Reduction of selected oligopeptides containing methionine induced by hydrated electrons.

Bimolecular rate constants for the reaction of the hydrated electron with zwitterionic forms of several linear oligopeptides containing methionine were determined using the pulse radiolysis technique. The rate constants were found to vary in the range of (0.2-1.2) x 10(9) M(-1) s(-1). The reactivity of the peptides toward e(aq)- is governed by the pKa of the N-terminal amino group and the number of peptide bonds. At a fixed number of peptide bonds, the reactivity increases with the pKa, and for a given N-terminal amino acid residue, it increases with the number of peptide bonds. For the linear peptides the observed transient spectra are assigned to deaminated oligopeptide radicals, *CHRCONH approximately, obtained due to rapid deamination of the corresponding electron adducts formed initially. The radicals derived from oligopeptides containing methionine at the N-terminus absorb at approximately 400 nm with extinction coefficients of approximately 1300+/-150 M(-1) cm(-1). Their absorption maxima are shifted hypsochromically by approximately 30 nm with respect to those derived from oligopeptides with glycine at the N-terminus. The e(aq)- reacts with cyclic Met-Met, k = 2.0 x 10(9) M(-1) s(-1), probably by addition to the carbonyl bond, forming an adduct absorbing below 250 nm with epsilon250 = 2300+/-250 M(-1) cm(-1).

Pulse radiolysis studies of intramolecular electron transfer in model peptides and proteins. 7. Trp-->TyrO radical transformation in hen egg-white lysozyme. Effects of pH, temperature, Trp62 oxidation and inhibitor binding.

Intramolecular long-range electron transfer (LRET) in hen egg-white lysozyme (HEWL) accompanying Trp-->TyrO radical transformation was investigated in aqueous solution by pulse radiolysis as a function of pH (5.2-7.4) and temperature (283-328 K). The reaction was induced by highly selective oxidation of Trp with N3 radicals under low concentration of the reactants but at a high HEWL/N3 molar ratio, so that more than 99% of the oxidized protein molecules contained only a single tryptophyl radical. Synchronous decay of Trp and build-up of TyrO conformed satisfactorily to first-order kinetics, indicating that LRET involved either one or more Trp./Tyr redox pairs characterized by similar rate constants. The rate constant of LRET, k5, increased monotonously with decreasing pH showing the following characteristics: (i) in the pH range 7.4-5.2 the plot of k5 against pH was sigmoidal in shape, reflecting protonation of Glu35 (pKa approximately 6) and pointing to involvement of conformational control of the kinetics of LRET, (ii) below pH 5.2 a sharp increase in k5 was observed due to the protonation of Trp to form TrpH.+, which is known to oxidize tyrosine faster than does Trp.. Arrhenius plots of the temperature-dependence of k5 showed that the activation energy of LRET varies both with temperature and the protonation state of the enzyme. The activation energies are in the range 7.6-56.0 kJ mol-1, and are similar to those for activation of amide hydrogen exchange in native HEWL below its denaturation temperature. Selective oxidation by ozone of the Trp62 indole side-chain in HEWL to N'-formylkynurenine (NFKyn62-HEWL) caused a large drop in the initial yield of Trp. radicals, G(Trp.)i. This was accompanied by a relatively small decrease in k5 but selective oxidation by ozone had a pronounced effect on its temperature-dependence. Taken together these observations indicate that of the six tryptophans present in HEWL Trp62 contributes about 50% to the yield of the observed LRET. In the enzyme-inhibitor complex, HEWL(GlcNAc)3, where Trp62 and Trp63 are completely shielded from the solvent by the bound triacetylchitotriose, G(Trp.)i was lower than in NFKyn62-HEWL, and both the kinetic and energetic characteristics of LRET, observed at pH 5.2, were again somewhat different than in HEWL alone. Considering known solvent accessibilities of tryptophans in the complex, the observed LRET process in HEWL(GlcNAc)3 was assigned to Trp123. Theoretical evaluation of the electronic coupling for the dominant LRET pathways between all the potential Trp/Tyr redox couples in HEWL, with help of the PATHWAYS model, enabled Trp623/Tyr53, Trp63/Tyr53 and Trp123/Tyr23 to be identified as the pairs involved in the experimentally observed electron transfer.

Intramolecular electron transfer in peptides containing methionine, tryptophan and tyrosine: a pulse radiolysis study.

The kinetics of pulse radiolytically induced intramolecular radical transformations: Trp/N.----Tyr/O., Tyr/O.----Trp/N., Met/S therefore Br----Trp/N. and Met/S therefore Br----Tyr/O. has been investigated at various pH levels and temperatures in model peptides: Trp-(Pro)n-Tyr, Trp-(Gly)n-Tyr, Trp-(Pro)n-Met (n = 0-3), Tyr-Phe-Met-Arg-Phe-NH2 x 2AcOH, Met5-enkephaline and [D-Ala]2Met5-enkephaline. The rate constants of the Trp/N.----Tyr/O. transformation at pH 8 were found to decrease exponentially with the distance between Trp and Tyr in the proline peptides, while in the glycine peptides the rate of the transformation is less dependent on the number of bridging glycine residues. The activation energies determined fall into the range 10-20 kJ mol-1 irrespective of: (i) the ionization state of tryptophyl radical and tyrosine, (ii) type of bridging amino acids, and (iii) reversal of the direction of the electron transfer upon tyrosine OH group ionization. The activation entropies at 298 K for the peptides of the glycine and proline series are negative and rather high, and fall into the relatively narrow range of -90 to -140 J mol-1 deg-1. These activation parameters seem to indicate that a tunnelling mechanism is involved in the electron transfer between strictly oriented aromatic moieties of Trp and Tyr. The variation of the activation parameters with average separation distance in the case of Trp-(Pro)n-Tyr shows a predominance of the electronic factor over the nuclear in determining the distance dependence of the electron transfer rate. The intramolecular Met/S therefore Br----Tyr/O. transfer proceeds with the rate constant of 1.1 x 10(5) s-1 in Met5-enkephaline and 5.7 x 10(4) s-1 in [D-Ala]2Met5-enkephaline. The activation parameters for this transformation Ea = 30 kJ mol-1 and delta S298 = -62 J mol-1 deg-1 are close to those of the Trp/N.----Tyr/O. transformation, suggesting a similar mechanism for the electron transfer.

Pulse radiolysis studies of intramolecular electron transfer in model peptides and proteins. IV. Met/S:.Br-->Tyr/O. radical transformation in aqueous solution of H-Tyr-(Pro)n-Met-OH peptides.

The intramolecular radical transformation Met/S:.Br-->Tyr/O. in aqueous peptides H-Tyr-(Pro)n-Met-OH, n = 0-3, was investigated in the temperature range of 283-328 K by pulse radiolysis. Corresponding first-order rate constants and thermodynamic parameters of activation of electron transfer, Ea and delta S++, were determined from kinetic data. The rate constants of the reaction were found to decrease exponentially with the number of Pro units and the distance between CR atoms of the terminal amino acids, with a correlation coefficient alpha = 3.2 +/- 0.5 nm-1 at 298 K. Its value appeared to be temperature dependent suggesting the occurrence of thermally induced conformational changes in the peptides. Analysis of experimental data in terms of known conformational properties of the peptides indicates that apparent values of alpha, Ea and delta S++ are probably complicated functions of conformation and thermodynamic stability of the oligoproline bridge, varying with the number of Pro residues, and of intramolecular hydrophobic interactions between side chains of tyrosine and methionine. Estimation of the relative efficiency of electron transfer pathways through the peptide backbone and through direct and/or water mediated contact between groups bearing radical sites led to the conclusion that partitioning of electron transfer along these pathways is likely to occur.

Formation of three-electron bonds in one-electron oxidized methionine dipeptides: a pulse radiolytic study.

One electron oxidation of methionine dipeptides (Met-X and X-Met, where X = Gly or Ser) was carried out using the pulse radiolysis technique. It was apparent that the mode of oxidative action of OH radicals on methionine dipeptides was governed by the sequence of amino acids. Spectral evidence suggests that an intramolecular three-electron bond between nitrogen and sulphur atoms is not formed if these two atoms are separated by a peptide bond.

Temperature dependence of intramolecular electron transfer as a probe for predenaturational changes in lysozyme.

Intramolecular electron transfer in hen egg-white lysozyme between tryptophan and tyrosine units was investigated by means of pulse radiolysis in the temperature range 288-333 K. An Arrhenius plot for the kinetics of this process shows a sharp break at approximately 303 K (30 degrees C) compatible with the trend noted earlier (cf P. Jolles, et al. BBA, 491, 354, (1977)) on the Arrhenius plot for kinetics of bacterial substrate digestion by lysozyme. The departure from linearity of the Arrhenius plot for intramolecular electron transfer is interpreted in terms of local intralobe fluctuations of the native structure of lysozyme. It is suggested that such an approach can be useful for probing predenaturational changes in proteins.

The interaction between Cu(I) superoxide dismutase and hydrogen peroxide.

The interaction between superoxide dismutase (SOD) and peroxide, under anaerobic conditions in the presence of an OH radical scavenger, formate, and an indicator, nitro blue tetrazolium, involves five reactions and an equilibrium: (table; see text) Reaction 3 occurs at a rate that is proportional to both peroxide and enzyme with no kinetic evidence for any intermediate peroxide-enzyme complex. Rate studies as a function of pH corroborate previously published work (Fuchs, H. J. R., and Borders, C. L., Jr. (1983) Biochem Biophys. Res. Commun. 116, 1107-1113; Blech, D. M., and Borders, C. L., Jr. (1983) Arch. Biochem. Biophys. 224, 579-586) suggesting that HO2-, and not H2O2, is the active species in this system: k(HO2- + superoxide dismutase-Cu+) = 2.6 x 10(3) M-1 s-1. Evidence is presented which suggests that HO2-, like O2-, reacts at rates that are affected by the electrostatic forces of the enzyme.