The nuclear modulation effect in pulsed EPR spectroscopy was used to study the type 2 copper binding site in the mercury derivative of laccase (MDL) in which the type 1 copper is substituted by Hg(II). By comparing the three-pulse electron spin-echo modulations and Fourier transform spectra of MDL and several model compounds, we conclude that the imidazole groups of two histidyl amino acid residues are equatorially coordinated to Cu(II) in the type 2 site. Computer simulations of these data suggest that the remote nonbonding nitrogens of the two imidazoles possess nuclear quadrupole parameters e2qQ = 1.47 MHz and eta = 0.83. A(iso) values of these two nitrogens are not identical, being 1.5 and 2.0 MHz. We have also used samples of the enzyme exchanged with D2O to examine the coordination of the water to the type 2 copper site. The deuterium modulation that is resolved by taking the ratio of the time domain ESEEM data from native and D2O-exchanged enzyme indicates that there is an equatorial water ligand, and further data show that this water is displaced by azide.

Temperature-dependent structural changes involving the type 2 site in laccase are probed by EPR studies of a derivative of laccase in which the type 1 Cu has been replaced by Hg(II) [Morie-Bebel, M. M., Morris, M. C., Menzie, J. L., & McMillin, D. R. (1984) J. Am. Chem. Soc. 106, 3677-3678]. At the temperature extremes (123 and 299 K), single well-defined species are present, but at intermediate temperatures (between 213 and 253 K), the presence of multiple structures is indicated. For the first time, the room temperature EPR spectrum of the type 2 copper has been resolved. Azide binding and fluoride binding have also been studied as a function of temperature. The results suggest that each anion preferentially interacts with the type 3 site in fluid solution and that these adducts can be trapped by rapidly cooling the sample to 123 K. Annealing the adducts at 253 K permits rearrangement and binding at an equatorial position of the type 2 Cu. This pathway to anation at the type 2 site contrasts sharply with previous studies which required a large excess of anions, and it reveals important insight into the flexibility of the type 2/type 3 cluster in laccase.

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