The soluble domain of the subunit II of cytochrome c oxidase from Paracoccus versutus was cloned, expressed, and studied by H-1 NMR at 600 MHz. The properties of the redox-active dinuclear Cu-A 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 H-1 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 H-beta 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 B-2(3u) 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 ?B-2u, excited state is thermally accessible at room temperature (Delta E approximate to kT).
Understanding the electronic properties of the CuA site from cytochrome c oxidase through paramagnetic 1H NMR
BUBACCO, LUIGI;
1998
Abstract
The soluble domain of the subunit II of cytochrome c oxidase from Paracoccus versutus was cloned, expressed, and studied by H-1 NMR at 600 MHz. The properties of the redox-active dinuclear Cu-A 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 H-1 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 H-beta 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 B-2(3u) 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 ?B-2u, excited state is thermally accessible at room temperature (Delta E approximate to kT).Pubblicazioni consigliate
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