CRYSTALLIZATION & CRYSTAL STRUCTURE OF BEEF HEART CYTOCHROME C OXIDASE. K. Shinzawa-Itoh, R. Nakashima, R. Yaono S. Yoshikawa, Department of Life Science, Himeji Institute of Technology, Kamigohri Akoh, Hyogo 678-12, Japan; T. Tsukihara, H. Aoyama, E. Yamashita, T. Tomizaki, H. Yamaguchi, Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita 565 Japan

Crystals of bovine heart cytochrome c oxidase, which diffract X-rays to 2.5 Å resolution, have been obtained in the presence of decyl maltoside. Three dimensional structure of the enzyme, determined at 2.8 Å resolution with an R value of 20.4 % reveals that the monomer of the enzyme is composed of 13 subunits, different from any other, five phosphatidyl ethanolamines, three phosphatidyl glycerols and two cholates, two hemes A, and three Cu, one Mg, and one Zn. Each structure of all the residues of the monomer (1780 in total) except for 23 residues has been converged to a reasonable structure by structural refinement. A dimeric structure in the crystal in which the two monomers are bridged by one of the subunits in each monomer suggests the stability of the dimer under physiological conditions. Essentially all the transmembrane part of the protein moiety is folded to a-helical conformation and contains many alcoholic amino acids, serine and threonine. A concaved surface which could accept cytochrome c is located very near one of the copper site, Cu_A, suggesting that the initial electron acceptor from cytochrome c is Cu_A. A hydrogen bond system including a propionate of a heme A(heme a), peptide unit and an imidazole bound to Cu_A could provide much faster electron transfer between Cu_A and heme a than between Cu_A and the other heme A at the oxygen reduction site (heme a₃), though the Cu_A-heme a distance (20.6 Å) is only 2.6 Å shorter than the Cu_A-heme a₃ (23.2 Å)

Two structures likely to serve as proton transfer path, which include hydrogen bonds and internal cavities likely to contain water molecules are spanned from the matrix surface to the cytosolic. The O₂ reduction site is equipped with a possible proton transfer path from the matrix surface to produce H₂O from O₂ and a structure likely to be a water channel to the cytosolic surface. The crystal structure shows three possible O₂ paths to the O₂ reduction site which are composed of hydrophobic amino acid side chains loosely packed.