CRYSTAL STRUCTURE ANALYSIS OF BOVINE HEART CYTOCHROME C OXIDASE. H. Yamaguchi, T. Tsukihara, H. Aoyama, E. Yamashita, T. Tomizaki, K. Shinzawa-Itoh^*, R. Nakashima,^* R. Yaono^* and S. Yoshikawa^*. Institute for Protein Research, Osaka University, Suita, Osaka565, Japan; and ^*Faculty of Science, Himeji Institute of Technology, Kamigohori, Ako-gun, Hyogo678-12, Japan

Bovine cytochrome c oxidase is a large membrane protein complex with molecular size of 200KDa comprising 13 different subunits and six metal centers, heme a, heme a₃, Cu_A, Cu_B, Mg and Zn. As the terminal enzyme of biological oxidation, the enzyme catalyzes O₂ reduction to H₂O at an active site with the four redox active transition metals coupling to a proton pumping process across the mitochondrial inner membrane.

The structure of the enzyme has been determined at 2.8Å resolution. Electron transfer pathway has been elucidated by the structural study. A clear electron density map at 2.8Å resolution was obtained by m.i.r. method followed by a density modification method. Out of 3606 amino acid residues in an asymmetric unit composed of a dimmer, structural models of 3560 residues as well as those of metal centers were successfully built.

The electron density map indicates a dinuclear copper center of Cu_A with an unexpected structure of [2Cu-2S[[gamma]]] similar to a [2Fe-2S] center. Zinc site is located at a nuclear encodes subunit on the matrix side of the membrane. Magnesium site is situated between heme a₃ and Cu_A. The O₂ binding site contains heme a₃ iron and copper (Cu_B) atoms with a distance of 4.5Å. There is no amino acid ligand bridging between the iron and the copper atoms in spite of a strong anti-ferromagnetic coupling between them. However, such a small bridging ligand between them as an oxygen atom is not excluded at present. The heme a and the heme a₃ are bridged by a polypeptide segment of His-Phe-His of which one histidine coordinates to the heme a iron and the other histidine to the heme a₃ iron.

The electron transfer path within the molecule has been established as follows: cytochrome c -> Cu_A -> heme a -> the O₂ binding site, which includes heme a₃ and Cu_B. Several hydrogen bonds providing electron transfer pathway were found between the Cu_A site and the hemes a. There exist several hydrogen bonds around the Cu_A, the heme a and the heme a₃. The structure implicates that Cu_A-His²⁰⁴-CO of Arg⁴³⁸-NH of Arg⁴³⁹-a propionic group of the heme a is the primary electron transfer pathway between Cu_A and the heme a.