copper binding peptides

Many of us may be accustomed to getting suspicious sounding science news on our cell phones.  A customer sent Mitosynergy this link .   Sure enough, I got the same story on my cheap smart phone.  Fake News!!  Unfortunately the new clip did not do a very interesting study justice.  The author did provide a link to the online publication.

Summary/abstract for Scientists

“The amino‐terminal copper and nickel/N‐terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards CuII ions and have been implicated in human copper physiology….One of these novel intermediates, characterized by two‐nitrogen coordination, t_1/2 ≈100 ms at pH 6.0 and the ability to maintain the Cu^II/Cu^I redox pair is the best candidate for the long‐sought reactive species in extracellular copper transport.”

Why I think this is a cool paper for Mitosynergy and its customers

• Charlie Barker asked me if one could see  Cu^II/Cu^I redox cycling color changes of Cu in cytochrome C oxidase in the mitochondria.  My reply was that if you could build a spectrophotometer small enough one could.
• Charlie Barker made the big deal that oxidation and reduction are dependent on pH.  This is true.   This is what charged amino acid side chains do in enzymes.  They control the local pH, never you mind what the pH is in the bulk environment.
• This is the problem with these Cu^II/Cu^I redox cycling peptides.  There is absolutely no local pH control.  There is absolutely no control of where they may pick up electrons Shown there are Fenton and  Haber Weiss reactions.  Hydrogen peroxide and superoxide may be products of the mitochondria.

What is stopped flow? 

Stopped flow is a way of measuring the kinetics of a reaction.  I’ve taken much liberty in embellishing supplemental figures so that you, the lay reader, can understand this very interesting work.  In panel A we have the basic stopped flow design.  The one I used in grad school had a trigger that released compressed air on the drive that pushed a set amount of contents in syringe A and syringe B and sent them to the mixer.  The same trigger that released the compressed air also started measuring fluorescence in the optical cell.  The same trigger that released the compressed air also started the recording.  of the reaction going on in the optical cell.  These authors measured absorbance. In Panel B I added the structure of the buffers they used to set the pH of the reactions.  Note that MES and HEPES have very similar structures.   These authors are careful and know their stuff.

In panel C we have a time dependent increase in the absorbance of what Kotuniak concluded to be the final product, 4N.  Naturally, the absorbance of the intermediate decreased with time.  Note that all of this happens in less than a second.   If our eyes were fast enough, we’d see a cyan colored complex turn to a sort of magenta color, based on its absorbance of green light.  Note that the higher the pH, the faster the reaction with  Cu²⁺.  No reaction was seen at stomach pH.  If this reaction occurs in humans, it is as peptides and Cu²⁺ enter the duodenum.   Now that we’ve covered what stopped flow is, now we need to cover what a peptide is.

Some peptide basics

Amino acids link together to form peptides and proteins just like pop beads link together to form strings. The “carboxy terminus” of one bead fits into the “amino terminus” of another bead, and so on.Peptides are made up of amino acids.  Amino acids have an NH₂ group on the amino terminus and a -COOH group on the carboxy terminus.  By abstracting an H₂0 a “peptide bond” is formed.

What is this  amino‐terminal copper and nickel/N‐terminal site (ATCUN/NTS) ?

We now know what a peptide bond and N-terminus are.  The Cu/Ni  site is any two N-terminal amino acids and a histine in the number 3 position.  The N-terminal sequence woudl be X-X-His where X = any amino acid.   The authors used Glycine because it is the simplest amino acid.  Panel A shows the structures of the amino acids Glycine and Histidine.  The carboxy terminus of Gycine and hte amino terminus of Histidine have been circled.  Panel B shows the formation of a peptide bond between two glycines  Panel C shows the structure of the Gly-Gly-His peptide. Heavy lines denote the peptide bond. The histidine side chain is circled panel D.

The authors mentioned human serum albumin in their review of the literature.Let’s take a closer look and see what they mean.

Based on pH alone, we can expect this peptide to bind copper in the blood.

How does  Cu²⁺  bind to the  ATCUN/NTS motif?

We should add that Kotuniak were also able to freeze the reactions and use EPR to solve the structures of the reaction intermediates and product.

And what about redox cycling?

Kotuniak (2020) used cyclic voltametry to measure the redox potential of the complexes.  Wikipedia gives a rather nice overview of this rather complicated physical technique.

This brief overview is not giving the publication or the ample supplemental information that supports the publication.  The authors referenced previous work on scanning voltammetry work performed on Azheimer’s Disease Aβ peptides.

Implications of this work for Mitosynergy

Kotuniak demonstrated that the copper complexes do not form at pH 1-3 of the stomach.  As partially digested food leaves our stomach and enters our duodenum, our pancreas secretes bicarbonate and digestive enzymes.

Kotuniak (2020) were careful to use chemically related buffers.  What if we were to repeat these experiments with physiological buffers?

• We could have peptides and a standard Cu²⁺ supplement and peptides in one stopped flow syringe and a bicarbonate solution in another.
• Does this “toxic copper” form complexes in duodenum conditions that can redox cycle?
• What about Cu(I)NA₂?
• Can Cu(I)NA₂ donate Cu⁺ to peptides?

Kotuniak R, Strampraad MJF, Bossak-Ahmad K, Wawrzyniak UE, Ufnalska I, Hagedoorn PL, Bal W.(2020)Key Intermediate Species Reveal the Copper(II)-Exchange Pathway in Biorelevant ATCUN/NTS Complexes. Angew Chem Int Ed Engl. 2020 Apr 8. doi: 10.1002/anie.202004264. [Cross Ref]