Low-temperature magnetic circular dichroism studies of native laccase: spectroscopic evidence for exogenous ligand bridging at a trinuclear copper active site.

The detailed nature of N-3 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-3 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-3 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-3 bridges the paramagnetic type 2 and diamagnetic type 3 binuclear Cu(II) 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(II) sites. In these sites, N-3 has displaced the endogenous bridge, thereby rendering the site paramagnetic and detectable by both LTMCD and EPR spectroscopy. High-affinity N-3 is found to bind to the paramagnetic type 2 Cu(II) site in a limited fraction of the protein molecules that contains reduced type 3 sites. Finally, the possible role of this trinuclear (type 2-type 3) Cu(II) active site in enabling the irreversible reduction of dioxygen to water is considered.