The pentapeptide NHSFM, derived from the surface-exposed region of the metal ion binding loop in subunit II of cytochrome c oxidase, has been investigated for its role in the maturation of the binuclear purple CuA center. Using a combination of experimental and computational techniques, we demonstrate that copper ions form a 1:1 complex with NHSFM, exhibiting a binding constant in the range of 10⁴ to 10⁵ M⁻¹. Spectroscopic and structural analyses reveal a type 2 copper coordination environment involving four ligands—histidine (H), methionine (M), the N-terminal amine of asparagine (N), and a carbonyl oxygen from the asparagine side chain—with water molecules also participating in the coordination sphere. pH-dependent studies indicate a pKa of approximately 10, suggesting deprotonation of the N-terminal amine plays a key role in modulating copper binding.

Extended X-ray absorption fine structure (EXAFS) measurements combined with density functional theory (DFT) calculations confirm pH-dependent changes in metal-ligand bond distances. Time-dependent DFT (TDDFT) simulations reproduce the experimentally observed UV–visible absorption profiles, showing a distinct blue shift at higher pH due to deprotonation of the N-terminal amine. Restrained molecular dynamics (RMD) simulations further support a distorted square planar geometry around copper, where the asparagine carbonyl oxygen and two water molecules coordinate alongside H, M, and the deprotonated N-terminus. These findings suggest that copper binding induces conformational rigidity and compaction in the peptide backbone, likely facilitating sequential copper uptake during CuA center assembly.

The structural data, including B-factor analysis and interatomic contact mapping, indicate that the apo-form of the coordinating loop is more flexible, while the holo-form exhibits reduced backbone mobility. This transition implies that copper binding triggers a structural rearrangement that brings distant residues into closer proximity, enabling formation of the final Cu₂S₂ core.RAD9A Antibody manufacturer The conserved nature of the NHSFM sequence across eukaryotes suggests it functions as an initial recognition site for incoming copper ions during the conversion of apo-CuA to its mature holo-state.Actin Muscle Specific Antibody site Together, these results highlight the importance of protonation states and dynamic backbone restructuring in directing metal ion delivery to the active site.PMID:34811469

Keywords: Cytochrome c oxidase; Copper binding peptide; TDDFT; Restrained molecular dynamics (RMD) simulations; EXAFSMedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com