432 IRON-CONTAINING PROTEINS AND ENZYMES
cytochrome c oxidase complex, important questions include (1) the nature of
the metal binding sites — metal ions bound, oxidation – reduction behavior, and
so on, (2) the metal centers ’ amino acid ligands, (3) the mechanism that
couples O 2 binding and reduction to proton pumping (translocation), and (4)
the electron and proton pathways through the protein and how these are
facilitated by the metal centers, channels, and amino acids.
7.8.2 Metal-Binding Sites in Cytochrome c Oxidase,
X - ray crystallographic structures determined in the mid - 1990s established the
nature and positions of the metal centers in bovine heart cytochrome c
oxidase.^137 Two heme a centers (see Figure 7.38 ), three copper ions, one mag-
nesium ion, and one zinc ion were located in the crystal structures. Starting
from the membrane cytoplasmic side and working toward the matrix side, one
fi rst encounters a bimetallic copper site, Cu A. The bimetallic Cu A site resides
within CcO ’ s subunit II in the region protruding into the mitochondrial mem-
brane ’ s cytosolic side (intermembrane space) approximately 8 Å above the
membrane ’ s surface. The two copper ions in the Cu A site are held in place by
six subunit II amino acid residue ligands — two cysteine sulfurs of cys196 and
cys200, two imidazole nitrogens of histidine residues, his161and his204, a
methionine sulfur of met207, and the backbone carbonyl of a glutamate,
glu198. The sulfur atoms of cys196 and cys200 bridge the two copper ions
yielding a short Cu – Cu distance of 2.38 Å in PDB: 1OCC 137b and 2.20 Å in
PDB: 2OCC 137c (both bovine heart enzyme complexes). The other ligands form
an overall tetrahedral coordination sphere for the two Cu A ions. While the
histidine and cysteine ligands have normal bond lengths and envelop the
copper ions in a triangular plane, the methionine and glutamate ligands are
loosely coordinated with long bond lengths (2.75 and 2.41 Å , respectively). The
CuA and heme a sites (described next) function to transport electrons from
external reductant moieties, cytochrome c for one, to the catalytic site. The
CuA and heme a sites may also function as electron storage locations within
the enzyme. In PDB: 1OCC the distances between the Cu A site and Fe ion in
heme a is equal to 20.6 Å , while for PDB: 2OCC the Cu A site to Fe ion in heme
a 3 is equal to 22.3 Å.
The heme a and heme a 3 – Cu B centers, located at the same depth within the
mitochondrial membrane, are found about 13 Å below the cytosolic membrane
surface and∼ 13 Å apart (see Figure 7.39 , or Figure 2 of reference 137a ).
The heme units, lying approximately perpendicular to the membrane plane,
are ligated to residues within subunit I of CcO: (a) the low - spin Fe 2+ (S = 0)
ion within heme a to his61 (subunit I, helix II) and his378 (subunit I, helix X)
and (b) the high - spin Fe 2+ pentacoordinate (S = 2) ion within heme a 3 to his376
(subunit I, helix X). Heme a is bridged to heme a 3 through a his378 – phe377 –
his376 linkage. Cu B , located 4.70 Å (PDB: 1OCC) or 4.85 Å (PDB: 2OCC)
away from the heme a 3 Fe iron ion, is ligated by imidazole nitrogens of three
subunit I histidine ligands: his240 (helix VI) and his290 and his291, both in a