Oxidation of protein disulfide bonds by singlet oxygen gives rise to glutathionylated proteins

Abstract

Disulfide bonds play a key function in determining the structure of proteins, and are the most strongly conserved compositional feature across proteomes. They are particularly common in extracellular environments, such as the extracellular matrix and plasma, and in proteins that have structural (e.g. matrix) or binding functions (e.g. receptors). Recent data indicate that disulfides vary markedly with regard to their rate of reaction with two-electron oxidants (e.g. HOCl, ONOOH), with some species being rapidly and readily oxidized. These reactions yielding thiosulfinates that can react further with a thiol to give thiolated products (e.g. glutathionylated proteins with glutathione, GSH). Here we show that these ‘oxidant-mediated thiol-disulfide exchange reactions’ also occur during photo-oxidation reactions involving singlet oxygen ( 1 O 2 ). Reaction of protein disulfides with 1 O 2 (generated by multiple sensitizers in the presence of visible light and O 2 ), yields reactive intermediates, probably zwitterionic peroxyl adducts or thiosulfinates. Subsequent exposure to GSH, at concentrations down to 2 μM, yields thiolated adducts which have been characterized by both immunoblotting and mass spectrometry. The yield of GSH adducts is enhanced in D 2 O buffers, and requires the presence of the disulfide bond. This glutathionylation can be diminished by non-enzymatic (e.g. tris-(2-carboxyethyl)phosphine) and enzymatic (glutaredoxin) reducing systems. Photo-oxidation of human plasma and subsequent incubation with GSH yields similar glutathionylated products with these formed primarily on serum albumin and immunoglobulin chains, demonstrating potential in vivo relevance. These reactions provide a novel pathway to the formation of glutathionylated proteins, which are widely recognized as key signaling molecules, via photo-oxidation reactions.

Formation of protein cross-links by singlet oxygen-mediated disulfide oxidation

“…This indicates that the initial exposure of these proteins to 1 O 2 results in significant, and sufficient, structural changes to allow reaction at the oxidized disulfide bond of B2M or CRP, with the one of the Cys residues on GAPDH, and consequent B2M-GAPDH and CRP-GAPDH cross-link formation. These data suggest that the time-dependent formation of such cross-links depends on initial modification of the protein structure and the formation of intermediate(s) with significant life-times as shown for the corresponding reactions with GSH [ 22 , 23 , 25 ].…”

“…The former is believed to be more likely, as the generation of a thiosulfinate requires further reactions. Both zwitterionic peroxides and thiosulfinates appear to have lifetimes of up to several hours [ 22 , 23 , 25 ], though this is structure dependent [ 51 ]. The oxidation of the disulfide bond as a result of these photo-oxidation reactions has been confirmed in the current study for B2M where a marked time-dependent loss of the cross-linked peptide containing the disulfide bond was detected by MS analysis.…”

“…Our previous studies on the reaction of peptides and proteins that contain disulfide bonds with oxidants (HOCl, ONOOH, H 2 O 2 , 1 O 2 ) have provided evidence for the incorporation of multiple oxygen atoms (as determined by the detection of a large series of ions with m/z increases of +16 in MS analyses) and for subsequent adduction of GSH (and other thiols) to peptide/protein-derived reactive intermediates [ 22 , 23 , 25 ]. Whilst some of the oxygen atom adduction is likely to occur at Met, His, Tyr and Trp residues (which are known targets for these oxidants [ 17 , [52] , [53] , [54] ]), data has also been obtained for oxygen atom addition at disulfide bonds to give intermediates that react with GSH [ 22 , 23 , 25 ]. The formation of these peptide-/protein-GSH adducts is (at least partially) reversible, and requires both the initial disulfide bond and an endogenous or added free thiol [ 22 , 23 , 25 ].…”

“…Studies on the reaction of 1 O 2 with low-molecular-mass disulfides have provided evidence for the formation of zwitterionic peroxides [RS + (OO − )SR’], thiosulfinates (disulfide-S-monoxides, RS(=O)SR′) and thiosulfonates (disulfide-S-dioxides, RS(=O) 2 SR′) [ 20 , 21 ] (reaction 3). Both the zwitterionic peroxides [RS + (OO − )SR’] and thiosulfinates are unstable and can undergo further reaction with another thiol to give a new disulfide bond with cleavage of the original disulfide [ 22 , 23 ] (reaction 4). In the absence of added thiol, cleavage of the disulfide bond can also occur with formation of sulfinic (RSO 2 H) and sulfonic acids (RSO 3 H) (reaction 5).…”

“…glutathionylated) species have been detected with a range of other oxidants (e.g. hypochlorous acid, peroxynitrous acid, H 2 O 2 ) [ [22] , [23] , [24] , [25] ], indicating that these ‘oxidant-mediated thiol-disulfide exchange reactions’ are a common pathway after initial oxidation of a disulfide bond (reaction 6).

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