10 reusability cycles. The utility of HheCCLEAs as a valuable biocatalyst was exhibited by the kinetic resolution of azide-mediated ring-opening reaction of rac-1,2-epoxy-2-methylbutane. These results indicated that HheCCLEAs overcame some disadvantages of free enzymes to become biocatalysts. Together with further engineering of the enzyme, HheCCLEAs could become a promising biocatalyst for the synthesis of valuable chiral compounds.

Ultrafast spectroscopy studies on the mechanism of electron transfer and energy conversion in the isolated pseudo ginseng, water hyacinth and spinach chloroplasts

The spectroscopy characteristics and the fluorescence lifetime for the chloroplasts isolated from the pseudo ginseng, water hyacinth and spinach plant leaves have been studied by absorption spectra, low temperature steady-state fluorescence spectroscopy and single photon counting measurement under the same conditions and by the same methods. The similarity of the absorption spectra for the chloroplasts at room temperature suggests that different plants can efficiently absorb light of the same wavelength. The fluorescence decays in PS II measured at the natural QA state for the chloroplasts have been fitted by a three-exponential kinetic model. The three fluorescence lifetimes are 30, 274 and 805 ps for the pseudo ginseng chloroplast; 138, 521 and 1494 ps for the water hyacinth chloroplast; 197, 465 and 1459 ps for the spinach chloroplast, respectively. The slow lifetime fluorescence component is assigned to a collection of associated light harvesting Chl a/b proteins, the fast lifetime component to the reaction center of PS II and the middle lifetime component to the delay fluorescence of recombination of P+ 680 and Pheo-. The excitation energy conversion efficiency(η) in PS II RC is defined and calculated on the basis of the 20 ps electron transfer time constant model, 60%, 87% and 91% for the pseudo ginseng, water hyacinth and spinach chloroplasts, respectively. This interesting result is in unconformity with what is assumed to be 100% efficiency in PS II RC. Our result in this work stands in line with the 20 ps electron transfer time constant in PS II rather sound and the water hyacinth plant grows slower than the spinach plant does as envisaged on the efficiency. But, our results predict that those plants can perform highly efficient transfer of photo-excitation energy from the light-harvesting pigment system to the reaction center (closely to 100%). The conclusion contained in this paper reveals the plant growth characteristics expressed in the primary processes of photosynthesis and a relationship between a plant growing rate and its spectroscopy characteristics and fluorescence lifetimes, namely, the slower a plant grows, the less excitation energy conversation efficiency used might be anticipated.

Trans-membrane controlling of an amphiphilic metalloporphyrin

In order to perform a transfer process of the porphyrin moiety across membrane media, an amphiphilic porphyrin 5,10,15-tri(4-hydroxyphenyl)-20-(4-hexadecyloxy-phenyl) porphyrin (P1) and its metalloporphyrin (P1Zn) were selected to be solubilized in cetyltrimethyl ammonium bromide (CTAB) micellar solutions. By taking advantage of the microenvironment sensitivity of their Soret band, UV-Vis spectra were used to study the dependence of location of the porphyrins in CTAB micellar media on the bulk pH value or the salt concentration of bulk solutions. The red shift of the Soret band, the decrease of its absorbance and the increase of the full width at half-maximum (FWHM) of P1Zn and P1 with pH titration process from neutral to weak basicity were attributed to the transfer process of the porphyrin moiety from the inner core to the outer surface of the micelles. In the pH sensitive area of the Soret band of P1Zn and P1, the blue shift of the Soret band, the increase of its absorbance and decrease of FWHM were observed on decreasing the bulk pH value or increasing the salt bulk solution concentration, indicating that the porphyrin moiety was pushed into the inner core again. Therefore, we can control the trans-membrane transfer process of porphyrin moiety by changing the pH value or the salt concentration of the bulk solutions.

Interaction between flavin mononucleotide-containing azoreductase and azo dyes

ABSTRACT Azoreductase, a flavin mononucleotide-containing oxidoreductase from Escherichia coli, can catalyze the reduction of azo dyes to form aromatic amine compounds. Few spectroscopic studies have explored the binding mode of azo dyes or the role of the arginine at site 59 in Azoreductase. In this article, protein engineering strategy has been used to construct one mutant in which the arginine residue at site 59 was mutated to glycine. Fluorescence spectroscopic data showed that the addition of Methyl Red and Methyl Orange resulted in the fluorescence quenching of the cofactor flavin mononucleotide bound to Azoreductase. The association constant was fitted using the standard binding equation instead of the Stern-Volmer equation. The results showed that the mutation from the arginine to glycine at site 59 weakened the association constant from 2.21 × 105 L.mol−1 to 4.55 × 104 L.mol−1 at 25°C. A similar phenomenon was also observed when Methyl Orange was used as a substrate. In each case, the association constant tended to decrease as the temperature increased from 25°C to 37°C. Thermodynamic parameter analysis revealed that the interaction type changed from a van der Waals interaction (between Azoreductase and the dyes) to a hydrogen bonding interaction (between the mutant and the dyes). Moleculcar docking was also performed in this work to provide some support for the binding mode and binding stability between Azoreductase/mutant and azo dyes.

Role of the CPC Sequence in the Antioxidant Activity of GcGAST Protein in E.coli

Gibberellic acid stimulated transcriptional protein from Gymnadenia conopsea (GcGAST) is a novel member of GA-induced cysteine-rich protein family, which shared 12 highly conserved cysteine residues with other members in C-terminal domain. In the present paper, the recombinant plasmid, as well as two mutants Serine–Proline–Cysteine (SPC) and Cysteine–Proline–Serine (CPS), were constructed to investigate for the first time the effects of the cysteines in Cysteine–Proline–Cysteine (CPC) sequence on the antioxidant activity of GcGAST protein. It was found that E.coli expressing wt GcGAST exhibited significant resistance against exogenous H2O2. Similar phenomenon was observed for E.coli harboring SPC mutant. In contrast, the host cell overexpressing CPS mutant became more sensitive to H2O2. Some studies on the level of inclusion body revealed that wt GcGAST and SPC mutant embedded in Inclusion bodies (IB) could effectively eliminate H2O2, whereas the mutagenesis to Ser of the second Cys residue in CPC sequence gave rise to the compete loss of H2O2-eliminating ability. Fourier transform Infrared spectroscopy analysis indicated that the IB of CPS mutant contained more β-sheet secondary structure than wt and SPC mutant. Non-reducing SDS-PAGE combined western-blotting analysis revealed that the disulfide bonds were important for the formation of IBs of wt GcGAST and SPC mutant, whereas non-reducing SDS-PAGE of resolubilized IBs showed that hydrophobic interaction favored the aggregation of IBs in CPS mutant. Taken together, these results suggested that GcGAST possessed antioxidant activity in the level of IB, which made some contribution to cellular resistance to H2O2. More importantly, the second cysteine residue in CPC sequence was more essential for its antioxidant biological function.

Effects of pH on the peripheral light-harvesting antenna complex for Rhodopseudomonas palustris

In this work steady-state absorption spectroscopy, circular dichroism spectroscopy and sub-microsecond time-resolved absorption spectroscopy were used to investigate the effect of pH on the structures and functions of LH2 complex for Rhodopseudomonas palustris. The results revealed that: (1) B800 Bchla was gradually transformed to free pigments absorbing around 760 nm on the minutes timescale upon the induction of strong acidic pH, and subsequently there disappeared the CD signal for Qy band of B800 in the absence of B800. In addition, Carotenoids changed with the similar tendency to B850 BChl. (2) The introduction of strong basic pH gave rise to no significant changes for B800 Bchla, while B850 BChla experienced remarkable spectral blue-shift from 852 to 837 nm. Similar phenomenon was seen for the CD signal for Qy band of B850. Carotenoids displayed strong and pH-independent CD signals in the visible range. (3) In the case of both physiological and basic pH, broad and asymmetrical positive Tn ← T1 transient absorption appeared following the pulsed photo-excitation of Car at 532 nm. By contrast, the featureless and weak positive signal was observed on the sub-microsecond timescale in the acidic pH environment. The aforementioned experimental results indicated that acidic pH-induced removal of B800 Bchla prevented the generation of the carotenoid triplet state (3Car*), which is known to be essential for the photo-protection function. Nevertheless, carotenoids can still perform this important physiological role under the basic pH condition, where the spectral blue shift of B850 exerts little effect on the overall structure of the cyclic aggregate, therefore favoring the formation of carotenoid triplet state.