Rack‐induced bonding in blue copper proteins: Spectroscopic properties and reduction potential of the azurin mutant Met‐121 → Leu

Abstract

Site‐directed mutagenesis has been used to prepare azurin in which the methionine‐121 residue has been replaced by leucine. The oxidized mutant protein displays the strong blue color and characteristic EPR signal of a type 1 Cu(II) ion, showing that methionine is not an obligatory component of a blue copper site. The optical absorption maximum is shifted 5 nm towards longer wavelength and the extinction coefficient increased by about 10% compared to the wild‐type protein. In addition, there are small changes in the EPR parameters, in particular the copper hyperfine splitting. The reduction potential is increased by 70 mV. The results show that a small change in primary structure without any alteration in the three strong ligands can perturb the Cu(II) site and shift the reduction potential, in accord with the concept of rack‐induced bonding in blue copper proteins.