Glutathione traps formaldehyde by formation of a bicyclo[4.4.1]undecane adduct

Abstract

Glutathione forms complex reaction products with formaldehyde, which can be further modified through enzymatic modification. We studied the non-enzymatic reaction between glutathione and formaldehyde and identified a bicyclic complex containing two equivalents of formaldehyde and one glutathione molecule by protein X-ray crystallography (PDB accession number 2PFG). We have also used (1)H, (13)C and 2D NMR spectroscopy to confirm the structure of this unusual adduct. The key feature of this adduct is the involvement of the gamma-glutamyl alpha-amine and the Cys thiol in the formation of the bicyclic ring structure. These findings suggest that the structure of GSH allows for bi-dentate masking of the reactivity of formaldehyde. As this species predominates at near physiological pH values, we suggest this adduct may have biological significance.

Fig. 1

(a) Binding mode of CBR1·BiGF₂. Hydrogen bonding interactions and residues within a distance of 4.0 Å from the ligand are shown. Non-polar residues (green), polar residues (purple), and charged residues (purple/blue) are indicated. Distances are given (Å). (b) Electron density maps of CBR1·BiGF₂. The 2F_o – F_c map is contoured in gray at a level of 1 σ. The F_o – F_c map is contoured in red at a level of 2.75 σ. NADP (gray, blue and red) and BiGF₂ (grey, red, blue and yellow) are shown occupying the CBR1 active site.

Fig. 2

Structures of the previously reported GSH : formaldehyde 2 : 1 adduct (GF₂, (S)-1-((R,Z)-6-(carboxymethylimino)-1,3-oxathian-5-yl)-7-oxo-1,3-diazepane-4-carboxylic acid) and BiGF₂ are shown. Predicted and observed ¹³C chemical shifts for the formaldehyde-derived methylenes are indicated.

Fig. 3

Percent composition by ¹³C NMR of various GSH–¹³C formaldehyde adducts vs. pH for NMR samples containing an 8-fold excess of formaldehyde. BiGF₂ (-○-, ¹³C NMR = 61.5 and 62.2 ppm), HMGSH (-●-, ¹³C NMR = 66.7 ppm) and an unidentified constituent (-▾-, ¹³C NMR = 44.1 ppm).