Effects of amino acids substitution of hydrophobic residues on haem-binding properties of designed two-α-helix peptides

Abstract

We have designed and synthesized a series of amphiphilic two-α-helix peptides, which bound Fe^III-mesoporphyrin (haem) through a ligation of two His residues. In the designed structure, amino acid residues arranged around the axial ligands were systematically substituted by hydrophobic Phe, Ile, Leu, Val and Ala residues, in order to know how their hydrophobic and/or steric differences influenced the interaction between the peptides and haem. The binding constants of the peptides with haem, which were estimated from UV–VIS measurements, were significantly correlated with the hydrophobicity at the haem-binding site. Size-exclusion chromatography demonstrated that a tetrameric assembly of peptides was induced cooperatively by the haem-binding. Furthermore, computer-modeling studies suggested that van der Waals contacts between the haem and the side-chains of amino acids around His were important for effective haem-binding. Especially, a Phe residue introduced at an appropriate position contributed to effective haem-binding via the edge-to-face interaction between the aromatic Phe side-chain and the porphyrin ring. In addition to the haem-binding properties of the peptides, the catalytic activity of the haem bound to peptides, which was similar to that of peroxidase, varied significantly depending on the amino acid composition at the haem-binding site. The results obtained in this study demonstrated that the amino acid composition and arrangement at the haem-binding site affected the haem-binding properties of the artificially designed two-α-helix peptides and the catalytic activity of the haem bound to the peptides.