Axial ligand modulation of the electronic structures of binuclear copper sites: Analysis of paramagnetic H-1 NMR spectra of Met160Gln Cu-A

CUA is an electron-transfer copper center present in heme-copper oxidases a nd N2O reductases. The center is a binuclear unit. with two cysteine ligand s bridging the metal ions and two terminal histidine residues. A Met residu e and a peptide carbonyl group are located on opposite sides of the Cu2S2 p lane, these weaker ligands are fully conserved in all known CUA Sites. The Met160Gln mutant of the soluble subunit II of Thermus thermophilus ba3 oxid ase has been studied by NMR spectroscopy. In its oxidized form, the binucle ar 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 coordinat ion mode of His114 but does not affect His157, thereby confirming the propo sal that the axial ligand-to-copper distances influence the copper-His inte ractions (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 constan ts 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 addit ion. shift the thermally accessible excited states. thus influencing the sp ectral position of the Cys beta -CH2 resonances. The Cu-Cys bonds are not s ubstantially altered by the Cu-Gln160 interaction, in contrast to the situa tion found in the evolutionarily related blue copper proteins. It is possib le that regulatory subunits in the mitochondrial oxidases fix the relative positions of thermally accessible CUA excited states by tuning axial ligand interactions.