Understanding the Electronic Properties of the Cu_A Site from the Soluble Domain of Cytochrome c Oxidase through Paramagnetic ¹H NMR

Abstract

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).

pH-dependent transition between delocalized and trapped valence states of a Cu A center and its possible role in proton-coupled electron transfer

“…NMR studies on the Cu A center from soluble domain of CcO from Paracoccus versutus indicate a decrease in the intensities of all paramagnetic signals in the pH range between 6.5 and 10. It was interpreted that the formation of an electron-localized species and slow electronic relaxation make the observation of the paramagnetic signal impossible (36).…”

Alternative ground states enable pathway switching in biological electron transfer

“…1B) (16,17). NMR studies have revealed a fast interconversion between the σ u * and π u ground states (18)(19)(20). However, the σ u * ground state is considered as the only redox-active orbital in ET, based on (i) the low population of the π u ground state at physiological temperature, and (ii) the higher λ value predicted for the π u ground state, making its involvement in ET unlikely (16,17).…”

Different Interaction Modes of Two Cytochrome-c Oxidase Soluble CuA Fragments with Their Substrates

“…The two copper atoms are bound by two cysteine residues forming thiolate bridges, two histidine residues, and as further ligands a methionine sulfur and a glutamate peptide carbonyl. Soluble domains of several bacterial cytochrome-c oxidases have been prepared including P. denitrificans (3), T. thermophilus (4), Paracoccus versutus (5), and Bacillus subtilis (6). Following electron transfer to the Cu A center from cytochrome c, electrons are further transferred to the low-spin heme a (or b) in subunit I, in a very fast s time-scale process, and finally to the binuclear heme a 3 -Cu B site (on a ms time scale), where dioxygen is reduced to water.…”