Abstract
Interactions between hexacyanoiron(II/III) and a dinuclear, mixed valence CuA center in engineered CuA azurin have been investigated by UV–visible (UV–vis) and electron paramagnetic resonance (EPR) spectroscopic techniques. Addition of ferricyanide (hexacyanoiron(III)) to the CuA 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)4PF6 to ferricyanide or ferrocyanide, as well as gel filtration purification of the ferricyanide–CuA 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 CuA centers in proteins.
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