Enols of aldehydes in the peroxidase/oxidase-promoted generation of excited triplet species

General (acid and base) or specific (fluoride ion) catalysis generates the enol of isobutanal and propanal from the corresponding trimethylsilyl enol ethers. The enols are directly rapidly oxidized by peroxidase (acting as an oxidase) to triplet acetone or triplet acetaldehyde, respectively, and formic acid. Due to the faster rate of reaction and the absence of quenching by excess aldehyde, the excited carbonyl emits more strongly than when the aldehyde itself is the substrate. With both enols the emission is pure phosphorescence. Both triplet acetone and triplet acetaldehyde are generated within the enzyme, as shown by the different quenching by D- and L-tryptophan, and are somewhat protected from oxygen quenching, as attested by the very fact that phosphorescence is observed. The use of enol precursors as substrates opens wide possibilities for photochemical investigations in the absence of light over a much broader range of experimental conditions.     

Peroxidase-promoted aerobic oxidation of 2-nitropropane: mechanism of excited state formation

Using sensitized emission, the horseradish peroxidase-catalyzed aerobic oxidation of the toxic pollutant 2-nitropropane to nitrite and acetone is shown to produce the latter in the electronically excited triplet state. In turn, this chemiexcitation implies a hydroperoxide precursor. Taking into account the stoichiometry of the reaction and available isotopic data it is inferred that the hydroperoxide reacts with a second molecule of the substrate (aci form). While triplet acetone formed from isobutanal (enol form) is generated within the enzyme, in the present case triplet acetone is formed in the bulk solution.     

Peroxidase-catalyzed formation of triplet acetone and chemiluminescence from isobutyraldehyde and molecular oxygen

It has been established that the horseradish peroxidase/O2/isobutyraldehyde (IBAL) system leads to triplet acetone and formic acid formation followed by phosphorescence of the triplet acetone (see, for example, Bechara, E.J.H., Faria Oliveira, O.M.M., Durán, N., Casadei de Baptista, R., and Cilento, G. (1979) Photochem. Photobiol. 30, 101-110). In this paper many of the mechanistic details are established. The reaction is initiated by the autoxidation of IBAL to form the peracid (CH3)2CHC = O(OOH). The peracid converts horseradish peroxidase into compound I which in turn is converted into compound II by abstracting the alcoholic hydrogen atom from the enol form of IBAL. This creates a free radical with two resonance forms. (Formula: see text) Addition of molecular oxygen to the latter resonance form creates a peroxy radical which abstracts a hydrogen atom near the active site of the enzyme. The newly formed alpha-peroxide in turn forms a dioxetane-type of intermediate which rapidly decomposes into triplet acetone and formic acid. Compound II reacts with the enol by the same pathway as compound I. Thus native horseradish peroxidase is regenerated. The hydrogen atom abstraction near the enzyme active site may occur directly from ethanol, present to solubilize IBAL or from a group on the enzyme, in which case ethanol participates in a repair mechanism. Phosphate buffer is necessary because it catalyzes the keto-enol conversion of IBAL. Thus horseradish peroxidase participates in a normal peroxidatic cycle. The only chain reaction is the uncatalyzed autoxidation of IBAL, most of which occurs prior to the mixing of IBAL with the oxygenated horseradish peroxidase solution.     

Characterization of the transient species generated by the photoexcitation of C-phycocyanin from Spirulina platensis: a laser photolysis and pulse radiolysis study

Nanosecond laser flash photolysis and pulse radiolysis were used to generate and characterize the triplet state and cation radical of C-phycocyanin (C-PC) from Spirulina platensis. The transient absorption spectra of C-PC were measured from direct excitation and acetone sensitization in aqueous solution at room temperature by KrF (248 nm) laser flash photolysis. Laser-induced transient species have been characterized by the method of acetone sensitization and one-electron oxidation. In nitrous oxide-saturated phosphate buffer saline (pH = 7.0) of C-PC, the produced intermediates are assigned to the excited triplet state and the radical cation. Using acetone as photosensitizer, the C-PC excited triplet states produced via triplet-triplet energy transfer and the C-PC radical cation from electron transfer reaction were further confirmed. Furthermore, the corresponding kinetic parameters were determined. To our knowledge, the transient absorption spectra of C-PC have been reported for the first time.     

Enzymatic generation of triplet acetone by deglycosylated horseradish peroxidase

A study of the effects of deglycosylation of horseradish peroxidase on protein conformation, as well as on its catalytic activity of oxidation of isobutyraldehyde or its enol form to triplet acetone and formic acid, was performed. The loss of carbohydrates leads to structural modifications of this enzyme. This is confirmed by a change in the circular dichroism spectrum, an increase in tryptophan's environment polarity, and a loss of the chiral specificity toward D- and L-tryptophan. Deglycosylation does not destroy either the peptide backbone or the amino acid residues and does not affect the heme group content of the protein. The rates of oxygen uptake and light emission observed when horseradish peroxidase oxidizes isobutyraldehyde or the trimethylsilyl enol ether form of the latter are reduced when the enzyme is 70% deglycosylated. Concomitantly, the acting deglycosylated enzyme becomes inactivated during the course of the reaction. It appears that the carbohydrate moiety plays an important role in the protection of the peroxidase from damaging effects induced by triplet acetone and in the stabilization of the three-dimensional structure of this enzyme.     

Transient state kinetics of the reactions of isobutyraldehyde with compounds I and II of horseradish peroxidase

Elementary reactions have been studied quantitatively in the complex overall process catalyzed by horseradish peroxidase whereby isobutyraldehyde and molecular oxygen react to form triplet state acetone and formic acid. The rate constant for the reaction of the enol form of isobutyraldehyde with compound I of peroxidase is (8 +/- 1) X 10(6) M-1 s-1 and with compound II (1.3 +/- 0.3) X 10(6) M-1 s-1. Neither the enolate anion nor the keto form is reactive. The reactivity of enols with peroxidase parallels that of unionized phenols and a common mechanism is proposed. The overall catalyzed reaction of isobutyraldehyde and oxygen consists of an initial burst followed by a steady state phase. The burst is caused by the following sequence: 1) an initial high yield of compound I is formed from reaction of native enzyme with the autoxidation product of isobutyraldehyde, a peracid and 2) compound I rapidly depletes the equilibrium pool of enol which is present. After this burst a steady state phase is observed in which the rate-limiting step is the conversion of the keto to the enol form of the aldehyde catalyzed by phosphate buffer. The rate constant for the keto form reacting with phosphate is (8.7 +/- 0.6) X 10(-5) M-1 s-1. All constants were measured in dilute aqueous ethanol at 35 degrees C, pH 7.4, and ionic strength 0.67 M. Both the initial burst of light and the steady state emission from triplet acetone can be observed with the naked eye. Since the magnitude of the burst is a measure of the equilibrium amount of enol, the keto-enol equilibrium constant is readily calculated and hence also the rate constant for conversion of enol to keto. The keto-enol equilibrium constant is unaffected by phosphate which therefore acts as a true catalyst.     

太阳能电池反应模拟光合系统反应中心电荷复合形成三线态分子的过程

光合系统反应中心普遍存在电荷复合反应形成三线态分子的过程,并通过所形成的三线态β-胡萝卜素将剩余的能量经无辐射通道耗散给环境,实现光合系统的光保护功能.这一过程在人工合成系统中十分罕见,见诸报道的仅有少数由给体-受体组成的超分子体系.首次报道应用染料敏化TiO2胶体颗粒的人工太阳能电池反应,模拟光合系统三线态分子的形成过程,成功地观测到了视黄酸自由基正离子与TiO2表面束缚电子复合而形成的三线态视黄酸分子,并对其光谱和动力学过程进行了纳秒时间分辨光谱表征.     

Temperature-dependent triplet and fluorescence quantum yields of the photosystem II reaction center described in a thermodynamic model.

A key step in the photosynthetic reactions in photosystem II of green plants is the transfer of an electron from the singlet-excited chlorophyll molecule called P680 to a nearby pheophytin molecule. The free energy difference of this primary charge separation reaction is determined in isolated photosystem II reaction center complexes as a function of temperature by measuring the absolute quantum yield of P680 triplet formation and the time-integrated fluorescence emission yield. The total triplet yield is found to be 0.83 +/- 0.05 at 4 K, and it decreases upon raising the temperature to 0.30 at 200 K. It is suggested that the observed triplet states predominantly arise from P680 but to a minor extent also from antenna chlorophyll present in the photosystem II reaction center. No carotenoid triplet states could be detected, demonstrating that the contamination of the preparation with CP47 complexes is less than 1/100 reaction centers. The fluorescence yield is 0.07 +/- 0.02 at 10 K, and it decreases upon raising the temperature to reach a value of 0.05-0.06 at 60-70 K, increases upon raising the temperature to 0.07 at approximately 165 K and decreases again upon further raising the temperature. The complex dependence of fluorescence quantum yield on temperature is explained by assuming the presence of one or more pigments in the photosystem II reaction center that are energetically degenerate with the primary electron donor P680 and below 60-70 K trap part of the excitation energy, and by temperature-dependent excited state decay above 165 K. A four-compartment model is presented that describes the observed triplet and fluorescence quantum yields at all temperatures and includes pigments that are degenerate with P680, temperature-dependent excited state decay and activated upward energy transfer rates. The eigenvalues of the model are in accordance with the lifetimes observed in fluorescence and absorption difference measurements by several workers. The model suggests that the free energy difference between singlet-excited P680 and the radical pair state P680+l- is temperature independent, and that a distribution of free energy differences represented by at least three values of about 20, 40, and 80 meV, is needed to get an appropriate fit of the data.     

Photochemical oxidation of chlorpromazine in the dark induced by enzymically generated triplet carbonyl compounds

Enzymically generated triplet acetone and ethanal transfer energy to chlorpromazine as indicated by (i) suppression of the acetone chemiphosphorescence (ii) concomitant formation of chlo$\text{r}$ promazine photoproducts, that is the radical cation and the sulfoxide (iii) inhibition of photoproduct formation by a very efficient competition for triplet carbonyl energy using the sodium salt of 9,10-dibromoanthracene-2-sulfonic acid.This is the first report of a photooxidation in the dark.     

The triplet-state charge-transfer reaction between p-nitroacetophenone and guanosine monophosphate: a possible mechanism for electron-affinic radiosensitization.

Using flash photolysis it has been observed that the triplet state of p-nitroacetophenone reacts rapidly with guanosine monophosphate to give the p-nitroacetophenone radical anion, and presumably the guanine radical cation is also formed in this reaction. From this observation, it is predicted by theory that a similar reaction will occur between p-nitroacetophenone and the triplet state of guanosine monophosphate. Because guanine is excited to its triplet state when DNA is irradiated with X-rays, it is suggested that the charge transfer reaction between p-nitroacetophenone and the triplet state of guanine contributes to the radiosensitizing action of p-nitroacetophenone.     

EPR characterization of genetically modified reaction centers of Rhodobacter capsulatus

Electron paramagnetic resonance (EPR) has been used to investigate the cation and triplet states of Rhodobacter capsulatus reaction centers (RCs) containing amino acid substitutions affecting the primary donor, monomeric bacteriochlorophylls (Bchls), and the photoactive bacteriopheophytin (Bphe). The broadened line width of the cation radical in HisM200----Leu and HisM200----Phe reaction centers, whose primary donor consists of a Bchl-Bphe heterodimer, indicates a highly asymmetric distribution of the unpaired electron over the heterodimer. A T0 polarized triplet state with reduced yield is observed in heterodimer-containing RCs. The zero field splitting parameters indicate that this triplet essentially resides on the Bchl half of the heterodimer. The cation and triplet states of reaction centers containing HisM200----Gln, HisL173----Gln, GluL104----Gln, or GluL104----Leu substitutions are similar to those observed in wild type. Oligonucleotide-mediated mutagenesis has been used to change the histidine residues that are positioned near the central Mg2+ ions of the reaction center monomeric bacteriochlorophylls. Reaction centers containing serine substitutions at M180 and L153 or a threonine substitution at L153 have unaltered pigment compositions and are photochemically active. The cation and triplet states of HisL153----Leu reaction centers are similar to those observed in wild type. Triplet energy transfer to carotenoid is not observed at 100 K in HisM180----Arg chromatophores. These results have important implications for the structural requirements of tetrapyrrole binding and for our understanding of the mechanisms of primary electron transfer in the reaction center.     

Interaction between enzyme-generated triplet carbonyls and molecules intercalated into DNA

The phosphorescence from enzyme-generated and -protected triplet acetone is very efficiently quenched by dyes intercalated into DNA. The process is unlikely to be due to energy transfer and is tentatively ascribed to electron transfer occurring within the DNA helix complex with the acting enzyme. This quenching markedly protects DNA from breaks induced by triplet acetone. In the case of some barely emissive enzyme-generated triplet carbonyl species, it is possible to detect a weak emission resulting from the interaction with dye X DNA; this emission may be associated with back electron transfer.     

Bacteriochlorophyll fluorescence of purple bacteria at low redox potentials. The relationship between reaction center triplet yield and the emission yield.

This work describes fluorescence yield measurements in suspensions of strains of Rhodospirillum rubrum and Rhodopseudomonas sphaeroides in which the iron . quinone complex (X) was chemically reduced (state [PIX-]; P is the reaction center bacteriochlorophyll dimer, I is the long wavelength bacteriopheophytin), and compares these with the fluorescence observed when all the traps are open (state [PIX]) and with the fluorescence observed when all the traps are closed (state [P+IX]). At 77 K the amplitude and the shape of the fluorescence emission spectrum in [PIX-] are identical to those observed in [PIX]. This is a strong indication that all the extra fluorescence observed at room temperature in [PIX-] is, in fact, caused by an efficient back reaction [P+I-X-] leads to [P*IX-]. Using an equation similar to the original Vredenberg-Duysens relationship (Vredenburg, W.J. and Duysens, L.N.M. (1963) Nature 197, 355-357) but now assuming that a single reaction center has a probability pt of trapping an excitation and (1--pt) of re-emitting it to the surroundings, we are able to calculate pt as a function of the temperature by measuring the fluorescence in [PIX], [PIX-] and [P+IX] as a function of the temperature. The calculated pt values agree reasonably well with triplet yields measured in isolated reaction centers. Finally, we have measured the reaction center triplet yield (PTR) in intact systems and we have shown that the sum of the triplet yield and the remaining loss processes (PL) in the antenna bacteriochlorophyll including the bacteriochlorophyll dimer (such as fluorescence, internal conversion or direct triplet formation) is approximately constant; if we assume that at 77 K the only process which occurs in the reaction center is the formation of a reaction center triplet, than PTR + PL=1. The energy barrier between [P*IX-] and [P+I-X-] was estimated to be 0.11--0.15 eV for a set of preparations.     

Effect of SiO2 and zeolite surfaces on the excited triplet state of benzophenone, BT; a spectroscopic and kinetic study.

Laser flash photolysis has been used to study the triplet excited state of benzophenone B(T), on various surfaces, SiO(2), zeolites NaY, KY, NaX and KX, and in rigid media at room temperature, polyethylene and polymethylmethacrylate. The studies point to similarities of the spectroscopy and kinetics of B(T) in fluid solution, in a solid matrix (polymers) and on a SiO(2) surface. However, stark differences are observed for B(T) in zeolites where the absorption spectrum mimics that of the protonated ketone, and the reactivities of B(T) with C(6)H(12) and CH(3)OH are an order of magnitude smaller than those in liquid C(6)H(12) and CH(3)OH. Inclusion of ammonia, which blocks acidic sites in the zeolite, produces a triplet spectrum which is similar to that in polar solution. The reactivity of the triplet with ammonia in a zeolite is also comparable to that observed for this reaction in polar solution. These data are discussed in terms of the interaction of benzophenone with acidic sites in the zeolites, and to restrictions placed on the reactants in the zeolite cages. The blocking of the zeolite acidic sites by ammonia produces spectral and kinetic data (reactivity with NH(3)) of the triplet that are comparable to those observed in solution. This is one of the few cases where zeolites inhibit rather than promote reactions of a solute adsorbed in them.     

Damages induced in lambda phage DNA by enzyme-generated triplet acetone

Exposure of lambda phage to triplet acetone, generated via the oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only under alkaline conditions, and so true breaks do not occur. Also, no sites sensitive to UV-endonuclease from Micrococcus luteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed.     

Damages induced in λ phage DNA by enzyme-generated triplet acetone

Exposure of λ phage to triplet acetone, generated via the oxidation of isobutanal by peroxidase, leads to genome lesions. The majority of these lesions are detected as DNA single-strand breaks only under alkaline conditions, and so true breaks do not occur. Also, no sites sensitive to UV-endonuclease from Micrococcus leteus were found in DNA from treated phage. The participation of triplet acetone in the generation of such DNA damage is discussed.     

Triplet state of the primary donor in reaction centers of the phototrophic bacterium Rhodobacter sphaeroides R26 with active photoinduced electron transfer

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T0 mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.     

Low-temperature pulsed EPR study at 34 GHz of the triplet states of the primary electron Donor P865 and the carotenoid in native and mutant bacterial reaction centers of Rhodobacter sphaeroides

The photosynthetic charge separation in bacterial reaction centers occurs predominantly along one of two nearly symmetric branches of cofactors. Low-temperature EPR spectra of the triplet states of the chlorophyll and carotenoid pigments in the reaction center of Rhodobacter sphaeroides R-26.1, 2.4.1 and two double-mutants GD(M203)/AW(M260) and LH(M214)/AW(M260) have been recorded at 34 GHz to investigate the relative activities of the "A" and "B" branches. The triplet states are found to derive from radical pair and intersystem crossing mechanisms, and the rates of formation are anisotropic. The former mechanism is operative for Rb. sphaeroides R-26.1, 2.4.1, and mutant GD(M203)/AW(M260) and indicates that A-branch charge separation proceeds at temperatures down to 10 K. The latter mechanism, derived from the spin polarization and operative for mutant LH(M214)/AW(M260), indicates that no long-lived radical pairs are formed upon direct excitation of the primary donor and that virtually no charge separation at the B-branch occurs at low temperatures. When the temperature is raised above 30 K, B-branch charge separation is observed, which is at most 1% of A-branch charge separation. B-branch radical pair formation can be induced at 10 K with low yield by direct excitation of the bacteriopheophytin of the B-branch at 590 nm. The formation of a carotenoid triplet state is observed. The rate of formation depends on the orientation of the reaction center in the magnetic field and is caused by a magnetic field dependence of the oscillation frequency by which the singlet and triplet radical pair precursor states interchange. Combination of these findings with literature data provides strong evidence that the thermally activated transfer step on the B-branch occurs between the primary donor, P865, and the accessory bacteriochlorophyll, whereas this step is barrierless down to 10 K along the A-branch.     

Excited carbonyl formation in the combination and disproportionation of free radicals

The pyrolyisis of di-tert-butyl peroxyoxalate in the presence of para-substituted benzaldehydes produces almost quantitatively the corresponding p,p′-disubstituted benzils. The formation of these products is accompanied by chemiluminescence arising from excited triplets. From the quantum yield of excited triplet generation and the rate constants for the triplet photocleavage it is possible to obtain the change in Gibbs free energy associated with triplet formation. The values obtained are ?5.6, ?5.7 and ?8.1 kcal/mol for benzil, p,p′-dimethylbenzil and p,p′-dimethoxybenzil, respectively. The pyrolysis of di-tert-butyl peroxyoxalate in the presence of isopropanol or benzoin leads to the formation of acetone and benzil. These products are generated in disproportionation processes involving the α-hydroxy radical produced by hydrogen abstraction. The luminescence observed in these reactions constitutes the first experimental indication of excited species generation in the disproportionation of uncorrelated free radicals.     

Chemiluminescent determination of esterases in monocytes

Esterase from monocytes promotes the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) yielding 2-methyl-1-propenol, which is oxidized by horseradish peroxidase/H₂O₂ producing triplet acetone. The chemiluminescence of this reaction can be enhanced by the addition of 9,10-dibromoanthracene-2-sulphonate. The non-specific esterase present in monocytes is responsible for MPB hydrolysis, since (a) the chemiluminescence of the reaction was inhibited by fluoride, and (b) cells that do not contain a significant amount of non-specific esterases, e.g. lymphocytes and neutrophils, did not trigger light emission. The analytical application of this reaction is considered. © 1998 John Wiley & Sons, Ltd.