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

AbstractSite-directed mutagenesis has been used to prepare azurin in which the methionine-121 residue has been replaced by leucine.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