Molecular basis of intramolecular electron transfer in proteins during radical-mediated oxidations: computer simulation studies in model tyrosine-cysteine peptides in solution

Abstract

Experimental studies in hemeproteins and model Tyr/Cys-containing peptides exposed to oxidizing and nitrating species suggest that intramolecular electron transfer (IET) between tyrosyl radicals (Tyr-O(·)) and Cys residues controls oxidative modification yields. The molecular basis of this IET process is not sufficiently understood with structural atomic detail. Herein, we analyzed using molecular dynamics and quantum mechanics-based computational calculations, mechanistic possibilities for the radical transfer reaction in Tyr/Cys-containing peptides in solution and correlated them with existing experimental data. Our results support that Tyr-O(·) to Cys radical transfer is mediated by an acid/base equilibrium that involves deprotonation of Cys to form the thiolate, followed by a likely rate-limiting transfer process to yield cysteinyl radical and a Tyr phenolate; proton uptake by Tyr completes the reaction. Both, the pKa values of the Tyr phenol and Cys thiol groups and the energetic and kinetics of the reversible IET are revealed as key physico-chemical factors. The proposed mechanism constitutes a case of sequential, acid/base equilibrium-dependent and solvent-mediated, proton-coupled electron transfer and explains the dependency of oxidative yields in Tyr/Cys peptides as a function of the number of alanine spacers. These findings contribute to explain oxidative modifications in proteins that contain sequence and/or spatially close Tyr-Cys residues.

Figure 1. Electron transfer and oxidation reactions in Tyr-Cys-containing peptides

The figure shows reactions involved in uni- and bimolecular processes related to radical-mediated oxidations in Tyr-Cys-containing peptides in the presence of oxygen, nitric oxide and nitrogen dioxide.The numbers in red indicate discrete reactions as follows. 1. Intramolecular electron transfer; 2. Combination reaction between tyrosyl radical and ^●NO₂ to yield 3-nitrotyrosine peptide; 3. Tyrosyl radical dimerization yielding 3,3′-di-tyrosine peptide; 4. Thiyl radical dimerization yielding disulfide peptide; 5. Combination reaction between cysteinyl radical and ^●NO to yield S-nitrosocysteine peptide; 6-7. Thiyl radical reaction with oxygen to yield thiylperoxyl radical peptide (6) and subsequent rearrangement and reduction tosulfinic acid derivative (7); 8. Intermolecular electron transfer; 9. Reaction between tyrosyl radical and superoxide yielding tyrosine hydroperoxide; 10-11. Reaction of cysteinyl radical peptide with tyrosine-cysteine peptide to yield the disulfide radical anion (10), followed by the reduction of molecular oxygen to yield O₂^●- and the corresponding disulfide peptide (11).

Figure 2. Possible mechanisms for the intramolecular electron transfer (IET) reaction in the Tyr-Cys-containing peptides

The figure shows four possible mechanisms studied in the present work. A. Involves a direct hydrogen- or time-concerted proton-coupled electron transfer (PCET) as described by reaction 2. B. Involves a water-bridged time concerted PCET as described by reaction 3. C. Consists of a first IET, i.e. Cys oxidation (reaction 4) followed by proton release and uptake steps (reaction 5) and D. involves the acid-base equilibrium of both residues and IET in the charged peptides in a solvent mediated non concerted PCET, as described by reactions 6, 7 and 8. The structures indicated in square brackets and the ^‡symbol, correspond to proposed transition states.

Figure 3. Proposed mechanism of intramolecular electron transfer in tyrosine-cysteine-containing peptides

The mechanism involves three discrete steps that may occur significantly separated in time: (1) deprotonation of the cysteine thiol; (2) intramolecular electron transfer from cysteine to tyrosine which determines K_IET; and (3) protonation of the tyrosine phenolate.

Figure 4. Selected snapshots for alternative charge redistribution pathways in the Tyr-(Ala₄)-Cys peptide

The figure shows the three paths for ET as predicted with the pathways algorithm and taken from the explicit water MD simulation (see Methods for details). The peptide is shown as bold sticks. (A) Through-bond path, (B) through-space jump and (C) through two consecutive jumps involving a bridging water molecule. The predicted ET paths are shown in orange arrows. Surrounding waters were omitted for clarity. The relative contribution of each pathway (A, B or C) to intramolecular electron transfer in the shown studied peptides for one, two and four alanine spacers, are indicated in the text. The percent contribution for each path type in each peptide was computed by visually inspecting and counting the corresponding path type in one hundred snapshots.