Amide neighbouring-group effects in peptides: phenylalanine as relay amino acid in long-distance electron transfer

In nature, proteins serve as media for long‐distance electron transfer (ET) to carry out redox reactions in distant compartments. This ET occurs either by a single‐step superexchange or through a multi‐step charge hopping process, which uses side chains of amino acids as stepping stones. In this study we demonstrate that Phe can act as a relay amino acid for long‐distance electron hole transfer through peptides. The considerably increased susceptibility of the aromatic ring to oxidation is caused by the lone pairs of neighbouring amide carbonyl groups, which stabilise the Phe radical cation. This neighbouring‐amide‐group effect helps improve understanding of the mechanism of extracellular electron transfer through conductive protein filaments (pili) of anaerobic bacteria during mineral respiration

Chlorination and oxidation of the extracellular matrix protein laminin and basement membrane extracts by hypochlorous acid and myeloperoxidase

Basement membranes are specialized extracellular matrices that underlie arterial wall endothelial cells, with laminin being a key structural and biologically-active component. Hypochlorous acid (HOCl), a potent oxidizing and chlorinating agent, is formed in vivo at sites of inflammation via the enzymatic action of myeloperoxidase (MPO), released by activated leukocytes. Considerable data supports a role for MPO-derived oxidants in cardiovascular disease and particularly atherosclerosis. These effects may be mediated via extracellular matrix damage to which MPO binds. Herein we detect and quantify sites of oxidation and chlorination on isolated laminin-111, and laminin in basement membrane extracts (BME), by use of mass spectrometry. Increased modification was detected with increasing oxidant exposure. Mass mapping indicated selectivity in the sites and extent of damage; Met residues were most heavily modified. Fewer modifications were detected with BME, possibly due to the shielding effects. HOCl oxidised 30 (of 56 total) Met and 7 (of 24) Trp residues, and chlorinated 33 (of 99) Tyr residues; 3 Tyr were dichlorinated. An additional 8 Met and 10 Trp oxidations, 14 chlorinations, and 18 dichlorinations were detected with the MPO/H2O2/Cl- system when compared to reagent HOCl. Interestingly, chlorination was detected at Tyr2415 in the integrin-binding region; this may decrease cellular adhesion. Co-localization of MPO-damaged epitopes and laminin was detected in human atherosclerotic lesions. These data indicate that laminin is extensively modified by MPO-derived oxidants, with structural and functional changes. These modifications, and compromised cell-matrix interactions, may promote endothelial cell dysfunction, weaken the structure of atherosclerotic lesions, and enhance lesion rupture. Keywords: Extracellular matrix, Hypochlorous acid, Laminin, Protein oxidation, 3-chlorotyrosine, Myeloperoxidas

Oxidative Damage of Biomolecules by the Environmental Pollutants NO₂^• and NO₃^•

ConspectusAir pollution is responsible for the premature death of about 7 million people every year. Ozone (O₃) and nitrogen dioxide (NO₂^•) are the key gaseous pollutants in the troposphere, which predominantly result from combustion processes. Their inhalation leads to reactions with constituents in the airway surface fluids (ASF) of the respiratory tract and/or lungs. ASF contain small molecular-weight antioxidants, which protect the underlying epithelial cells against oxidative damage. When this defense system is overwhelmed, proteins and lipids present on cell surfaces or within the ASF become vulnerable to attack. The resulting highly reactive protein and lipid oxidation products could subsequently damage the epithelial cells through secondary reactions, thereby causing inflammation. While reactions of NO₂^• with biological molecules are considered to proceed through radical pathways, the biological effect of O₃ is attributed to its high reactivity with π systems. Because O₃ and NO₂^• always coexist in the polluted ambient atmosphere, synergistic effects resulting from in situ formed strongly oxidizing nitrate radicals (NO₃^•) may also require consideration. For example, in vitro product studies revealed that phenylalanine, which is inert not only to oxidants produced through biochemical processes, but also to NO₂^• or O₃ in isolation, is damaged by NO₃^•. The reaction is initiated by oxidation of the aromatic ring and, depending on the availability of NO₂^•, leads to formation of nitrophenylalanine or β-nitrooxyphenylalanine, which could serve as marker for NO₃^•-induced oxidative damage in peptides. More easily oxidizable aromatic amino acids are directly attacked by NO₂^• and are converted to the same products independent of whether O₃ is also present. Remarkably, NO₂^•-induced oxidative damage in peptides occurs not only through the well-established radical oxidation of peptide side chains, but also through an unprecedented fragmentation/rearrangement of the peptide backbone. This process is initiated by a nonradical N-nitrosation of a peptide bond involving the dimer of NO₂^•, i.e., N₂O₄, and contracts the peptide chain in the N → C direction by expelling one amino acid residue with simultaneous fusion of the remaining molecular termini, thereby forming a new peptide bond. This peptide cleavage could potentially be highly relevant for peptide segments with “nonvulnerable” side chains closer to the terminus that are not tied up in complex secondary and tertiary structures and therefore accessible for environmental oxidants. Likewise, NO₂^• reacts with cholesterol at the CC moiety through an ionic mechanism, which leads to formation of 6-nitrocholesterol in the presence of moisture. Contrary to common belief, this clearly shows that ionic chemistry, in particular nitrosation reactions by intermediately formed NO⁺, requires consideration when assessing NO₂^• toxicity. This conclusion is supported by recent work by Colussi et al. (Enami, S.; Hoffmann, M. R.; Colussi, A. J. Absorption of inhaled NO₂. <i>J. Phys. Chem. B.</i> <b>2009</b>, <i>113</i>, 7977–7981), who showed that anions in the airway surfaces fluids mediate NO₂^• absorption by catalyzing its hydrolytic disproportionation into NO₂^–/HNO₂ and NO₃^–. These findings could be the key to our understanding why NO₂^•, despite its low water solubility, has such pronounced biological effects in vivo

Synthesis and cellular evaluation of click-chemistry probes to study the biological effects of alpha, beta-unsaturated carbonyls

Humans are commonly exposed to α,β-unsaturated carbonyls as both environmental toxins (e.g. acrolein) and therapeutic drugs (e.g. dimethylfumarate, DMFU, a front-line drug for the treatment of multiple sclerosis and psoriasis). These compounds undergo rapid Michael addition reactions with amine, imidazole and thiol groups on biological targets, with reaction at protein Cys residues being a major reaction pathway. However, the cellular targets of these species (the ‘adductome’) are poorly understood due to the absence of readily identifiable tags or reporter groups (chromophores/fluorophores or antigens) on many α,β-unsaturated carbonyls. Here we report a ‘proof of concept’ study in which we synthesize novel α,β-unsaturated carbonyls containing an alkyne function introduced at remote sites on the α,β-unsaturated carbonyl compounds (e.g. one of the methyl groups of dimethylfumarate). The presence of this tag allows ‘click-chemistry’ to be used to visualize, isolate, enrich and characterize the cellular targets of such compounds. The probes show similar selectivity and reactivity to the parent compounds, and compete for cellular targets, yielding long-lived (stable) adducts that can be visualized in intact cells (such as primary human coronary artery smooth muscle cells), and extracted and enriched for subsequent target analysis. It is shown using this approach that dimethylfumarate forms adducts with multiple intracellular targets including cytoskeletal, organelle and nuclear species, with these including the rate-limiting glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This approach should be amenable to use with multiple α,β-unsaturated carbonyls and a wide variety of targets containing nucleophilic sites

Effect of macromolecular crowding on protein oxidation:Consequences on the rate, extent and oxidation pathways

Biological systems are heterogeneous and crowded environments. Such packed milieus are expected to modulate reactions both inside and outside the cell, including protein oxidation. In this work, we explored the effect of macromolecular crowding on the rate and extent of oxidation of Trp and Tyr, in free amino acids, peptides and proteins. These species were chosen as they are readily oxidized and contribute to damage propagation. Dextran was employed as an inert crowding agent, as this polymer decreases the fraction of volume available to other (macro)molecules. Kinetic analysis demonstrated that dextran enhanced the rate of oxidation of free Trp, and peptide Trp, elicited by AAPH-derived peroxyl radicals. For free Trp, the rates of oxidation were 15.0 ± 2.1 and 30.5 ± 3.4 μM min(−1) without and with dextran (60 mg mL(−1)) respectively. Significant increases were also detected for peptide-incorporated Trp. Dextran increased the extent of Trp consumption (up to 2-fold) and induced short chain reactions. In contrast, Tyr oxidation was not affected by the presence of dextran. Studies on proteins, using SDS-PAGE and LC-MS, indicated that oxidation was also affected by crowding, with enhanced amino acid loss (45% for casein), chain reactions and altered extents of oligomer formation. The overall effects of dextran-mediated crowding were however dependent on the protein structure. Overall, these data indicate that molecular crowding, as commonly encountered in biological systems affect the rates, and extents of oxidation, and particularly of Trp residues, illustrating the importance of appropriate choice of in vitro systems to study biological oxidations