Immobilization of cytochrome c oxidase into electrode-supported lipid bilayer membranes for in vitro cytochrome c sensing

The crystal structure of bovine heart cytochrome c oxidase at 2.8 A resolution with an R value of 19.9 percent reveals 13 subunits, each different from the other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three copper, one magnesium, and one zinc. Of 3606 amino acid residues in the dimer, 3560 have been converged to a reasonable structure by refinement. A hydrogen-bonded system, including a propionate of a heme A (heme a), part of peptide backbone, and an imidazole ligand of CuA, could provide an electron transfer pathway between CuA and heme a. Two possible proton pathways for pumping, each spanning from the matrix to the cytosolic surfaces, were identified, including hydrogen bonds, internal cavities likely to contain water molecules, and structures that could form hydrogen bonds with small possible conformational change of amino acid side chains. Possible channels for chemical protons to produce H2O, for removing the produced water, and for O2, respectively, were identified.

The high resolution three-dimensional x-ray structure of the metal sites of bovine heart cytochrome c oxidase is reported. Cytochrome c oxidase is the largest membrane protein yet crystallized and analyzed at atomic resolution. Electron density distribution of the oxidized bovine cytochrome c oxidase at 2.8 A resolution indicates a dinuclear copper center with an unexpected structure similar to a [2Fe-2S]-type iron-sulfur center. Previously predicted zinc and magnesium sites have been located, the former bound by a nuclear encoded subunit on the matrix side of the membrane, and the latter situated between heme a3 and CuA, at the interface of subunits I and II. The O2 binding site contains heme a3 iron and copper atoms (CuB) with an interatomic distance of 4.5 A; there is no detectable bridging ligand between iron and copper atoms in spite of a strong antiferromagnetic coupling between them. A hydrogen bond is present between a hydroxyl group of the hydroxyfarnesylethyl side chain of heme a3 and an OH of a tyrosine. The tyrosine phenol plane is immediately adjacent and perpendicular to an imidazole group bonded to CuB, suggesting a possible role in intramolecular electron transfer or conformational control, the latter of which could induce the redox-coupled proton pumping. A phenyl group located halfway between a pyrrole plane of the heme a3 and an imidazole plane liganded to the other heme (heme a) could also influence electron transfer or conformational control.