The angiotensin metabolite His-Leu is a strong copper chelator forming highly redox active species

His-Leu is a hydrolytic byproduct of angiotensin metabolism, whose concentration in the bloodstream could be at least micromolar. This encouraged us to investigate its Cu(II) binding properties and the concomitant redox reactivity. The Cu(II) binding constants were derived from isothermal titration calorimetry and potentiometry, while identities and structures of complexes were obtained from ultraviolet–visible, circular dichroism, and room-temperature electronic paramagnetic resonance spectroscopies. Four types of Cu(II)/His-Leu complexes were detected. The histamine-like complexes prevail at low pH. At neutral and mildly alkaline pH and low Cu(II):His-Leu ratios, they are superseded by diglycine-like complexes involving the deprotonated peptide nitrogen. At His-Leu:Cu(II) ratios of ≥2, bis-complexes are formed instead. Above pH 10.5, a diglycine-like complex containing the equatorially coordinated hydroxyl group predominates at all ratios tested. Cu(II)/His-Leu complexes are also strongly redox active, as demonstrated by voltammetric studies and the ascorbate oxidation assay. Finally, numeric competition simulations with human serum albumin, glycyl-histydyl-lysine, and histidine revealed that His-Leu might be a part of the low-molecular weight Cu(II) pool in blood if its abundance is >10 μM. These results yield further questions, such as the biological relevance of ternary complexes containing His-Leu

Aβ5−xPeptides: N‑Terminal Truncation Yields Tunable Cu(II)Complexes

The Aβ5−x peptides (x = 38, 40, 42) are minor Aβ species in normal brains but elevated upon the application of inhibitors of Aβ processing enzymes. They are interesting from the point of view of coordination chemistry for the presence of an Arg-His metal binding sequence at their N-terminus capable of forming a 3-nitrogen (3N) three-coordinate chelate system. Similar sequences in other bioactive peptides were shown to bind Cu(II) ions in biological systems. Therefore, we investigated Cu(II) complex formation and reactivity of a series of truncated Aβ5−x peptide models comprising the metal binding site: Aβ5−9, Aβ5−12, Aβ5−12Y10F, and Aβ5−16. Using CD and UV−vis spectroscopies and potentiometry, we found that all peptides coordinated the Cu(II) ion with substantial affinities higher than 3 × 1012 M−1 at pH 7.4 for Aβ5−9 and Aβ5−12. This affinity was elevated 3-fold in Aβ5−16 by the formation of the internal macrochelate with the fourth coordination site occupied by the imidazole nitrogen of the His13 or His14 residue. A much higher boost of affinity could be achieved in Aβ5−9 and Aβ5−12 by adding appropriate amounts of the external imidazole ligand. The 3N Cu-Aβ5−x complexes could be irreversibly reduced to Cu(I) at about −0.6 V vs Ag/AgCl and oxidized to Cu(III) at about 1.2 V vs Ag/AgCl. The internal or external imidazole coordination to the 3N core resulted in a slight destabilization of the Cu(I) state and stabilization of the Cu(III) state. Taken together these results indicate that Aβ5−x peptides, which bind Cu(II) ions much more strongly than Aβ1−x peptides and only slightly weaker than Aβ4−x peptides could interfere with Cu(II) handling by these peptides, adding to copper dyshomeostasis in Alzheimer brains

Metal assisted peptide bond hydrolysis: Chemistry, biotechnology and toxicological implications

Metal-assisted hydrolysis of peptide bond is a promising alternative for enzymatic cleavage of proteins with prospective applications in biochemistry and bioengineering. Many metal ions and complexes have been tested for such reactivity with a number of targets, from dipeptides through oligopeptides through proteins. The majority of reaction mechanisms reported so far is based on the Lewis acidity of a given metal ion. In the alternative hydrolysis reaction the metal ion, Cu(II), Ni(II) or Pd(II), plays a structural role by forming a square planar complex with Ser/Thr–His or Ser/Thr–Xaa–His sequence, which enables a N → O rearrangement of the acyl moiety in the peptide bond downstream from the Ser/Thr residue. Both Lewis acid and N → O acyl rearrangement reaction types are discussed in detail, including molecular mechanisms, the chemical character of hydrolytic agents, reaction conditions, and the origins of differences between the results obtained for peptide and protein models. Toxicological implications and practical applications of metal assisted peptide bond hydrolysis are also presented, with a focus on the Ni(II) assisted N → O acyl rearrangement in Ser/Thr–Xaa–His sequences

cis-Urocanic acid as a potential nickel(ii) binding molecule in the human skin

cis-Urocanic acid, a derivative of histidine, is one of the essential components of human skin. We found that it can bind nickel(II) ions in a pH-dependent manner, with the dissociation constant in the low millimolar range, as revealed by potentiometry, and confirmed by isothermal titration calorimetry and UV-vis spectroscopy. The binding occurs within the physiological skin pH range. Considering the fact that cis-urocanic acid is present in the human skin in concentrations as high as millimolar, this molecule may be a physiologically important player in nickel trafficking in the human organism

Cu(II) Binding to the N-Terminal Model Peptide of the Human Ctr2 Transporter at Lysosomal and Extracellular pH

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Ni(II) ions cleave and inactivate human alpha-1 antitrypsin hydrolytically, implicating nickel exposure as a contributing factor in pathologies related to antitrypsin deficiency

Human alpha-1 antitrypsin (AAT) is an abundant serum protein, present at a concentration of 1.0–1.5 g x L−1. AAT deficiency is a genetic disease, manifesting itself with emphysema and liver cirrhosis, due to accumulation of a misfolded AAT mutant in hepatocytes. Lung AAT amount is inversely correlated with chronic obstructive pulmonary disease (COPD), a serious and often deadly condition, with increasing frequency in the aging population. Exposure to cigarette smoke and products of fossil fuel combustion aggravates AAT deficiency and COPD according to mechanisms that are not fully understood. Taking into account that these fumes contain particles that can release nickel to human airways and skin, we decided to investigate interactions of AAT with Ni(II) ions within the paradigm of Ni(II)-dependent peptide bond hydrolysis. We studied AAT protein derived from human blood using HPLC, SDS-PAGE, and mass spectrometry. These studies were aided by spectroscopic experiments on model peptides. As a result, we identified three hydrolysis sites in AAT. Two of them are present in the N-terminal part of the molecule next to each other (before Thr-13 and Ser-14 residues) and effectively form one N-terminal cleavage site. The single C-terminal cleavage is located before Ser-285. The N-terminal hydrolysis was more efficient than the C-terminal one, but both abolished the ability of AAT to inhibit trypsin in an additive manner. Nickel ions bound to hydrolysis products demonstrated an ability to generate ROS. These results implicate Ni(II) exposure as a contributing factor in AAT-related pathologies

Cysteine and glutathione trigger the Cu-Zn swap between Cu(ii)-amyloid-β4-16 peptide and Zn7-metallothionein-3

Cysteine and glutathione are able to reduce Cu(ii) coordinated to the peptide amyloidβ4-16, and shuttle the resulting Cu(i) to partially replace Zn(ii) in the protein Zn7-metallothionein-3. The released Zn(ii) in turn binds to amyloid-β4-16. Thus cysteine and glutathione are modulators of Cu/Zn-distribution between metallothionein-3 and amyloid-β4-16

Ni(II) Ions May Target the Entire Melatonin Biosynthesis Pathway-A Plausible Mechanism of Nickel Toxicity.

Nickel is toxic to humans. Its compounds are carcinogenic. Furthermore, nickel allergy is a severe health problem that affects approximately 10-20% of humans. The mechanism by which these conditions develop remains unclear, but it may involve the cleavage of specific proteins by nickel ions. Ni(II) ions cleave the peptide bond preceding the Ser/Thr-Xaa-His sequence. Such sequences are present in all four enzymes of the melatonin biosynthesis pathway, i.e., tryptophan 5-hydroxylase 1, aromatic-l-amino-acid decarboxylase, serotonin N-acetyltransferase, and acetylserotonin O-methyltransferase. Moreover, fragments prone to Ni(II) are exposed on surfaces of these proteins. Our results indicate that all four studied fragments undergo cleavage within tens of hours at pH 8.2 and 37 °C, corresponding with the conditions in the mitochondrial matrix. Since melatonin, a potent antioxidant and anti-inflammatory agent, is synthesized within the mitochondria of virtually all human cells, depleting its supply may be detrimental, e.g., by raising the oxidative stress level. Intriguingly, Ni(II) ions have been shown to mimic hypoxia through the stabilization of HIF-1α protein, but melatonin prevents the action of HIF-1α. Considering all this, the enzymes of the melatonin biosynthesis pathway seem to be a toxicological target for Ni(II) ions

Human Annexins A1, A2, and A8 as Potential Molecular Targets for Ni(II) Ions

Nickel is harmful for humans, but molecular mechanisms of its toxicity are far from being fully elucidated. One of such mechanisms may be associated with the Ni(II)-dependent peptide bond hydrolysis, which occurs before Ser/Thr in Ser/Thr-Xaa-His sequences. Human annexins A1, A2, and A8, proteins modulating the immune system, contain several such sequences. To test if these proteins are potential molecular targets for nickel toxicity we characterized the binding of Ni(II) ions and hydrolysis of peptides Ac-KALTGHLEE-am (A1-1), Ac-TKYSKHDMN-am (A1-2), and Ac-GVGTRHKAL-am (A1-3), from annexin A1, Ac-KMSTVHEIL-am (A2-1) and Ac-SALSGHLET-am (A2-2), from annexin A2, and Ac-VKSSSHFNP-am (A8-1), from annexin A8, using UV-vis and circular dichroism (CD) spectroscopies, potentiometry, isothermal titration calorimetry, high-performance liquid chromatography (HPLC), and electrospray ionization mass spectrometry (ESI-MS). We found that at physiological conditions (pH 7.4 and 37 °C) peptides A1-2, A1-3, A8-1, and to some extent A2-2 bind Ni(II) ions sufficiently strongly in 4N complexes and are hydrolyzed at sufficiently high rates to justify the notion that these annexins can undergo nickel hydrolysis in vivo. These results are discussed in the context of specific biochemical interactions of respective proteins. Our results also expand the knowledge about Ni(II) binding to histidine peptides by determination of thermodynamic parameters of this process and spectroscopic characterization of 3N complexes. Altogether, our results indicate that human annexins A1, A2, and A8 are potential molecular targets for nickel toxicity and help design appropriate cellular studies