Intramolecular hydrogen transfer reactions of thiyl radicals from glutathione: formation of carbon-centered radical at Glu, Cys, and Gly

Abstract

Glutathione thiyl radicals (GS(•)) were generated in H(2)O and D(2)O by either exposure of GSH to AAPH, photoirradiation of GSH in the presence of acetone, or photoirradiation of GSSG. Detailed interpretation of the fragmentation pathways of deuterated GSH and GSH derivatives during mass spectrometry analysis allowed us to demonstrate that reversible intramolecular H-atom transfer reactions between GS(•) and C-H bonds at Cys[(α)C], Cys[(β)C], and Gly[(α)C] are possible.

Figure 1

MS spectra of GSH-NEM derivatized (m/z 433.14) obtained after exposure of GSH (500 uM) to AAPH (2 mM) at 37 °C during 1 hour in H₂O-buffer (A) and D₂O-buffer (B). The table summarizes the percentage of GSH-NEM derivatized molecules having incorporated covalently one deuteron atom after reaction of GSH with AAPH at different pH/pD in H₂O/D₂O-buffers.

Figure 2

Overlay of the CID spectra of GSH-NEM derivatized (m/z 433.14) obtained after exposure of GSH (500 µM) to AAPH (2 mM) at 37 °C during 1 hour in H₂O-buffer (black) and D₂O-buffer (gray). The table summarizes the percentage of fragment ions having incorporated covalently one deuteron atom after reaction of GSH with AAPH at different pH/pD in H₂O/D₂O-buffers.

Figure 3

MS³ spectra of the y2 fragment ions of product 2-d-X after incorporation of A) two deuterons, B) three deuterons. X stands for deuterium incorporation at Cys[^αC], Cys[^βC], or Gly[^αC].

Figure 4

MS³ spectra of the y2 fragment ions of A) product 3-h and B) product 3-d-X after incorporation of three deuterons. X stands for deuterium incorporation at Cys[^αC], Cys[^βC], or Gly[^αC].

Figure 5

Overlay of the MS spectra of 2-h and 2-d-X (m/z 433.14) generated after 10 min of UV-irradiation (λ = 253.7 nm) of GSSG (500 µM) in H₂O (black) and D₂O (gray). X stands for deuterium incorporation at Cys[^αC], Cys[^βC], or Gly[^αC].

Figure 6

MS³ spectra of the b2 fragment ions of A) product 4-h-S and B) product 4-d-S. MS⁴ of the fragment ion with m/z 199.0 is presented in panel C.

Figure 7

MS³ spectra of the y2 fragment ions of A) products 4-h-S and 5-h-Y, B) products 4-d-S and 5-d-Y, and C) products 6-h-S and 7-h-Y. The label Y refers to the acetylation of GSH or GSH* at the ^αC carbon of Cys, at the ^αC carbon of Gly/Gly* or at the ^βC carbon of Cys.

Scheme 1

Formation of glutathione thiyl radical using three different protocols, i) the thermal decomposition of AAPH, ii) the photochemistry of disulfide bond, iii) the photochemistry of acetone (X stands for H or D), and subsequent reactions of the thiyl radical.

Scheme 2

Reaction pathways for the MS³ fragmentation of the y2 fragment ions of product 2-d-X after incorporation of two or three deuterons during the reaction of AAPH with GSH/GSH* in D₂O. X stands for deuterium incorporation at Cys[^αC], Cys[^βC], or Gly[^αC].

Scheme 3

Reaction pathways for the MS³ fragmentation of the y2 fragment ions of product 3-h and 3-d-X after incorporation of three deuterons during the reaction of AAPH with GSH/GSH* in D₂O. X stands for deuterium incorporation at Cys[^αC], Cys[^βC], or Gly[^αC].

Scheme 4

Postulated fragmentation pathway for the MS³ fragmentation of the b2 fragment ions of products 4-h-S and 4-d-S. Color code for m/z: X=H (black), X=D (blue).

Scheme 5

Postulated fragmentation pathways for the MS³ fragmentation of the b2 fragment ions of products 4-h-S and 4-d-S (continued). The dashed frame indicates the MS⁴ fragmentation mechanism of the ion with m/z 199.0. Color code for m/z: X=H (black), X=D (blue).

Scheme 6

Acetylation of GSH and GSH*.

Scheme 7

Postulated fragmentation pathways for the MS³ fragmentation of the y2 fragment ions of the following products: 4-h-S (m/z are black colored, and X=H), 4-d-S (m/z are blue colored, and X=D), 6-h-S (m/z are red colored, and X=H). The red-colored atoms indicate the position of ¹⁵N and ¹³C when GSH* is used to explain the fragmentation mechanism.

Scheme 8

Postulated fragmentation pathways for the MS³ fragmentation of the y2 fragment ions of the following products: 5-h-Gly[^αC] (m/z are black colored), 5-d-Gly[^αC] (m/z are blue colored), 7-h-Gly[^αC] (m/z are red colored). The red-colored atoms indicate the position of ¹⁵N and ¹³C when GSH* is used to explain the fragmentation mechanism. The blue-colored atoms indicate either the use of a protonated acetyl radical (X=H) or a deuterated acetyl radical (X=D).

Chart 1

Structures of glutathione (GSH, GSH*) and GSSG.