EPR spectra for the purple mixed-valence [Cu1.5+...Cu1.5+], S = 1/2, site (CuA) in nitrous-oxide reductase (N2OR) were obtained after insertion of either 63Cu or 65Cu or both 65Cu and [15N]histidine. The spectrum of 65Cu- and [15N]histidine-enriched N2OR improved the resolution of the Cu hyperfine lines, but no lines from nitrogen and proton couplings were resolved. The Cu hyperfine parameters obtained by a theory analogous to that of Maki and McGarvey were indicative of a highly covalent Cu site. The total Cu character (CuA1 + CuA2) in the ground state wave function required to describe the spin density distribution was 31−37% compared to 41% for type-1 Cu in plastocyanin. This value does not completely account for the reduction of gmax from 2.23 of type-1 Cu in plastocyanin to 2.18 of CuA. Remaining discrepancies were discussed in terms of different alignments of the principal axes for the hypothetical monomeric CuA1 and CuA2 in [Cu1.5+...Cu1.5+]. This effect appeared in the simulations of the EPR spect...