Characterization of cucumber ascorbate oxidase and its reaction with hexacyanoferrate (II)

The spectroscopic features of cucumber ascorbate oxidase (AOase) and its type-2 copper-depleted (T2D) derivative, and the electron pathway among the copper sites in the enzyme have been investigated. The electronic and CD spectra of native and T2D AOase in the visible region bear a striking resemblance to those of plastocyanin or azurin, which contain type-1 copper alone. The electronic absorption shoulder of the native enzyme at around 330 nm for the native enzyme which has been assigned to type-3 copper disappears with the depletion of the type-2 copper. The reduction of AOase with a large excess of hexacyanoferrate(II) results in a selective reduction of the type-2 Cu, giving rise to an additional EPR-detectable species which is considered to be originated from partly reduced type-3 copper. The type-1 copper is, however, not reduced even in the presence of excess hexacyanoferrate(II). The redox potential of type-1 Cu was determined to be +350 mV, which is distinctly lower than that of hexacyanoferrate(II-III). Type-2 copper was supposed to be a mediator of the electron transfer between type-1 and type-3 coppers in consideration of the extremely low activity of the T2D enzyme under the same condition. A comparison of the electron pathway in AOase with that in laccase is also argued.