thiols and sulfur

Copper Two and homocysteine

Homocysteine and copper, who’d have thought? A group from Sofia Bulgaria were thinking about such things back in 2003. These authors gave some reasons for caring.

  1. Having too much comocysteine in our blood, hyperhomocystemia, is associated with B12 and folate deficiency as well as cerebrovascular, neuronal, and renal diseases.
  2. Homocysteine can form disulfide bonds with itself, other small thiol containing molecules, and protein thiols. Homocysteine can form a disulfide bond with itself or with cysteine. These authors claim 70% of the total homocysteine!

Reading these molecular structures can be difficult at first. Red letters indicate atoms with a partial to outright negative charge. Oxygen has a big nucleus and is an electron hog. Nitrogen tends to have a positive charge. Sulfurs are always colored yellow. Hydrogens and carbons don’t count in the charge tallying. Carbons are never drawn out. We assume that there is a carbon at each kink. Cysteine has three kinks and three carbons. Homocysteine has four carbons and four kinks. Note the two red oxygens in both homocysteine and cysteine. The one H can come off leaving an electron that is shared between the two oxygens. This is indicated by a dashed line.

From left to right: cysteine has one less carbon than homocysteine. Homocystine is homocysteine disulfide bonding (S-S) to another cysteine. Homocysteine can form a mixed disulfide by forming a disulfide bond with cysteine. Protein such as the U shaped blob may have cysteines in their sequences. These cysteines can form disulfide bonds with homocysteine too.

Glutathione peroxidase protects cells and enzymes from oxidative damage, by catalyzing the reduction of hydrogen peroxide, lipid peroxides and organic hydroperoxide, by glutathione.

A peroxide is any molecule with two oxygens (R-O-OR) connected by a single bond. R can be a hydrogen, lipids, or other organic molecules. Cells control the activity of GPx by how much messenger RNA that transcribe they make and translate into proteins or other post translational modifications of the GPx protein. Glutatione is a tripeptide of glutamate, cysteine, and glycine.

All of these thiol compounds are inter-connected. What happens when eh endothelial cells are loaded up with hCys and allowed 72 hours, three days, to transform the hCys into something else. Or not. Maybe the cells convert homosyteine to cysteine and make some glutathione. Or not.

Human endothelial cells (cell line EA.hy 926) and pro-monocytes (cell line U937) were treated with 100μM homocysteine, 100μM CDuCl2, or a combination of the two. Monocytes, an immune cell, may push their way through endothelial cells, a type of cells in our blood vessels. How does homocysteine with and without copper…. with and without hydrogen peroxide, H2O2 affect this process? This was examined in Figures 4 and 5.

This post is honoring copyright rules and estimating some numbers off the bar graphs from the journal article. [1]

FigassaycontHCyCuHCyH2O2CuHCy Cu
2GSH peroxidase activity17a17a8b9b16a
GSHPx mRNA100a50b40c30bc30c
3homocysteine, μM extracelular0a20b40c30d0a
glutathione, μM28a32b50c40d45c
cysteine, μM180a180a110b150c110b
5A monocyte round on top %80a40b35c70d75a
B spread on top5a30b25c7a10a
C transmigrating %5a15b20c7a5a
D underneath %1a7b12c5b1a
These numbers were estimated from bar graphs. The reader is invited to read the public access publication [1] for themselves. The superscripts indicate significant differences at p<0.01. This means we are 99% certain that the observed differences are not due to random chance.
  • Figure 2, The authors saw a big hit in glutathione peroxidase activity with copper/homocysteine chombos.The GSH peroxidase mRNA messages waiting to be translated into protein also decreased.
  • Figure 3 Extracellular concentrations of homocysteine, glutathione and cysteine following 72 h treatment with 100 mM hCys, Cu or Cu–hCys complexes. So these endothelial cell monolayers were sucking in the Cu(II)-hCys complexes and then spitting them back out in a variety of different forms three days later.
  • Figure 5 data suggest that the Cu(II)-hCys complex makes monocytes particularly invasive when there’s a little bit of H2O2 around.

Apostolova and coauthors cited a 1983 with some stunning electron microscope images of monocytes invading the protective endothelial layer of aortas of rats on a high cholesterol diet. Joris 2018 free paper. This invasion is thought to be part of the atherosclerosis process.

In 2006 Catalina Carrasco-Pozo published a study that suggested CuI)-hCys complexes are “redox inactive.” In this stable form Cu(I) <=> Cu(I) cannot shuttle back and forth dumping electrons onto O2 to form super oxide in the process. [2] Depending
on the molar Hcys:Cu (II) ratio, the Interaction resulted in two types of complexes

  • ratios up to 2:1 hCys:Cu(II) are time stable and ascorbate reducible.
  • ratios up to 4:1 are ascorbate- and oxygen-redox-Inactive
  • > 4:1 super oxide generating activity.

There is still a claim of a complex, but the authors offer no cartoons as to what the complex might look like.

Left, a rendition of the Apostovolova 2003 propped structure. Each Cu(II) atom forms bond with the thiol, the amine nitrogen (N) and the carboxyl group oxgen (O).

Carrasco-Pozo and coauthors never really told us what these “up to 2:1” hCys:Cu(II) structures might look like. The APostolova 2003 structure is 1:1. The reactants that this post is about to discuss actually contain two hCys. The structure shown here is 4:1.

In 2021 Megha Gupta and coauthors asked the question of why the Cu(II) and hCys complex is so much more dangerous Cu(II) with cysteine, methionine (another sulfur containing amino acid), and glutathione, a cysteine containing anti-oxidant tripeptide. [3] These authors have some images of blue Cu(II) complexed with glutathione, cysteine, and methione. It’s a nice Cu(I) brown complexed to homosyteine. [3] Let’s take a very superficial journey of the chemistry revealed in this publication. Speculation on this post’s part will be duly noted.

  1. Meet Cu(I) and Cu(II). Copper has 29 protons and some neutrons. In the metallic state there is an extra outer shell with just one electron. It is speculated that this is why copper metal makes such a great conductor of electricity. Cu(I) has 28 electrons, the outer shell is full with 18 electrons. Cu(II) has only 27 electrons. The outer shell is one electron short of being full.
  2. This is homocysteine in its official structure
  3. The sulfur group can lose a H to become a thiyl radical. A radical is an unpaired electron. Nature drives electrons to pair up making radicals very reactive.
  4. The unpaired electron on the sulfur can migrate to the carbon that is bound to the nitrogen (N). This is because the Hcy thiyl radical can undergo a kinetically favored hydrogen atom transfer (HAT) reaction to afford a “captodatively” stabilized Cα-radical.
  5. Cu(II) looks at this situation as an opportunity to fill up its outer shell with electrons. It steals the radical electron from homocysteine becoming Cu(I) in the process. Note that what is shown here is a combination of Figure 1 and Scheme 1. There’s a slightly different scheme 2 version of what could explain the new species
  6. We get some inter molecular reactions that transform two homocysteines that look quite a bit different to the point of no longer being homocysteine. The arrows going in both direction indicate an equilibrium

It is speculation that Cu(II) is far more likely than Cu(I) to steal electrons from radical forms of homocysteine.

This post is only scratching the surface of why Cu(II) are a heart disease causing combination. We have ignored most of the literature of protein mixed disulfides with homocysteine that may occur without Cu(II) We didn’t much get into pro- and anti- antioxidant duality. One would hope that Gupta and coauthors are following up on their work. With a little bit of Cu(II) can hCys form permanent structures with proteins that are not reversible like mixed disulfides?

  1. Apostolova MD, Bontchev PR, Ivanova BB et al (2003) Copper-homocysteine complexes and potential physiological actions. J Inorg Biochem 95:321–333. SciHub free article
  2. Carrasco-Pozo C, Álvarez-Lueje A, Olea-Azar C et al (2006) In vitro interaction between homocysteine and copper ions: Potential redox implications. Exp Biol Med 231:1569–1575. Sci-Hub free article
  3. Gupta M, Meehan-Atrash J, Strongin RM. (2021) Identifying a role for the interaction of homocysteine and copper in promoting cardiovascular-related damage. Amino Acids. 2021 May;53(5):739-744. Sci-Hub free article

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