The dinuclear Cuᴀ centre of cytochrome oxidase

Abstract

Cytochrome c oxidase is the last enzyme in mitochondrial and many bacterial respiratory chains. It catalyzes electron transfer from cytochrome c to molecular oxygen and transfers free energy of this reaction into a transmembrane proton electrochemical gradient. In bacteria, there are two main classes of terminal oxidases, cytochrome c - and quinol oxidases. The main difference between these oxidases lies in subunit II that is also believed to be the electron entry site. Cytochrome c oxidases contain a redox-active copper centre (Cuᴀ) in subunit II, whereas this copper centre has been lost from quinol oxidases during evolution. In the present work, the C-terminal domain of subunit II from various bacterial terminal oxidases was expressed in Escherichia coli as a soluble protein. Using site-directed mutagenesis, it was possible to engineer copper centres similar to Cuᴀ and blue copper into the originally copperless quinol oxidase domain. This shows that subunit II in quinol- and cytochrome c oxidases must have a similar three-dimensional structure. Spectroscopic studies on the isolated Paracoccus denitrificans Cuᴀ-binding domain showed that it is similar to the centre A of nitrous oxide reductase. The spectrum of the isolated Cuᴀ centre is sensitive to pH, which is not the case for the Cuᴀ in the intact oxidase. The biochemical copper quantification and electrospray mass spectrometry showed that the engineered Cuᴀ -like centre as well as the isolated Paracoccus Cuᴀ centre contain two coppers. The EPR spectra, together with the quantification of EPR-detectable coppers, indicated that the two coppers form a dinuclear, mixed valence [Cu(l.5) ... Cu(l.5)] centre. Site-directed mutagenesis showed that these coppers are ligated by two cysteine and two histidine residues. The interaction of the oxidized Cuᴀ-binding domain with reduced cytochrome c was studied by stopped-flow spectroscopy. The reaction follows monophasic kinetics, indicating the presence of only one catalytically active cytochrome c binding site in this domain. The kₒₙ rates and dissociation constants were similar to the ones that have been obtained earlier with the intact cytochrome c oxidase. This indicates that the Cuᴀ-binding domain contains a complete cytochrome c binding site. By site-directed mutagenesis, five conserved residues (two aspartates, two glutamates and a glutamine) in the Cuᴀ-binding domain were identified as cytochrome c binding residues.