Trinuclear Cu II Cluster Sites

Trinuclear Cu clusters play a central role in catalytic O2 reduction in the multicopper oxidases [1,76]. These enzymes contain at least four Cu centers that are necessary to catalyze the 4e reduction of O2 to H2O with concomitant le oxidations of various substrates (Fig. 6). The electrons are taken up at the T1 blue copper site and transferred 13 A to the trinuclear copper cluster site that is comprised of a T2 and a T3 copper center where O2 reduction occurs [100]. Reaction of the fully reduced enzyme with O2 involves two 2e intermediate stages, where the first 2e reduction step is rate determining (A 2 x 10 M s ) while the second is fast (k 350 s ), therefore effectively resulting in a single 4e process [101,102]. 

When the fully reduced enzyme is reacted with O2, the 4e reduced native intermediate (NI) is formed. This intermediate has been trapped using a rapid freeze-quench technique and spectroscopically characterized using EPR, absorption, CD, VTVH MCD, and XAS [22,105]. It was shown that the NI is a fully oxidized species with the three Cu(II) centers in the trinuclear site mutually bridged by the product of the full 4e O2 reduction with cleavage of the 0-0 bond. 

Orbach analysis suggests the presence of a low-lying excited doublet state at —150 above the ground state, which was confirmed by variable-temperature MCD experiments [22], 

Earlier studies have attributed this new signal to a three-electron reduced oxyl or hydroxyl species with the T2 Cu center still reduced, based on EPR studies using isotopically enriched O2 [106]. However, all four Cu centers in the NI have been determined to be oxidized by XAS edge studies [22], In addition, a pseudo-A term is observed in the CT region of flic MCD spectrum of the Nl that is very different from what is observed in the MCD spectrum of a hydroxyl radical [22,105]. Thus, XAS and MCD studies have provided definitive evidence that the Nl form is not a radical species produced by a 3e reduction of O2, but rather a 4e reduced product of O2 reduction with all four Cu centers oxidized. 

In the NI, on the otoer hand, the inequvalent Cu centers of the protein active site would allow a dominant contribution of the symmetry lowering effect into the 

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