Spectroscopic evidence for interactions between hexacyanoiron(II/III) and an engineered purple Cu_A azurin

Interactions between hexacyanoiron(II/III) and a dinuclear, mixed valence Cu_A center in engineered Cu_A azurin have been investigated by UV-visible (UV-vis) and electron paramagnetic resonance (EPR) spectroscopic techniques. Addition of ferricyanide (hexacyanoiron(III)) to the Cu_A azurin resulted in a new absorption band around 500 nm in the UV-vis and an isotropic line at g=2.16 in the EPR spectra. Control experiments, including additions of Cu(II)SO₄ or Cu(I)(CH₃CN)₄PF ₆ to ferricyanide or ferrocyanide, as well as gel filtration purification of the ferricyanide-Cu_A azurin adduct indicate complex formation between cupric ion and ferrocyanide ion in the protein. Solvent or small molecule accessibility, metal oxidation state and the presence of more than one metal ion are potential factors important for the complex formation. These findings must be taken into consideration when using ferricyanide or ferrocyanide as redox agents for studying Cu_A centers in proteins.