Loop Stability in the Engineered Potassium Binding Site of Cytochrome c Peroxidase

The Trp191 containing flexible loop of cytochrome c peroxidase (CcP) exists in equilibrium between open and closed conformers. The open conformer creates a cavity in the loop, which enables it to bind protonated forms of imidazole derivatives such 1,2-dimethylimidazolium (DMI). In the present study we have engineered the K+ binding site into CcP and find the equilibrium of the conformer shifted in favor of the open form probably due to electrostatic destabilization. Subsequent changing of a hinge residue in the loop, Asn195, to Pro stabilizes the loop in the presence of the bound K+.