Photoinduced double proton transfer in a model hydrogen bonded base pair. Theoretical study

A theoretical study of the 7-azaindole dimer confirms the photoinduced biprotonic transfer mechanism in the first ($\text{π, π}{}^1$) excited singlet state. Emission is forbidden from the first excited singlet state (A_g) of the normal form and it is allowed from the same state (B_u) of a tautomeric form. Proton transfer depends very strongly on the distance between the two monomer molecules, and extension of this mechanism to explain U.V. induced mutation in DNA is conditioned to the possibility of its basis attaining the optimal separation.     

An anomalous distance dependence of intraprotein chlorophyll-carotenoid triplet energy transfer

In the light-harvesting chlorophyll pigment-proteins of photosynthesis, a carotenoid is typically positioned within a distance of ~4 Å of individual chlorophylls or antenna arrays, allowing rapid triplet energy transfer from chlorophyll to the carotenoid. This triplet energy transfer prevents the formation of toxic singlet oxygen. In the cytochrome b₆f complex of oxygenic photosynthesis that contains a single chlorophyll a molecule, this chlorophyll is distant (14 Å) from the single β-carotene, as defined by x-ray structures from both a cyanobacterium and a green alga. Despite this separation, rapid (<8 ns) long-range triplet energy transfer from the chlorophyll a to β-carotene is documented in this study, in seeming violation of the existing theory for the distance dependence of such transfer. We infer that a third molecule, possibly oxygen trapped in an intraprotein channel connecting the chlorophyll a and β-carotene, can serve as a mediator in chlorophyll-carotenoid triplet energy transfer in the b₆f complex.     

Ethidium bromide–DNA complex: Wavelength dependence of pyrimidine dimer inhibition and sensitized fluorescence as probes of excited states

Ethidium bromide inhibits the formation of ultraviolet-induced pyrimidine dimers in DNA. The efficiency of dimer inhibition increases with increasing energy of the exciting photons. The efficiency of energy transfer from the DNA singlet to the dye singlet, as monitored by sensitized fluorescence, is independent of wavelength. The efficiency of singlet–singlet transfer agrees with that for dimer inhibition at photon energies corresponding to excitation of the lowest singlet state of DNA. Our results support a model in which dimers are formed both directly from the singlet state and also from the triplet state, with triplets arising from higher vibrational levels of the singlet.     

Singlet oxygen quenchers and the photodynamic inactivation of E. coli ribosomes by methylene blue

$\text{E. coli}$ ribosomes are readily photoinactivated by methylene blue in the presence of air. A variety of singlet oxygen quenchers like NaN₃, 2,5-dimethylfuran, hydroquinone and ascorbic acid provide about 60% protection against this photoinactivation indicating that a major mechanism of ribosome inactivation proceeds through the formation of singlet oxygen, with small contributions (<40%) from other mechanisms. The singlet oxygen quenchers, 1,4-diazabicyclo [2.2.2] octane and triethylamine give unexpected results, in that they show no protection against photoinactivation.     

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     

Triplet states of bacteriochlorophyll and carotenoids in chromatophores of photosynthetic bacteria

Chromatophores from photosynthetic bacteria were excited with flashes lasting approx. 15 ns. Transient optical absorbance changes not associated with the photochemical electron-transfer reactions were interpreted as reflecting the conversion of bacteriochlorophyll or carotenoids into triplet states. Triplet states of various carotenoids were detected in five strains of bacteria; triplet states of bacteriochlorophyll, in two strains that lack carotenoids. Triplet states of antenna pigments could be distinguished from those of pigments specifically associated with the photochemical reaction centers. Antenna pigments were converted into their triplet states if the photochemical apparatus was oversaturated with light, if the primary photochemical reaction was blocked by prior chemical oxidation of P-870 or reduction of the primary electron acceptor, or if the bacteria were genetically devoid of reaction centers. Only the reduction of the electron acceptor appeared to lead to the formation of triplet states in the reaction centers.In the antenna bacteriochlorophyll, triplet states probably arise from excited singlet states by intersystem crossing. The antenna carotenoid triplets probably are formed by energy transfer from triplet antenna bacteriochlorophyll. The energy transfer process has a half time of approx. 20 ns, and is about 1 × 10³ times more rapid than the reaction of the bacteriochlorophyll triplet states with O₂. This is consistent with a role of carotenoids in preventing the formation of singlet O₂ in vivo. In the absence of carotenoids and O₂, the decay half times of the triplet states are 70 μs for the antenna bacteriochlorophyll and 6–10 μs for the reaction center bacteriochlorophyll. The carotenoid triplets decay with half times of 2–8 μs.With weak flashes, the quantum yields of the antenna triplet states are in the order of 0.02. The quantum yields decline severely after approximately one triplet state is formed per photosynthetic unit, so that even extremely strong flashes convert only a very small fraction of the antenna pigments into triplet states. The yield of fluorescence from the antenna bacteriochlorophyll declines similarly. These observations can be explained by the proposal that singlet-triplet fusion causes rapid quenching of excited singlet states in the antenna bacteriochlorophyll.     

DNA strand scission in E.coli by electronically excited state molecules generated by enzymatic systems

$\text{E.}\text{coli}$ containing pAT 153 plasmid undergoes strand scission when exposed to the indole-3-acetic acid/peroxidase/D₂ system. Neither the initial components of this reaction nor the final stable products are responsible for this effect. Indole-3-aldehyde in its triplet state and singlet oxygen have been recently identified in this system. That singlet oxygen is one of the species acting on the plasmid in $\text{E.}\text{coli}$ cells was suggested by protective effect of histidine and guanosine which are singlet oxygen quenchers. Similar effect on plasmid with malonaldehyde/peroxidase/O₂ system was observed, which is an excellent singlet oxygen generator. This is the first report of a biological system where it is possible to detect a DNA scission in the intact cell by a bioenergized process. This presumably is related to spontaneous mutagenesis.     

Electric-field dependence of the quantum yield in reaction centers of photosynthetic bacteria

Multilayer Langmuir-Blodgett films of reaction centers from the photosynthetic bacterium Rhodopseudomonas sphaeroides have been fabricated with partial net orientation. The films showed substantial electrical response under pulsed illumination. From measurements of the light-induced voltage generated across the Langmuir-Blodgett film, we have succeeded in quantitating the electric-field dependence of the quantum yield of charge separation in photosynthesis. The results presented here are compared with our previous determination of the field effect on quantum yield, in which flash-activated charge separation as a function of the applied field was assayed by the extent of bacteriochlorophyll dimer, (BChl)₂, oxidation measured optically at 860 nm. The two methods provided consistent dependencies of quantum yield on applied electric field. Analysis of the data reveals that the quantum yield of (BChl)^⨥₂BPhQ^⨪_A formation decreases from a value of 0.96 at zero applied field to about 0.75 for a field of 120 mV/nm vectorially directed to hinder light-activated electron transfer. For oppositely applied fields, the quantum yield saturates at unity. The source of the effects is considered to reside in the electric field dependence of the free-energy difference between the energy levels that are involved in the initial charge separation between the (BChl)₂ in the first singlet excited state, (BChl)1₂, through the bacteriopheophytin, BPh, to the primary ubiquinone, Q_A. Possible contributions to the field-induced loss of quantum yield of (BChl)^⨥₂BPhQ^⨪_A formation are: (1) a decrease in the free-energy gap between the states (BChl)1₂ and (BChl)^⨥₂BPh^⨪Q_A, leading to an increased rate of decay via the excited singlet state back to the ground state; (2) a stimulated return from (BChl)^⨥₂BPh^⨪Q_A directly or via the (BChl)₂ triplet state to the ground state and (3) an impeded electron transfer from (BChl)^⨥₂BPh^⨪Q_A to (BChl)^⨥₂BPhQ^⨪_A. These possibilities are discussed. Correlation of the electrical response with measurements of the photo-induced absorbance change allows determination of the projection of the electron-transfer distance on the normal to the plane of the film, which is in good agreement with previous measurements using different techniques.     

Singlet and triplet excited carotenoid and antenna bacteriochlorophyll of the photosynthetic purple bacterium Rhodospirillum rubrum as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation pulse and kinetics of absorbance changes were measured in chromatophores of the photosynthetic purple bacterium Rhodospirillum rubrum after chemical oxidation of the primary electron donor P-875. Kinetics and spectra were measured of the excited singlet states of carotenoid and bacteriochlorophyll a and also of the triplet state of the carotenoid. The excited singlet state of carotenoid, produced by direct excitation at 532 nm, is characterized by a bleaching of the ground state absorption bands in the region 450–490 nm and by an absorbance increase with a maximum near 570 nm. Its lifetime was calculated to be 0.6 ± 0.1 ps in vitro and less than 1 ps in vivo. The triplet state of carotenoid in vivo is formed within 100 ps after direct carotenoid excitation via a pathway that does not involve excited states of bacteriochlorophyll. Singlet excitation of a bacteriochlorophyll a molecule causes the bleaching of its Q_x and Q_y absorption bands, and is probably associated with blue shifts of the Q_y absorption band of about six neighboring bacteriochlorophyll molecules. Upon increasing the excitation density, the average lifetime of the singlet excitations on bacteriochlorophyll decreased from about 350 ps to about 10 ps or less. The results are in quantitative agreement with the known effect of singlet-singlet annihilation upon the fluorescence yield, and furthermore show that no bacteriochlorophyll or carotenoid triplet formation is associated with this annihilation.     

Energy transfer in polynucleotide homopolymers and copolymers

Using the fluorophore Tb³⁺ as a reporter, the effect of thallium (Tl⁺¹) on the transfer of energy in polyribonucleotides and polydeoxynucleotides at room temperature has been studied. In p(G), p(G, I), and pd(G)₆ thallium greatly increases the transfer of uv energy absorbed by the bases to Tb³⁺. In DNA, p(G, U), p(G, A), p(A, U), p(X), p(A), p(U), p(I), pd(G, A)₆, and pd(G, T)₅ thallium has little or no effect. Thallium increases intersystem crossing to the triplet states only in p(G), p(G, I), and pd(G)₆, and the triplets overlap the excited singlet state in Tb³⁺. In those G- and X-containing polymers showing little thallium effect, the evidence suggests that intersystem crossing is comparatively high to begin with. These polymers, including DNA, appear to transfer absorbed energy to triplet states efficiently at room temperature.     

Energy upconversion theory of the primary photochemical reaction in plant photosynthesis

In this paper we suggest a basic mechanism for the utilization of light quanta in photosynthesis. Through interactions between the lowest lying triplet state of the reaction-center chlorophylls and the first excited singlet state of the antenna chlorophylls, absorbed light quanta are upconverted to a higher-lying charge transfer state of the reaction-center Chl molecules. It is shown that the efficiency of the upconversion process is maximized by the parallel configuration of the two Chl porphyrin rings in the reaction-center water adduct proposed by the writer. Steady-state solutions are obtained, and the theoretical results are shown to account for a variety of crucial experimental observations including (1) the doubling (in whole cells) of in vivo fluorescence quantum yield of system II in strong light, (2) the observation by Dutton et al. of the light-induced triplet-state reaction-center bacteriochlorophyll when the primary electron acceptor is reduced and (3) despite the apparent involvement of two excitations in the energy upconversion process, only one quantum is needed for the transfer of one electron in the primary photo-chemical reaction, satisfying the eight-quanta requirement for the evolution of one O₂ molecule in photosynthesis.     

Formation of Schiff-base for photoreaction mechanism of red shift of GFP spectra

We have proposed the formation of Schiff-base between R96 and chromophore (CRO) to elucidate the reaction mechanism for the irreversible red shift of green fluorescent protein (GFP) spectra under the absence of oxygen. The difference between absorption energies of reactant and product for our GFP models with CIS(D)/6-31G* level is 0.21 eV, which is in reasonable agreement with the corresponding experimental value of 0.25 eV. We have suggested the irreversible photoreaction mechanism, where the CRO excited from ground (S₀) state to first excited singlet (S₁) state immediately turns to the first excited triplet (T₁) state, and the nucleophilic addition reaction occurs on the T₁ state.     

Light-induced binding of riboflavin to lysozyme

Summary The photodynamic inactivation of lysozyme in air saturated H₂O and D₂O (phosphate buffer 0.05 M, pH 7.0) in the presence of methylene blue and riboflavin has been studied. When H₂O was replaced by D₂O a great increase in the rate of photoinactivation of lysozyme was observed. This finding, together with the fact that photooxidation is inhibited by singlet oxygen quenchers like NaN₃, suggests that these reactions occur via a singlet oxygen mechanism.During the course of the studies of the riboflavin sensitized photoinactivation of lysozyme, it was found that riboflavin is strongly bound to the enzyme as a result of illumination. This finding would explain the higher quantum yield observed when riboflavin is used, although this dye is bleached during irradiation.     

Picosecond and microsecond pulse laser studies of exciton quenching and exciton distribution in spinach chloroplasts at low temperatures

Studies of the fluorescence quantum yield and decay times, determined at the emission maxima of 685 and 735 nm, using picosecond laser pulses for excitation, indicate that the pigments which are responsible for the 735 nm emission derive their energy by transfer of singlet excitons from the light-harvesting pigments and not by direct absorption of photons. Microsecond pulse laser studies of the fluorescence quantum yields at these two fluorescence wavelengths indicate that long lived quenchers (most probably triplet states), which quench singlet excitons, accumulate preferentially within the long wavelength pigment system which gives rise to the 735 nm emission band.     

Lipid and lipoprotein oxidation: basic mechanisms and unresolved questions in vivo

Abstract

The kinetics and mechanistic aspects of the riboflavin-photosensitised oxidation of the topically administrable ophthalmic drugs Timolol (Tim) and Pindolol (Pin) were investigated in water–MeOH (9:1, v/v) solution employing light of wavelength > 400 nm. riboflavin, belonging to the vitamin B₂ complex, is a known human endogenous photosensitiser. The irradiation of riboflavin in the presence of ophthalmic drugs triggers a complex picture of competitive reactions which produces the photodegradation of both the drugs and the pigment itself. The mechanism was elucidated employing stationary photolysis, polarographic detection of dissolved oxygen, stationary and time-resolved fluorescence spectroscopy, and laser flash photolysis. Ophthalmic drugs quench riboflavin-excited singlet and triplet states. From the quenching of excited triplet riboflavin, the semireduced form of the pigment is generated, through an electron transfer process from the drug, with the subsequent production of superoxide anion radical (O₂^?–) by reaction with dissolved molecular oxygen. Through the interaction of dissolved oxygen with excited triplet riboflavin, the species singlet oxygen (O₂(¹Δ_g)) is also generated to a lesser extent. Both O₂^?– and O₂(¹Δ_g) induce photodegradation of ophthalmic drugs, Tim being ~3-fold more easily photooxidisable than Pin, as estimated by oxygen consumption experiments.     

Excited states and primary charge separation in the pigment system of the green photosynthetic bacterium Prosthecochloris aestuarii as studied by picosecond absorbance difference spectroscopy

Picosecond absorbance difference spectra at a number of delay times after a 35 ps excitation flash and kinetics of absorbance changes were measured of the membrane vesicle preparation Complex I from the photosynthetic green sulfur bacterium Prosthecochloris aestuarii. After chemical oxidation of the primary donor the excitation pulse produced singlet and triplet excited states of carotenoid and bacteriochlorophyll a. With active reaction centers present also the flash-induced primary charge separation and subsequent electron transfer were observed. The singlet excited state of the carotenoid, formed by direct excitation at 532 nm, is characterized by an absorbance band peaking at 590 nm. Its average lifetime was calculated to be about 1 ps. Excited singlet states of bacteriochlorophyll a were characterized by a bleaching of their ground state Q_y absorption bands. Singlet excited states, localized on the so-called core complex, were produced by energy transfer from excited carotenoid. Their lifetime was about 70 ps. A decay component of about 280 ps was ascribed to singlet excited bacteriochlorophyll a in the bacteriochlorophyll a protein. These singlet excitations were partly converted to the triplet state. With active reaction centers, oxidation of the primary donor, P-840, characterized by the bleaching of its Q_y and Q_x absorption bands, was observed. This oxidation was accompanied by a bleaching between 650 and 680 nm and an absorbance increase between 680 and 750 nm. These changes, presumably due to reduction of bacteriopheophytin c (Van Bochove, A.C., Swarthoff, T., Kingma, H., Hof, R.M., Van Grondelle, R., Duysens, L.N.M. and Amesz, J. (1984) Biochim. Biophys. Acta 764, 343–346), were attributed to the reduction of the primary electron acceptor. Electron transfer to a secondary acceptor occurred with a time-constant of 550 ± 50 ps. Since no absorbance changes due to reduction of this acceptor were observed in the red or infrared region, we tentatively assume that this acceptor is an iron-sulfur center.     

Trapping of a reaction intermediate by tetranitromethane during catalysis by ribulose biphosphate carboxylase

Illumination at 230 K of dithionite-reduced particles results in the appearance of an EPR detectable radical 13 G wide with g = 2.0033. This radical is formed in a ratio of 2.28 (±0.5)/P700. Investigation of the time course of formation shows two components are present. One (A₁) has g = 2.0051 and the other (A_og= 2.0024. Reduction of A₁ results in an increase in reaction centre triplet formation, subsequent reduction of A_o results in a decrease of triplet formation to the base level. We propose that these components function sequentially in the transfer of electrons from P700 to the iron—sulphur acceptors.