CUA is an electron-transfer copper center present in heme-copper oxidases and N2O reductases. The center is a binuclear unit. with two cysteine ligands bridging the metal ions and two terminal histidine residues. A Met residue and a peptide carbonyl group are located on opposite sides of the Cu2S2 plane, these weaker ligands are fully conserved in all known CUA Sites. The Met160Gln mutant of the soluble subunit II of Thermus thermophilus ba3 oxidase has been studied by NMR spectroscopy. In its oxidized form, the binuclear copper is a fully delocalized mixed-valence pair, as are all natural CUA centers. The faster nuclear relaxation in this mutant suggests that a low-lying excited state has shifted to higher energies compared to that of the wild-type protein. The introduction of the Gin residue alters the coordination mode of His114 but does not affect His157, thereby confirming the proposal that the axial ligand-to-copper distances influence the copper-His interactions (Robinson, H. Ang, M. C.; Gao, Y. G.: Hay, M. T.; Lu, Y.; Wang, A. H. Biochemistry 1999, 38, 5677). Changes in the hyperfine coupling constants of the Cys beta -CH2 groups are attributed to minor geometrical changes that affect the Cu-S-C-beta-H-beta dihedral angles. These changes, in addition. shift the thermally accessible excited states. thus influencing the spectral position of the Cys beta -CH2 resonances. The Cu-Cys bonds are not substantially altered by the Cu-Gln160 interaction, in contrast to the situation found in the evolutionarily related blue copper proteins. It is possible that regulatory subunits in the mitochondrial oxidases fix the relative positions of thermally accessible CUA excited states by tuning axial ligand interactions.