The effect of axial ligand mutation on the Cu-A Site in the recombinant water soluble fragment of subunit II of Thermus thermophilus cytochrome c oxidase bas has been investigated. The weak methionine ligand was replaced by glutamate and glutamine which are stronger ligands. Two constructs, M160T0 and M160T9, that differ in the length of the peptide were prepared. M160T0 is the original soluble fragment construct of cytochrome bas that encodes 135 amino acids of subunit II, omitting the transmembrane helix that anchors the domain in the membrane. In M160T9 nine C-terminal amino acids are missing, including one histidine. The latter has been used to reduce the amount of a secondary T2 copper which is most probably coordinated to a surface histidine in M160T0. The changes in the spin density in the Cua site, as manifested by the hyperfine couplings of the weakly and strongly coupled nitrogens, and of the cysteine P-protons, were followed using a combination of advanced EPR techniques. X-band (similar to9 GHz) electron-spin-echo envelope modulation (ESEEM) and two-dimensional (2D) hyperfine sublevel: correlation (HYSCORE) :spectroscopy were employed to measure the weakly coupled N-14 nuclei, and X- and W-band (95 GHz) pulsed electron-nuclear double resonance (ENDOR) spectroscopy for probing the strongly coupled N-14 nuclei and the P-protons. The high field measurements were extremely useful as they allowed us to resolve the T2 and CUA signals in the gl. region and gave H-1 ENDOR spectra free of overlapping N-14 signals. The effects of the M160Q and M160E mutations were: (i) increase in A(\ \)((CU)-C-63,65), (ii) larger hyperfine coupling of the weakly coupled backbone nitrogen of C153, (iii) reduction in the isotropic hyperfine interaction, ai,,, of some of the P-protons making them more similar, (iv) the ai,, value of one of the remote nitrogens of the histidine residues is decreased, thus distinguishing the two histidines,;and finally, (v) the symmetry of the g-tensor remained axial. These effects were associated with an increase in the Cu-Cu distance and subtle changes in the geometry of the Cu2S2 core which are consistent with the electronic structural model of Gamelin et al.