ABSTRACT
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.
Subject(s)
Copper , Copper/chemistry , Buffers , Hydrogen-Ion Concentration , Kinetics , Coordination Complexes/chemistry , Peptides/chemistry , Oligopeptides/chemistry , Ions/chemistryABSTRACT
Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV-vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter.
Subject(s)
Coordination Complexes/metabolism , Copper/metabolism , Glutathione/metabolism , Oligopeptides/metabolism , Sulfhydryl Compounds/metabolism , Coordination Complexes/blood , Coordination Complexes/chemistry , Copper/blood , Copper/chemistry , Glutathione/blood , Glutathione/chemistry , Humans , Molecular Structure , Oligopeptides/blood , Oligopeptides/chemistry , Oxidation-Reduction , Sulfhydryl Compounds/blood , Sulfhydryl Compounds/chemistryABSTRACT
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. Little is known, however, about the rate and exact mechanism of formation of such complexes. We used the stopped-flow and microsecond freeze-hyperquenching (MHQ) techniques supported by steady-state spectroscopic and electrochemical data to demonstrate the formation of partially coordinated intermediate CuII complexes formed by glycyl-glycyl-histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two-nitrogen coordination, t1/2 ≈100â ms at pHâ 6.0 and the ability to maintain the CuII /CuI redox pair is the best candidate for the long-sought reactive species in extracellular copper transport.
Subject(s)
Copper/chemistry , Ion Transport , Proteins/chemistry , Spectrophotometry, Ultraviolet , Surface Plasmon ResonanceABSTRACT
The influence of cation-π interactions on the electrochemical properties of copper(II) complexes with synthesized pentapeptide C-terminal fragment of Atrial Natriuretic Factor (ANF) hormone was studied in this work. Molecular modeling performed for Cu(II)-NSFRY-NH2 complex indicated that the cation-π interactions between Tyr and Cu(II), and also between Phe-Arg led to specific conformation defined as peptide box, in which the metal cation is isolated from the solvent by peptide ligand. Voltammetry experiments enabled to compare the redox properties and stability of copper(II) complexes with NSFRY-NH2 and its analogues (namely: NSFRA-NH2, NSFRF-NH2, NSAAY-NH2, NSAAA-NH2, AAAAA-NH2) as well as to evaluate the contribution of individual amino acid residues to these properties. The obtained results led to the conclusion, that cation-π interactions play a crucial role in the effective stabilization of copper(II) complexes with the fragments of ANF peptide hormone and therefore could control the redox processes in other metalloproteins.
