UNDERSTANDING THE ELECTRONIC-PROPERTIES OF THE CU-A SITE FROM THE SOLUBLE DOMAIN OF CYTOCHROME-C-OXIDASE THROUGH PARAMAGNETIC H-1-NMR

The soluble domain of the subunit II of cytochrome c oxidase from Para coccus versutus was cloned, expressed, and studied by H-1 NMR at 600 M Hz. The properties of the redox-active dinuclear Cu-A site in the para magnetic mixed-valence Cu(I)-Cu(II) state were investigated in detail. A group of relatively sharp signals found between 30 and 15 ppm in th e H-1 NMR spectrum correspond to the imidazole protons of the coordina ted histidines (H181 and H224). A second group of broader and farther shifted signals between 50 and 300 ppm are assigned to H-beta protons of the bridging cysteines (C216 and C220); the protons from the weak M 227 and E218 ligands do not shift outside of the diamagnetic envelope. About 40% of the total spin density appears delocalized over the cyst eine-bridging ligands while a much smaller amount is delocalized on th e two ligand histidines. The latter have similar spin density distribu tions. Analysis of the pattern of the hyperfine shifts of the Cys H-be ta protons shows that the ground state bears B-2(3u) character, in whi ch the sulfur lobes in the singly occupied molecular orbital are align ed with the Cu-Cu axis. Analysis of the temperature dependence of the shifts of the Cys H-beta signals leads to the conclusion that the ?B-2 u, excited state is thermally accessible at room temperature (Delta E approximate to kT).