X‐ray crystallographic characterisation of type‐2‐depleted ascorbate oxidase from zucchini

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Abstract
The type‐2 depleted form of ascorbate oxidase from zucchini has been prepared in crystals and characterised by X‐ray crystallography and EPR spectroscopy. The X‐ray structure analysis by difference‐Fourier techniques and refinement shows that, on average, about 1.3 Cu atoms/ascorbate oxidase monomer are removed. The copper is lost from the trinuclear site whereby the EPR‐active type‐2 copper is depleted most; type‐1 copper is not affected. This observation indicates preferential formation of a 1 Cu‐depleted form with the hole equally distributed over all three copper sites. Each of these 1 Cu‐depleted species may represent an anti‐ferromagnetically coupled copper pair which is EPR‐silent and could explain the disappearance of the type‐2 EPR signal.