¹H, ¹³C‐NMR and X‐ray Absorption Studies of Copper(I) Glutathione Complexes

Abstract

The tripeptide glutathione (y-~-Gl~-~-Cys-Gly, GSH) is an important intracellular reducing agent for Cu(I1) and complexation agent for Cu(1). We have studied the complexation of Cu(1) to GSH in aqueous solution at a range of pH values and Cu(1):GSH molar ratios by 'H-NMR and 'IC-NMR spectroscopy and X-ray absorption spectroscopy. The NMR data are consistent with formation of a complex with approximate 1 : 1 stoichiometry [Cu(SG)] as the major species with only thiolate sulfur of GSH binding to Cu(1). The rate of exchange of GSH with GS-Cu was determined to be 13 s-' at 283 K, pH 6.8. X-ray absorption spectroscopic measurements showed that Cu(1) is coordinated to 3.1 +-0.3 sulfur atoms at approximately 0.222 nm in solutions (and solids) containing GSH:Cu in 1 : 1 and 2: 1 mol ratios. The possible structures of polymeric Cu(1)-glutathione complexes are discussed. The high thermodynamic stability of Cu(1)-S bonds in Cu(1)-glutathione complexes coupled with their kinetic lability may provide efficient and specific pathways for the transport of copper in cells.Keywords: glutathione ; copper; NMR ; X-ray absorption near-edge structure ; extended X-ray absorption fine structure.Glutathione (GSH) is present in all living cells and is frequently the most abundant cytosolic thiol compound with concentrations in the range 0.1-10 mM. Usually the ratio between the oxidised (GSSG) and the reduced (GSH) forms in the body is maintained at a low value of approximately 0.1 [l].-0,c c0,- SH 0 GSHGSH is a tlexible peptide, the X-ray structure showing no internal H-bonds [ 21, and has several potential metal-binding groups : two peptide bonds, two carboxylic acid groups, one amino group and one thiol group. However, its structure is such that few of these functional groups are likely to coordinate simultaneously to the same metal ion.In cells, one of the roles of GSH is as a substrate for GSHperoxidase, an enzyme capable of both removing hydrogen peroxide from cells and repairing peroxidativel y damaged membranes 131. It is also involved in metal detoxification, and an Correspondence to P. J. Sadler,

Formation constants of copper(i) complexes with cysteine, penicillamine and glutathione: implications for copper speciation in the human eye

“…6 which shows a mixture of predominantly Cu(GS)H  and a small amount of Cu(GS) 2 H 2 3 at this pH. However, these parallels might just be fortuitous given that Corazzo et al 15 used phosphate buffers in D 2 O (without applying corrections to pH meter readings), up to 10 times greater GSH and much lower Cl -concentrations than those employed in the present study.…”

“…Glutathione is a tri-peptide that also occurs in blood plasma 13 at concentrations of several mol dm 3 (M, where M is introduced as an abbreviation for the concentration unit mol dm 3 ) but at much higher levels (1 to 6 mM) in cells 14 . Glutathione is involved in the transport of Cu(I) among proteins and enzymes 15,16 and in the way cells respond to heavy metal poisoning. 17 The drug penicillamine has been commonly employed as a treatment for Wilson's disease 18,19 and has also been widely used to treat rheumatoid arthritis 20 .…”

“…Both Corazzo et al 15 and Poger et al 16 performed X-ray absorption measurements (mostly at just one GSH concentration, one or two GSH:Cu(I) ratios, one pH and low or zero Clconcentration) and concluded that Cu(I) is coordinated by 3 sulfur atoms. To account for measured GSH:Cu(I) ratios of 1.2 15 and 2.5 16 in the Cu(I)GSH complexes, they proposed polymeric 15 and a dinuclear species, Cu 2 (GS) 5 , 16 respectively.…”

“…To account for measured GSH:Cu(I) ratios of 1.2 15 and 2.5 16 in the Cu(I)GSH complexes, they proposed polymeric 15 and a dinuclear species, Cu 2 (GS) 5 , 16 respectively. Such species were not detected in the present study, although GSH:Cu(I) ratios ranging from 1:1 to 3:1 were investigated.…”

“…The Cu(I)GSH species containing fewer GSH ligands found in the present study appear plausible because Cu(I) is expected to be significantly coordinated to Cl -at 1 M NaCl. However, Corazzo et al 15 investigated solutions without and with Cl  (albeit at much lower concentration resulting from CuCl(s) used for sample preparation) and concluded that Cl -can be discounted as a ligand for Cu(I). We therefore cannot explain the differences between our findings and those reported by them.…”