Re‐evaluation of intramolecular long‐range electron transfer between tyrosine and tryptophan in lysozymes

Abstract

One-electron oxidation of six different c-type lysozymes from hen egg white, turkey egg white, human milk, horse milk, camel stomach and tortoise was studied by gammaand pulse-radiolysis. In the first step, one tryptophan side chain is oxidized to indolyl free radical, which is produced quantitatively. As shown already, the indolyl radical subsequently oxidizes a tyrosine side chain to the phenoxy radical in an intramolecular reaction. However this reaction is not total and its stoichiometry depends on the protein. Rate constants also vary between proteins, from 120AEs )1 to 1000AEs )1 at pH 7.0 and room temperature [extremes are hen and turkey egg white (120AEs) and human milk (1000AEsIn hen and turkey egg white lysozymes we show that another reactive site is the Asn103-Gly104 peptidic bond, which gets broken radiolytically. Tryptic digestion followed by HPLC separation and identification of the peptides was performed for nonirradiated and irradiated hen lysozyme. Fluorescence spectra of the peptides indicate that Trp108 and/or 111 remain oxidized and that Tyr20 and 53 give bityrosine. Tyr23 appears not to be involved in the process. Thus new features of long-range intramolecular electron transfer in proteins appear: it is only partial and other groups are involved which are silent in pulse radiolysis.Keywords: gamma and pulse radiolysis; intramolecular longrange electron transfer; lysozyme; one-electron oxidation.The early suggestions [1,2] of intramolecular long-range electron transfer (LRET) have now been verified by numerous observations of LRET between donor/acceptor redox centres with known separation distances in proteins, peptides and other small rigid organic molecules. This property exists in polymers and especially in biopolymers (proteins and DNA). Thus these macromolecules are considered as candidates for nanoelectronics components. In this view, several studies were devoted to a better understanding of the factors leading to modulation of LRET (for a review, see [3] and references therein [4-6]). As part of a program to unravel the mechanistic basis of LRET in proteins, systematic investigations on electron transfer between tyrosine and tryptophan in proteins and peptides have been conducted [7][8][9][10]. In this experimental system, developed first by Pru¨tz et al. [11], pulse radiolytically generated azide radical and dibromide radical anion preferentially oxidize the side chain of tryptophan (Trp) to the indolyl radical (Trp AE ) in molecules that also contain tyrosine (Tyr).The initiating oxidation (1) can be effected by other radiolytically generated species such as the dibromide (Br 2 -) and dichloride (Cl 2 -) radical anions [12]. The tryptophan neutral radical, with a midpoint reduction potential that is near 1 V at pH 7.0 [13], rapidly oxidizes the tyrosine side chain to the phenoxy radical (TyrO AE ) in the intramolecular equilibration:Both TyrO AE and Trp AE absorb strongly in different parts of the visible region. One of the first observations of reaction (2) in proteins was obtained ...

Inactivation of chosen dehydrogenases by the products of water radiolysis and secondary albumin and haemoglobin radicals

“…GAPDH and ADH which are effectively inactivated by secondary protein radicals have cysteine residues at the active site: ADH has two cysteine residues, Cys-43 and Cys-153, while GAPDH has only one cysteine residue, Cys-149, close to which (about 7 Å ) Cys-153 is located (PDB, code 2HCY for ADH and code 1JOX for GAPDH). Although secondary protein radicals cannot react directly with these groups due to steric reasons, they can be oxidized by radical transfer, involving especially aromatic residues (tyrosine and tryptophan) and cysteine (Bobrowski et al 1997, Stuart-Audette et al 2003. In GAPDH, Tyr-311 is located about 4.2 Å from Cys 149 at the active site.…”

Investigation of radical‐based damage of RNase A in aqueous solution and lipid vesicles

“…OH species is generally considered the primary radical responsible for Tyr modifications, although H atoms also can attack these aromatic residues, giving rise to intra-and intermolecular reactions including possible dimerization of RNase radicals. 12,15,46,47 Since the formation of covalent bonds between the Tyr moieties could be one of the major pathways of protein aggregation, [47][48][49] the spectral modifications of the Tyr bands can be connected to the formation of higher molecular weight aggregates evidenced by HPLC and SDS-PAGE analyses.…”

Tryptophan reduces creatine kinase activity in the brain cortex of rats

“…Tryptophan is ordinarily considered an antioxidant agent (Watanabe et al, 2002), but urate, a known antioxidant agent, may be oxidized by tryptophan neutral radical (Santus et al, 2001). On the other hand, when oxidized, the indoxyl radical formed from tryptophan subsequently oxidizes a tyrosine side chain of lysozymes to the phenoxyl radical (Stuart-Audette et al, 2003). Besides, free radicals derived from tryptophan oxidation may form stable adducts with proteins (Domingues et al, 2003).…”