Spectroscopic evidence for interactions between hexacyanoiron(II/III) and an engineered purple CuA 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)SO(4) or Cu(I)(CH(3)CN)(4)PF(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.