Spectroscopic Differentiation of the Electron‐Accepting Sites in Fungal Laccase

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Abstract
The difference specrum between oxidized and reduced fungal laccase at pH 5.5 shows an absorption band at 330 nm in addition to the visible absorption band at 610 nm. The 610 nm band and the 330 nm band are reduced together in a linear fashion with the addition of 3.5 electron equivalents of ascorbate. Titration of laccase in the presence of 3 mM fluoride leads to a differentiation of the electron‐accepting site in the molecule. The 610 nm band is found to be associated with a single electron‐accepting site and the 330 nm band is associated with a two electron‐accepting site. Electron paramagnetic resonance analysis during the titration indicates that neither the Type 1 Cu2+(the “blue” Cu2+) nor the Type 2 Cu2+ (the “non‐blue” Cu2+) are associated with the 330 nm band. Titration of laccase at pH 8.3 with quinol also associates the 330 nm band with a two electron‐accepting site. Preliminary stopped‐flow kinetic measurements on the absorbance changes at 330 nm and 610 nm are described and the results suggest an involvement of the component responsible for the 330 nm band in the catalytic reaction. The nature of the component responsible for the 330 nm band is discussed in the light of these results and our previous titration results. The findings are consistent with the 330 nm band being associated with the two additional copper atoms in the molecule and agree with our previous suggestion that these copper atoms exist as an electron‐accepting Cu2+ ‐Cu2+ pair in the oxidized protein.

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