Glutathione the Copper Chaperone

This post examines a report from the laboratory of Dr Christopher Fahrni at Georgia Tech University. What this group is saying is that the small molecule glutathione can chaperone intra cellular Cu+ to really low femptomole levels. [1]

This is a glutathione molecule. The carboxy C-terminus (had end) is a glycine. A carboxy group is -COOH. The “butt end” N-terminus of glycine is –NH2, an amino group. The middle amino acid is cysteine with the yellow –SH side chain. The Carboxy group of csyteine can come behind the butt end of glycine and take a big ole bite and kick off a H2O in the process. An amide bond is formed. The tail end of glutathione is formed when the -COOH of the side chain of glutamine comes in and takes a big ole bite of the butt of cysteine. Again, a H2O is formed from the OH of the glutamine side chain and a Hof the -NH2 amino terminus of cysteine. The The pKa values are from UCLA.

a molecule of glutathione (GSH) . Note, biochemists call the butt end the N-terminus. The “head” end is called the C-terminus.

The pKa in very simple terms is the pH in which 50% of the green circled “H” are there are dissociated leaving no charge in case of the amino (butt) terminus -NH2 group or the carboxy terminus -COO group.

The official macroscopic pKa values corresponding to sequential deprotonation of the Glu-α-COOH, Gly-COOH, –SH, and Glu-NH3+ groups, of GSH are 1.98, 3.49, 8.64, and 9.36. GSH exists primarily as a mono anion at neutral pH. [1]

The Fahrni group used three water soluble monovalent Cu ligands (MCL1-3) with known affinities for Cu+ to compete with GSH. We will not present the sophisticated optical techniques they used to use these competing MCL1-3 copper ligands to measure the interaction of GSH molecules for Cu+.

Quoted from the publication: “Free GSH exists primarily in mono anionic form at pH 7, whereas cluster formation presumably entails S-deprotonation to the corresponding dianion, which we represent as GS. An equilibrium between clusters differing in Cu(I)/glutathione ratio would therefore depend on [GS], which is influenced by both [GSH] and pH. Once the latter is significantly below the thiol pKa of 8.75, each further unit drop in pH will decrease the GS/GSH ratio by 10-fold. We therefore measured pairs of solutions containing either 1 mm GSH or 10 mm GSH at 1 unit lower pH.” The typical GSH-Cu complex was determined to be [Cu4(GS)6] as the dominant species over a wide pH range, from 5.5 to 7.5. [1]

MCL1-3 are probes the group used to compete with GSH for Cu+. Cu4S6, Cu5S7, and Cu5S6 are theoretical models based on other studies in the literature. Only the carbon next to the Cu binding S is shown for clarity.

Skipping some complicated technigues, the Fahrni group came up with an equation for “parts” Cu+ that is analogous to parts H+, or pH. pH is the –log base 10 of the concentration of H+ in molar. The log10 of 0.1 M H+ is “-1” Minus the log10 is pH 1. Going in the opposite direction, the concentration of H+ at pH 10 is 10-10 moles per liter H+, or 0.0000000001 moles per liter!

pCu=1.5log[GSH] + 1.5pH−0.25log[Cu(I)]total + 8.26

  • Say some catastrophic even occurs and results in 10mM GSH being oxidized to 4 mM GSSG leaving only 2 mM GSH. The first term goes from 1.5x -2 = -3 to 1.5x-2.7 = -4
  • For the 2nd term, say we go from 1.5 x pH 7.4 = 11.1 to 1.5 x acidic pH 6 = 8
  • 10uM total Cu = -0.25x (-5)= 2.5 and 1uM = -2.5x (-6)= 1.5

Good conditions: pCu= -3 + 11.1 – 2.5 + 8.26 = 14 10 -14 M free Cu is really low. We could be at f10-15 M or 1 fM free copper if we went even lower in total copper. Not so good conditions: pCu = -4 + 8 -1.5 + 8.26 = 10.8 = 10-11 M free Cu. pH and reduced glutathione are far more important determinants of free copper, than total copper.

Prior to the Fahrni group’s study, scientists had known for decades that Cu+, in the very reducing interior environment of the cell, formed complexes with reduced glutahione. This is the genius ov cuprous nicotinic acid that we call CopperOne. In this discussion we should not forget those that market NAD.

Why we need niacin and NADH/NADPH

Niacin is a precursor to nicotinamide, which is part of nicotinamide adenine dinucelotide (NAD) that may be phosphorylated shown as a phosphate not totally attached to a ribose group. The NADPH/NADP of source of reducing equivalents maintains the reduced state of the GSH thiol.

Bernt an coauthors have written a nice review on the role of thioredoxin and glutaredoxin in maintaining the reduced state of glutahione and protein thiols. [2] This figure only shows GSH in a GSSG disulfide bond that prevents proper buffering of intracellular copper. Further oxidation states are also possible. if it were not for NAD/NADP and thio and glutaredoxin. [2]

We become so worried about an increase in total copper in our cells and forget that free copper is more deleterious.

If we have a decrease in electron transport chain activity because complex IV lacks Cu, we have an increase in glycolysis and a decrease in pH. This increases free Cu. If we do not have fully active Cu/Zn superoxide dismutase, the same -S group in GSH might also become oxygenated to cysteine sulfenic acid -SOH that is a precursor to -S-S- disulfide bond. [2] Higher order cysteine oxidation products are possible. [2] Perhaps we should worry less on the affect of extra copper on free copper when we also should think about the role of niacin and NAD that contribute to this remarkable balance of nature that keeps the free copper in our cells insanely low!


  1. Morgan, M. T., Nguyen, L., Hancock, H. L., & Fahrni, C. J. (2017). Glutathione limits aquacopper(I) to sub-femtomolar concentrations through cooperative assembly of a tetranuclear cluster. The Journal of biological chemistry, 292(52), 21558–21567. PMC free article
  2. Berndt C, Lillig CH, Holmgren A. (2007) Thiol-based mechanisms of the thioredoxin and glutaredoxin systems: implications for diseases in the cardiovascular system. Am J Physiol Heart Circ Physiol. 2007 Mar;292(3):H1227-36. free article

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