The detailed nature of N-binding at the multi-copper active site in native laccase is investigated through a combination of low-temperature magnetic circular dichroism (LTMCD) and absorption spectroscopies. This combination of techniques allows charge-transfer spectral features associated with N-binding to the paramagnetic type 2 Cu(II) to be differentiated from those associated with binding to the antiferromagnetically coupled, and therefore diamagnetic, binuclear type 3 Cu(II) site. Earlier absorption titration studies have indicated that N-binds with two different binding constants, yielding a high-affinity and a low-affinity form. The studies presented here are interpreted as strong evidence that low-affinity N-bridges the paramagnetic type 2 and diamagnetic type 3 binuclear Cu(ll) sites in fully oxidized laccase. This assignment is further supported by features in the MCD spectrum whose intensity correlates with an EPR signal associated with uncoupled type 3 Cu(ll) sites. In these sites, N-has displaced the endogenous bridge, thereby rendering the site paramagnetic and detectable by both LTMCD and EPR spectroscopy. High-affinity N-is found to bind to the paramagnetic type 2 Cu(ll) site in a limiited fraction of the protein molecules that contains reduced type 3 sites. Finally, the possible role of this trinuclear (type 2-type 3) Cu(ll) active site in enabling the irreversible reduction of dioxygen to water is considered.

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