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In this paper a combined experimental and quantum chemical study of the geometry and opto-electronic properties of unsubstituted and dialkoxy-sustituted phenylene-vinylene oligomers (PV's) is presented. The optical absorption spectra for PV cations with different chain lengths and substitution patterns were measured using pulse radiolysis with time-resolved spectrophotometric detection from 1380 to 500 nm (0.9 to 2.5 eV). The geometries of the PV's studied were optimized using density functional theory (DFT) for both the neutral and singly charged molecule. The spectra for the PV radical cations were then calculated using singly excited configuration interaction with an intermediate neglect of differential overlap reference wave function method together with the DFT geometry. The agreement between experimental and theoretical absorption energies is excellent; most of the calculated radical cation absorption energies are within 0.15 eV of the experimental values. The pattern of dialkoxy-substitution is found to have a large effect on the optical absorption spectrum of the cation. Using the calculated charge distribution it is shown that the degree of delocalization of the charge correlates with the energy of the lowest absorption band. If alkoxy side chains are present on some of the rings the positive charge tends to localize at those sites. [ABSTRACT FROM AUTHOR]

Using the reduction potential of one-electron oxidized guanosine in water and the pKa values of the radical and of the parent, the N1-H bond energy of the 2'-deoxyguanosine moiety is determined to be (94.3+/-0.5) kcal mol(-1). Using the DFT method, the energy of the N1-centered guanosine radical is calculated and compared with those of the C1'- and C4'-radicals formed by H-abstraction from the 2'-deoxyribose moiety of the molecule. The result is that these deoxyribose-centered radicals appear to be more stable than the N1-centered one by up to 3 kcalmol(-1). Therefore, H-abstraction from a 2'-deoxyribose C-H bond by an isolated guanosine radical should be thermodynamically feasible. However, if the stabilization of a guanine radical by intrastrand pi-pi interaction with adjacent guanines and the likely lowering of the oxidation potential of guanine by interstrand proton transfer to the complementary cytosine base are taken into account, there is no more thermodynamic driving force for H-abstraction from a deoxyribose unit. As a further criterion for judging the probability of occurrence of such a reaction in DNA, the stereochemical situation that a DNA-guanosine radical faces was investigated utilizing X-ray data for relevant model oligonucleotides. The result is that the closest H-atoms from the neighboring 2'-deoxyribose units are at distances too large for efficient reaction. As a consequence, H-abstraction from 2'-deoxyribose by the DNA guanine radical leading subsequently to a "frank" DNA strand break is very unlikely. The competing reaction of the guanine radical cation with a water molecule which eventually yields 8-oxo-2'-deoxyguanosine (leading to "alkali-inducible" strand breaks) has thus a chance to proceed.

The reaction of *OH with 2'-deoxyguanosine yields two transient species, both identified as OH adducts (G*-OH), with strongly different reactivity towards O2, or other oxidants, or to reductants. One of these, identified as the OH adduct at the C-8 position (yield 17% relative to *OH), reacts with oxygen with k=4 x 10(9)M(-1)s(-1); in the absence of oxygen it undergoes a rapid ring-opening reaction (k = 2 x 10(5) s(-1) at pH4-9), visible as an increase of absorbance at 300-310 nm. This OH adduct and its ring-opened successor are one-electron reductants towards, for example, methylviologen or [Fe(III)(CN)6]3-. The second adduct, identified as the OH adduct at the 4-position (yield of 60-70% relative to *OH), has oxidizing properties (towards N,N,N',N'-tetra-methyl-p-phenylenediamine, promethazine, or [Fe(II)(CN)6]4-). This OH adduct undergoes a slower transformation reaction (k = 6 x 10(3) s(-1) in neutral, unbuffered solution) to produce the even more strongly oxidizing (deprotonated, depending on pH) 2'-deoxyguanosine radical cation, and it practically does not react with oxygen (k< or = 10(6)M(-1)s(-1)). The (deprotonated) radical cation, in dilute aqueous solution, does not give rise to 8-oxoguanosine as a product. However, it is able to react with ribose with k< or =4 x 10(3)M(-1)S(-1).

Photolysis of ferrocytochrome c by 248 nm laser light in aqueous solution at pH 7 generates hydrated electrons (eaq-) by a monophotonic process with quantum yield phi = 0.034. Approximately three-quarters of the eaq- originate from the heme, which is converted from the ferrous to the ferric state in < 100 ns. The conformational changes associated with the change in the redox state of cytochrome c are either not detectable spectrophotometrically or complete in < 100 ns. Also, under conditions where ferrocytochrome c is stable but ferricytochrome c is unfolded (3 M guanidine, pH 7, 40 degrees C), photoionization of ferrocytochrome c generated ferricytochrome c with similar quantum yield. Under these conditions, the lifetime of native ferricytochrome c is 67 microseconds; it decays via two intermediates with lambda max > 410 nm, neither of which is the thermodynamically favored, unfolded form. These species are putatively identified as unfolding intermediates with nonnative iron ligands, similar to those found during folding of ferrocytochrome c. The results suggest that unfolding, like folding, proceeds by intrachain diffusion and ligand exchange.

The 248 nm laser flash photolysis of myoglobin in various redox states (oxy, met and ferryl) in neutral aqueous solution yielded hydrated electrons with concurrent changes in the visible absorption spectrum of the heme. The results could be ascribed to the photoionization of both the peptide and the heme group, in approximately equal yields. The ionization of met- and ferrylmyoglobin was biphotonic, but that of oxymyoglobin was a mixture of mono- and biphotonic processes. Using appropriate electron and radical scavengers, the changes in the heme absorption could be investigated at times > or = 100 ns and were shown to be associated with a +1 increase of the formal oxidation state of the heme. Using this method, the formal iron (V) state of native myoglobin could be spectroscopically characterized for the first time. Its absorption, blue-shifted and less intense relative to the ferryl state, is reminiscent of that of the compound I of peroxidases, which contains a ferryl-oxo (iron[IV]) group and a porphyrin radical cation. On this basis, the same structure is proposed for the formal iron(V) state of native myoglobin. The transition from oxy- to metmyoglobin took approximately 5 microsecond, which may reflect the kinetics of exchange of oxygen with water as ligand. The transitions from the met to the ferryl state, and from ferryl to iron(V) states were faster (approximately 250 ns), consistent with processes that involve proton or electron movements but no changes in the iron coordination state.

The rates of oxidation of reducing substrates by heme peroxidases have previously been thought to be controlled only by their ease of oxidation. In the present study, we have compared the kinetics and thermodynamics of the oxidation of indole-3-acetic acid and derivatives and of phenols by horseradish peroxidase. Different dependencies of the reaction rates on the thermodynamic driving force reveal substrate specificity controlled by the enzyme-substrate complexes dissociation constants (Michaelis-Menten constants) and by the reorganization energies of electron-transfer within those complexes.

The objective of this study was to compare effects of quin2 and EDTA in iron-driven Fenton-type reactions. Seven different assays for detection of strong oxidants were used: the DMSO, deoxyribose, benzoate hydroxylation, and plasmid DNA strand breakage assays, detection of 8-oxo-deoxyguanosine in deoxyguanosine mononucleosides and calf thymus DNA, and electron spin resonance with the spin-trap (4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) in the presence of ethanol or DMSO. With H2O2 and Fe3+, quin2 generally strongly increased the formation of reactive species in all assays, whereas with EDTA the results varied between the assays from barely detectable to highly significant increases compared to H2O2 and unchelated Fe3+. We found that the species produced in the reaction between Fe3+-quin2 and H2O2 behaved like the hydroxyl radical in all assays, whereas with Fe3+-EDTA no clear conclusion could be drawn about the nature of the oxidant. The effect of quin2 on the formation of oxidants on Fe2+ autoxidation, varied from generally inhibiting to slightly promoting, depending on the assay used. EDTA had a promoting effect on the amount of oxidant detected by all but one assay. None of the autoxidation systems produced DMSO or ethanol radical adducts with 4-POBN. In the presence of either chelator, H2O2, and Fe2+ DMSO and ethanol radical adducts of 4-POBN were produced. Using the Fe2+ indicator ferrozine, evidence for direct reduction of Fe3+-quin2 by H2O2 was found. Superoxide anion radical appeared to be less efficient than H2O2 as reductant of Fe3+-quin2 as addition of superoxide dismutase in the ferrozine experiments only decreased the amount of Fe2+ available for Fenton reaction by 10-20%. The main conclusions from our study are that the reduction of Fe3+-quin2 can be driven by H2O2 and that Fe2+ in the following oxidation step produces a species indistinguishable from free hydroxyl radical.

In 1876, Fenton described a colored product obtained on mixing tartaric acid with hydrogen peroxide and a low concentration of a ferrous salt. Full papers in 1894 and 1896 showed the product was dihydroxymaleic acid. Haber, Weiss and Willstätter proposed in 1932-1934 the involvement of free hydroxyl radicals in the iron(II)/hydrogen peroxide system, and Baxendale and colleagues around 1950 suggested that superoxide reduces the iron(III) formed on reaction, explaining the catalytic nature of the metal. Since Fridovich and colleagues discovered the importance of superoxide dismutase in 1968, numerous studies have sought to explain the deleterious effects of cellular oxidative stress in terms of superoxide-driven Fenton chemistry. There remain questions concerning the involvement of free hydroxyl radicals or reactions of metal/oxo intermediates. However, these outstanding questions may obscure a wider appreciation of the importance of Fenton chemistry involving hypohalous acids rather than hydrogen peroxide as the oxidant.

The rates of reaction of seven indole-3-acetic acid derivatives with horseradish peroxidase compound 1 at pH 5 were measured by stopped flow, and the reduction potentials and pKa of their radical cations were determined by pulse radiolysis. Reasonable correlation of these properties with Hammett substituent parameters was found, but not with Brown-Okamoto (theta +) parameters. The rates of reaction with compound I correlate well with the reduction potentials under the same conditions, with rates of reaction that increase by ca. 2.5 orders of magnitude with a 100 mV decrease in the reduction potential. This relationship is in agreement with that previously estimated for the reaction of compound I with phenols and anilines, suggesting that the rate of reaction depends solely on the reduction potential of the substrate radical, even for compounds of dissimilar structure.

The peroxidation of liposomes by a haem peroxidase and hydrogen peroxide in the presence of indole-3-acetic acid and derivatives was investigated. It was found that these compounds can accelerate the lipid peroxidation up to 65 fold and this is attributed to the formation of peroxyl radicals that may react with the lipids, possibly by hydrogen abstraction. The peroxyl radicals are formed by peroxidase-catalyzed oxidation of the enhancers to radical cations which undergo cleavage of the carbon-carbon bond on the side-chain to yield CO2 and carbon-centred radicals that rapidly add oxygen. In competition with decarboxylation, the radical cations deprotonate reversibly from the N1 position. Rates of decarboxylation, pka values and rate of reaction with the peroxidase compound I indicate consistent substituent effects which, however, can not be quantitatively related to the usual Hammett or Brown parameters. Assuming that the rate of decarboxylation of the radical cations taken is a measure of the electron density of the molecule (or radical), it is found that the efficiency of these compounds as enhancers of lipid peroxidation increases with increasing electron density, suggesting that, at least in the model system, the oxidation of the substrates is the limiting step in causing lipid peroxidation.

Hypochlorous acid (HOCl) is a strong oxidant formed in neutrophils by the myeloperoxidase-catalyzed oxidation of chloride. Using stopped-flow with spectrophotometric detection, HOCl was found to react very rapidly with glutathione and ascorbate and less rapidly with taurine. No evidence could be found for the formation of reactive free-radical intermediates in these reactions, in support of an electrophilic mechanism. In contrast, the reaction with iron(II) aquo or citrate complexes (k approximately 10(4) dm3 mol-1 s-1 in acidic solution) yielded reactive intermediates distinguishable from hydroxyl radicals. The reaction between HOCl and ferrous ions, which is analogous to but faster than the Fenton reaction, is a potential source of free radicals in activated neutrophils.

Hypochlorous acid reacts with the model iron(II) complex, ferrocyanide (Fe(CN)6(4-)) in aqueous solution with the rate constant 220 +/- 15 dm3 mol-1 s-1. Free hydroxyl radicals are formed in this reaction in 27% yield as shown by the hydroxylation of benzoate to give a product distribution identical to that of free (radiolytically generated) hydroxyl radicals. This reaction is three orders of magnitude faster than the analogous reaction involving hydrogen peroxide (the Fenton reaction), suggesting that the hypochlorous acid generated by activated neutrophils may be a source of hydroxyl radicals.