Identification of a copper ion recognition peptide sequence in the subunit II of cytochrome c oxidase: a combined theoretical and experimental study

Abstract

The role of the pentapeptide, NHSFM, derived from the surface exposed part of the metal ion binding loop of the subunit II of cytochrome c oxidase on the maturation of the binuclear purple CuA center of the enzyme has been investigated using several experimental and computational methods. The copper ion was found to form 1:1 complex of the pentapeptide with a binding constant ~ 10⁴ M⁻¹ to 10⁵ M⁻¹, where a 4 ligand coordination from the peptide in a type 2 copper center was revealed. The pH dependence of the metal–peptide was associated with a \({\text{p}}K_{{\text{a}}}\) of ~ 10 suggesting deprotonation of the N-terminal amine. EXAFS studies as well as DFT calculations of the metal–peptide complexes revealed pH dependent changes in the metal–ligand bond distances. Spectroscopic properties of the metal peptides calculated from TDDFT studies agreed with the experimental results. Restrained molecular dynamics (RMD) simulations indicated coordination of a carbonyl oxygen from the asparagine (N) side chain and of water molecules apart from histidine (H), methionine (M) and terminal amine of asparagine (N) in a distorted square planar geometry of Cu–NHSFM. Analyses of the backbone distances as well as B-factors for the metal peptide suggested that the peptide backbone becomes more compact and rigid on binding of the metal ion. This indicated that binding of copper ion to this pentapeptide in the protein possibly cause movement of the protein backbone bringing other coordinating residues closer to the copper ion, and thus helping in sequential uptake of copper ions to the protein. Graphic abstract