Understanding the electronic properties of the CuA site from the soluble domain of cytochrome c oxidase through paramagnetic 1H NMR.

The soluble domain of the subunit II of cytochrome c oxidase from Paracoccus versutus was cloned, expressed, and studied by 1H NMR at 600 MHz. The properties of the redox-active dinuclear CuA site in the paramagnetic mixed-valence Cu(I)-Cu(II) state were investigated in detail. A group of relatively sharp signals found between 30 and 15 ppm in the 1H NMR spectrum correspond to the imidazole protons of the coordinated histidines (H181 and H224). A second group of broader and farther shifted signals between 50 and 300 ppm are assigned to Hbeta protons of the bridging cysteines (C216 and C220); the protons from the weak M227 and E218 ligands do not shift outside of the diamagnetic envelope. About 40% of the total spin density appears delocalized over the cysteine-bridging ligands while a much smaller amount is delocalized on the two ligand histidines. The latter have similar spin density distributions. Analysis of the pattern of the hyperfine shifts of the Cys H beta protons shows that the ground state bears 2B3u character, in which the sulfur lobes in the singly occupied molecular orbital are aligned 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 2B2u excited state is thermally accessible at room temperature (Delta E approximately kT).