John Butler

Reactions of nitrogen dioxide in aqueous model systems: Oxidation of tyrosine units in peptides and proteins

By application of pulse radiolysis it was demonstrated that nitrogen dioxide (NO2.) oxidizes Gly-Tyr in aqueous solution with a strongly pH-dependent rate constant (k6 = 3.2 X 10(5) M-1 S-1 at pH 7.5 and k6 = 2.0 X 10(7) M-1 S-1 at pH 11.3), primarily generating phenoxyl radicals. The phenoxyl can react further with NO2. (k7 approximately 3 X 10(9) M-1 S-1) to form nitrotyrosine, which is the predominant final product in neutral solution and at low tyrosyl concentrations under gamma-radiolysis conditions. Tyrosine nitration is less efficient in acidic solution, due to the natural disproportionation of NO2., and in alkaline solutions and at high tyrosyl concentrations due to enhanced tyrosyl dimerization. Selective tyrosine nitration by interaction of NO2. with proteins (at pH 7 to 9) was demonstrated in the case of histone, lysozyme, ribonuclease A, and subtilisin Carlsberg. Nitrotyrosine developed slowly also under incubation of Gly-Tyr with nitrite at pH 4 to 5, where NO2. is formed by acid decomposition of HONO. It is recalled in this context that NO2.-induced oxidations, by regenerating NO2-, can propagate NO2./NO2- redox cycling under acidic conditions. Even faster than with tyrosine is the NO2.-induced oxidation of cysteine-thiolate (k9 = 2.4 X 10(8) M-1 S-1 at pH 9.2), involving the transient formation of cystinyl radical anions. The interaction of NO2. with Gly-Trp was comparably slow (k approximately 10(6) M-1 S-1), and no reaction was detectable by pulse radiolysis with Met-Gly and (Cys-Gly)2, or with DNA. Slow reactions of NO2. were observed with arachidonic acid (k approximately 10(6) M-1 S-1 at pH 9.0) and with linoleate (k approximately 2 X 10(5) M-1 S-1 at pH 9.4), indicating that NO2. is capable of initiating lipid peroxidation even in an aqueous environment. NO2.-Induced tyrosine nitration, using 50 microM Gly-Tyr at pH 8.2, was hardly inhibited, however, in the presence of 1 mM linoleate, and was not affected at all in the presence of 5 mM dimethylamine (a nitrosamine precursor). It is concluded that protein modifications, and particularly phenol and thiol oxidation, may be an important mechanism, as well as initiation of lipid peroxidation, of action of NO2. in biological systems.

THE HABER‐WEISS CYCLE

Abstract— The Haber‐Weiss cycle:

Charge transfer between tryptophan and tyrosine in proteins

Abstract With numerous proteins, most of which are not involved in oxidation reduction reactions, azide radicals have been found to react with tryptophan units to give Trp. radicals. The Trp. radicals commonly transfer their electron deficiencies to tyrosine with rate constants in the region of 102-104 s-1, producing TyrO.. For β-lactoglobulin, which has been studied in most detail, the rate of transfer is independent of protein concentration, so the reaction must be intramolecular. Sodium perchlorate and SDS affect the rate, probably because of alterations in protein conformation. The activafion energy for the process is 45 kJ.mol-1, inconsistent with any mechanism involving hydrogen bounds or charge conduction throngh the chain, but consistent with tunnelling. With other proteins there is evidence of intermolecular transfer as well as intramolecular transfer, and there may be several different intramolecular steps corresponding to different tryptophan-tyrnosine pairs. Rates are not always the same as those initiated by photoionization of tryptophan, probably because different tryptophan units are involved. The possibility of transformation means that with enzymes the initial site of attnck by free radicals is not necessarily the site responsible for any consequent loss in activity. The oxidation-reduction potentials involved in the transfer are such that the process may be important in the mode of action of enzymes such as high-potential copper proteins and peroxidases, and perhaps also in the mode of action of other proteins if the environment is suitable.

The kinetics of the reduction of cytochrome c by the superoxide anion radical.

1. At neutral pH ferricytochrome c is reduced by the superoxide anion radical (O2-), without loss of enzymatic activity, by a second order process in which no intermediates are observed. The yield of ferrocytochrome c (82-104%), as related to the amount of O2- produced, is slightly dependent on the concentration of sodium formate in the matrix solution. 2. The reaction (k1 equals (1.1+/-0.1) - 10(6) M-1 - s-1 at pH 7.2, I equals 4 mM and 21 degrees C) can be inhibited by superoxide dismutase and trace amounts of copper ions. The inhibition by copper ions is removed by EDTA without interference in the O2- reduction reaction. 3. The second-order rate constant for the reaction of O2- with ferricytochrome c depends on the pH of the matrix solution, decreasing rapidly at pH greater than 8. The dependence of the rate constant on the pH can be explained by assuming that only the neutral form of ferricytochrome c reacts with O2- and that the alkaline form of the hemoprotein is unreactive. From studies at pH 8.9, the rate for the transition from the alkaline to the neutral form of ferricytochrome c can be estimated to be 0.3 s-1 (at 21 degrees C and I equals 4 mM). 4. The second-order rate constant for the reaction of O2- with ferricytochrome c is also dependent on the ionic strength of the medium. From a plot of log k1 versus I1/2-(I + alphaI1/2)-1 we determined the effective charge on the ferricytochrome c molecule as +6.3 and the rate constant at I equals 0 as (3.1+/-0.1) - 10(6) M-1 - s-1 (pH 7.1, 21 degrees C). 5. The possibility that singlet oxygen is formed as a product of the reaction of O2- with ferricytochrome c can be ruled out on thermodynamic grounds.

The Role of Sulphur Peptide Functions in Free Radical Transfer: A Pulse Radiolysis Study

Cascading transfers of free radical centres, involving sulphur and aromatic protein functions, have been studied in further detail. The disulphide radical anion appears to be an important terminus of both oxidative and reductive radical transfer. In deaerated solutions of cysteine (20 mmol dm-3) the yield of Cys2/SS.- closely resembles the yield of all primary free radicals generated by water radiolysis (.OH, H. and eaq-). The alanyl Ala/C beta., formed by electron addition to cysteine and subsequent SH- elimination, oxidizes cysteine with a rate constant of k8 = 5.0 x 10(6)dm3mol-1s-1 at pH 6 to 7 and 3.6 x 10(6)dm3mol-1s-1 at pH 9 to 10. In the case of glutathione (GSH) the eaq--induced carbon-centred radical oxidizes the parent thiol with rate constants k(G. + GSH) of 7.0 x 10(6) and 1.3 x 10(6)dm3mol-1s-1 at pH 8 and pH 10, respectively; and with dithiothreitol (D(SH)2) the corresponding reaction rate is k(.DSH + D(SH)2) = 5.5 x 10(6)dm3mol-1s-1 at pH 7.0. The decarboxylated methionyl Met/C. alpha, formed by reaction of .OH with methionine, is capable of electron transfer to cystine, indicating a reduction potential for decarboxylated methione more negative than -1.6 V. The ring-closed methionyl radical cation Met/SN.+, formed by reaction of .OH with Met-Gly, oxidizes azide via equilibration, Met/SN.+ + H+ + N3- in equilibrium Met + N3., which enables an estimate to be given for the one-electron reduction potential: E degrees (Met/SN.+ + H+; Met) = +1.42 +/- 0.3 V (pH 6.8). Some further reactions of oxidizing dimeric Met2/SS.+ species in neutral solution have been demonstrated. The direction and nature of the transfers can be expressed by the scheme: (formula; see text).

Interaction of Copper(I) with Nucleic Acids

Poly(dG-dC) and poly(I) form particularly stable complexes with Cu(I): thus characteristic UV absorbance changes enabled demonstration of Cu(I) transfer from poly(dA-dT) to poly(dG-dC), or from DNA to poly(I). Using pulse radiolysis to generate Cu(I), a rate constant of approximately 4 x 10(7) dm3 mol-1 s-1 (per base unit) was estimated for association of Cu(I) to native DNA, and slightly higher values were found for poly(dA-dT), poly(C), poly(dG-dC) and poly(G). For native DNA and for the models poly(dA-dT) and poly(dG-dC) the addition of Cu(I) was followed by secondary absorbance changes in the time scale of 10 ms, probably due to internal Cu(I) transfer; such secondary reactions were not detectable in heat-denatured DNA or in the homopolymers of A, C, G, and I. Extraction of Cu(I) from the DNA by EDTA is slow, 0.019 s-1, and independent of EDTA concentration, indicating that dissociation of the DNA-Cu(I) complex is the rate-determining step. A tentative value can hence be given for the DNA-Cu(I) stability constant: K = k (forward)/k (reverse) approximately 2 x 10(9) dm3 mol-1. Addition of H2O2 to solutions of gamma-radiolytically generated DNA-Cu(I), at Cu(I)/base less than 0.01, resulted in DNA degradation, comparable in yield to .OH-induced degradation. In the case of poly(dA-dT) and poly(dG-dC) the reaction of H2O2 with the corresponding Cu(I) complexes produced even more damage than the reaction of .OH. The formation of DNA-Cu(I), and the deleterious reaction with H2O2, were hardly affected by RNase or BSA, when added at equal (w/v) concentration. Dismutation of O2.- by (Cu,Zn)-SOD was partly inhibited by DNA and even more by poly(I) at pH 4.4, but not at pH 7, probably by competitive complexation of Cu(I), occurring in the catalytic cycle of SOD.

The repair of oxidized amino acids by antioxidants

Natural and synthetic antioxidants have been shown to repair tryptophan radicals produced from the one-electron oxidation of the free tryptophan amino acid. It has also been observed that both tryptophan and tyrosine radicals in lysozyme can be repaired by these antioxidants to varying degrees of efficiency. Although SDS-solubilized alpha-tocopherol efficiently repairs free tryptophan radicals, it is very inefficient in repairing the amino acids in lysozyme. The rigidity and immobility of solubilized alpha-tocopherol can explain this lack of efficiency.

Mechanism of reactions involving singlet oxygen and the superoxide anion

Since superoxide anions and hydrogen peroxide are formed in living systems and since singlet oxygen as well as the hydroxyl radical are considered to be harmful species, it is important to know in which reactions the formation or involvement of these species is kinetically feasible. For instance, does the dismutation of 0, yield ‘Z+, ‘A or %Z-02? In two recent papers by Khan [2,31 it zas calAlated that ‘Ci02 would be formed if 0, were surrounded by six or more water molecules. It is obvious that the calculations of Khan require the strict control of many parameters; therefore we maintain that the formation of ‘XL02 as a product of the dismutation reaction is most improbable [l] . To decide whether oxygen will be formed in its ‘Ag or “Cg state we must know the relative rates of the respective reactions.

Chemical mechanisms of the effects of high energy radiation on biological systems

Abstract The action of radiation on the molecules in living cells is partly direct but largely indirect, consisting of the formation of hydroxyl radicals, hydrated electrons and other species from the water, which then proceed to attack the organic molecules. The reactions of the species with amino acids and proteins, mitochondrial electron transport components, DNA bases, DNA itself and lipids are beginning to be thoroughly well established on a quantitative basis, as are the reactions of the organic radicals formed. Several aspects of radiobiology can now be understood in chemical terms. One such is the protective effect exerted by compounds like mercaptans. Another is the almost universal influence of oxygen. A number of different types of radiosensitisers have been discovered on a basis of expectations based on radiation chemistry. Future work will show how the chemical processes lead inevitably to the observable radiobiological response.

The glutathione free radical equilibrium, GS. + GS−⇌ GSS.−G, mediating electron transfer to FE(III) -cytochrome c

Abstract The GS.+GS− ⇌ GSS.− G equilibrium (1) was reinvestigated as a function of pH and ionic strength, using pulse radiolysis to oxidize GSH to GS.. All radicals formed by water radiolysis can be converted to populate equilibrium (1), which presents an interesting chemical junction between an electron acceptor (GS.) and an electron donor (GSS.−G). The secondary decay of the GS./GSS.− G couple into the reducing carbon-centred radical G(Cα.)SH, as observed in alkaline solution [10] (Grierson et al., Int. J. Radiat. Biol. 62 (1992) 265), seems to be of minor importance in neutral solution. Reduction of Fe(III)-cytochrome c at pH 6.8, after pulse radiolytic generation of the GS./GSS.− G couple in absence of oxygen, proceeds with half-lifes in the order of 100 to 200 μs. From the concentration dependent rate and efficiency of reduction it is concluded that GSS.− G is the reducing entity, k(GSS.− G + Fe(III)Cyt c) ≈ 6 x 1 07 M−1 s−1. The efficiency of reduction reaches about 72% (of GS.) for γ-radiolysis of deaerated solutions containing GSH and Fe(III)-cytochrome c at pH 6.8, even though the reverse reaction (-1) is favoured at this pH (where only few % of GS. equilibrate to GSS.−G). Reduction is less efficient under pulse radiolysis conditions due to competing radical-radical termination (e.g. GS.+ GS.-→ GSSG). In presence of oxygen the efficiency of reduction is even higher, 95% for γ-radiolysis, and the rate of reduction indicates that O2.− is the reluctant. Reversible formation of thiyl peroxy radicals (GS.+ O2 ⇌ GSOO.) seems to be overruled, via equilibrium (1), by irreversible electron transfer from GSS.− G to O2, for which a rate constant of 5.1 x 108 M−1 s−1 was estimated. The kinetics of copper-catalyzed reduction of Fe(III)-cytochrome c by GSH were investigated by stopped-flow techniques. The results presented indicate that the Cu(I)-thiolate complex is the reducing entity. It is concluded that Cu(II) does not interact with GSH to form the unbound GS. radical, and that reduction in this case is not mediated by equilibrium (1).