Nobody's Perfect: Choice of the Buffer and the Rate of Cu2+ Ion-Peptide Interaction.

The choice of correct pH buffer is crucial in chemical studies modeling biological processes involving Cu2+ ions. Popular buffers for physiological pH are known to form Cu(II) complexes, but their impact on kinetics of Cu(II) complexation has not been considered. We performed a stopped-flow kinetic study of Cu2+ ion interactions with four popular buffers (phosphate, Tris, HEPES, and MOPS) and two buffers considered as nonbinding (MES and PIPPS). Next, we studied their effects on the rate of Cu2+ reaction with Gly-Gly-His (GGH), a tripeptide modeling physiological Cu(II) sites, which we studied previously at conditions presumably excluding the buffer interference [Kotuniak, R.; Angew. Chem., Int. Ed. 2020, 59, 11234-11239]. We observed that (i) all tested pH 7.4 buffers formed Cu(II) complexes within the stopped-flow instrument dead time; (ii) Cu(II)-peptide complexes were formed via ternary complexes with the buffers; (iii) nevertheless, Good buffers affected the observed rate of Cu(II)-GGH complex formation only slightly; (iv) Tris was a competitive inhibitor of Cu(II)-GGH complexation; while (v) phosphate was a reaction catalyst. This is particularly important as phosphate is a biological buffer.

Reactive Cu2+-peptide intermediates revealed by kinetic studies gain relevance by matching time windows in copper metallomics.

The purpose of this essay is to propose that metallomic studies in the area of extracellular copper transport are incomplete without the explicit consideration of kinetics of Cu2+ion binding and exchange reactions. The kinetic data should be interpreted in the context of time constraints imposed by specific physiological processes. Examples from experimental studies of Cu2+ ion interactions with amino-terminal copper and nickel binding site/N-terminal site motifs are used to demonstrate that duration and periodicity of such processes as bloodstream transport or neurotransmission promote the reaction intermediates to the role of physiological effectors. The unexpectedly long lifetimes of intermediate complexes lead to their accumulation and novel reactivities. The emerging ideas are discussed in the context of other research areas in metallomics.

Electrospray-Induced Mass Spectrometry Is Not Suitable for Determination of Peptidic Cu(II) Complexes.

The toolset of mass spectrometry (MS) is still expanding, and the number of metal ion complexes researched this way is growing. The Cu(II) ion forms particularly strong peptide complexes of biological interest which are frequent objects of MS studies, but quantitative aspects of some reported results are at odds with those of experiments performed in solution. Cu(II) complexes are usually characterized by fast ligand exchange rates, despite their high affinity, and we speculated that such kinetic lability could be responsible for the observed discrepancies. In order to resolve this issue, we selected peptides belonging to the ATCUN family characterized with high and thoroughly determined Cu(II) binding constants and re-estimated them using two ESI-MS techniques: standard conditions in combination with serial dilution experiments and very mild conditions for competition experiments. The sample acidification, which accompanies the electrospray formation, was simulated with the pH-jump stopped-flow technique. Our results indicate that ESI-MS should not be used for quantitative studies of Cu(II)-peptide complexes because the electrospray formation process compromises the entropic contribution to the complex stability, yielding underestimations of complex stability constants.

Kinetics of Cu(II) complexation by ATCUN/NTS and related peptides: a gold mine of novel ideas for copper biology.

Cu(II)-peptide complexes are intensely studied as models for biological peptides and proteins and for their direct importance in copper homeostasis and dyshomeostasis in human diseases. In particular, high-affinity ATCUN/NTS (amino-terminal copper and nickel/N-terminal site) motifs present in proteins and peptides are considered as Cu(II) transport agents for copper delivery to cells. The information on the affinities and structures of such complexes derived from steady-state methods appears to be insufficient to resolve the mechanisms of copper trafficking, while kinetic studies have recently shown promise in explaining them. Stopped-flow experiments of Cu(II) complexation to ATCUN/NTS peptides revealed the presence of reaction steps with rates much slower than the diffusion limit due to the formation of novel intermediate species. Herein, the state of the field in Cu(II)-peptide kinetics is reviewed in the context of physiological data, leading to novel ideas in copper biology, together with the discussion of current methodological issues.

Probing the Structure and Function of the Cytosolic Domain of the Human Zinc Transporter ZnT8 with Nickel(II) Ions.

The human zinc transporter ZnT8 provides the granules of pancreatic ß-cells with zinc (II) ions for assembly of insulin hexamers for storage. Until recently, the structure and function of human ZnTs have been modelled on the basis of the 3D structures of bacterial zinc exporters, which form homodimers with each monomer having six transmembrane α-helices harbouring the zinc transport site and a cytosolic domain with an α,ß structure and additional zinc-binding sites. However, there are important differences in function as the bacterial proteins export an excess of zinc ions from the bacterial cytoplasm, whereas ZnT8 exports zinc ions into subcellular vesicles when there is no apparent excess of cytosolic zinc ions. Indeed, recent structural investigations of human ZnT8 show differences in metal binding in the cytosolic domain when compared to the bacterial proteins. Two common variants, one with tryptophan (W) and the other with arginine (R) at position 325, have generated considerable interest as the R-variant is associated with a higher risk of developing type 2 diabetes. Since the mutation is at the apex of the cytosolic domain facing towards the cytosol, it is not clear how it can affect zinc transport through the transmembrane domain. We expressed the cytosolic domain of both variants of human ZnT8 and have begun structural and functional studies. We found that (i) the metal binding of the human protein is different from that of the bacterial proteins, (ii) the human protein has a C-terminal extension with three cysteine residues that bind a zinc(II) ion, and (iii) there are small differences in stability between the two variants. In this investigation, we employed nickel(II) ions as a probe for the spectroscopically silent Zn(II) ions and utilised colorimetric and fluorimetric indicators for Ni(II) ions to investigate metal binding. We established Ni(II) coordination to the C-terminal cysteines and found differences in metal affinity and coordination in the two ZnT8 variants. These structural differences are thought to be critical for the functional differences regarding the diabetes risk. Further insight into the assembly of the metal centres in the cytosolic domain was gained from potentiometric investigations of zinc binding to synthetic peptides corresponding to N-terminal and C-terminal sequences of ZnT8 bearing the metal-coordinating ligands. Our work suggests the involvement of the C-terminal cysteines, which are part of the cytosolic domain, in a metal chelation and/or acquisition mechanism and, as now supported by the high-resolution structural work, provides the first example of metal-thiolate coordination chemistry in zinc transporters.