(1) Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita
565, Japan
(2) Department of Life Science, Himeji Institute of
Technology, Kamigohri Akoh, Hyogo 678-12, Japan.
Cytochrome c oxidase reduces molecular oxygen to water with the electrons from cytochrome c, coupled to pumping protons from matrix side of the mitochondrial membrane toward the intermembrane space. Crystal structure of bovine heart cytochrome c oxidase has been solved by the multiple isomorphous replacement method followed by density modification method at 2.8Å resolution. Positional refinement followed by temperature factor refinement with program X-PLOR (9) reduced R factor to 0.199 and Rfree to 0.252 at 2.8Å resolution. The root mean square deviations from standard values of bond length and angles for the refined structure were 0.012Å and 1.73Å, respectively. The crystal structure reveals 13 subunits, different from any other, eight phospholipids and two cholates, two hemes A, and three Cu, one Mg, and one Zn. 3560 amino acid residues out of 3606 residues in the dimer have been converged to a reasonable structure by refinement. The transmembrane part of the dimer consists of 56 alpha-helices. 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 chanels 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 which 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.
Eight phospholipids, five phosphatidyl ethanolamines and three phosphatidyl glycerols, have been clearly shown up in the multiple isomorphous replacement electron density distribution refined by the density modification method. All the eight phosphorus atoms are located at the membrane surface levels either on the cytosolic or matrix sides, and the hydrocarbon tails are directed toward the inside of the transmembrane region as would be expected if these phospholipids are in the lipid bilayer.
Specific electron density cages, in line with the cytosolic and the matrix locations of phospholipids in the enzyme molecule indicate specific bindings of two cholic acid molecules. The size and shape of ADP closely resembles that of cholate. The amino acid side chains surrounding the sterol moiety of the bound cholates and those near the carboxyl group are arranged so that ADP or other purine nucleotide with two inorganic phosphates can fit these sites.
A water channel for removing H2O produced at the O2 reduction site proceeds from the cytosolic side of heme a3 along the subunit I-II interface to the cytosolic surface of the enzyme. The magnesium site and the propionates of heme a3 form a hydrophilic environment that includes the two basic amino acids (Arg438 and His368 of subunit I), the two propionates of heme a3, Asp364 of subunit I and two fixed waters; all of them are connected with hydrogen bonds. The hydrophilic region is connected to a channel formed on the interface between subunits I and II with a loose arrangement of hydrophilic residues that could provide a water channel to the cytosolic surface with small conformational changes.