CYTOCHROME c OX IDASE 435
and tyr244 (Y244) (his240 N ε 2 – tyr244 C ε 2 = 1.35 Å ). The covalent bond would
bring the tyrosine – OH group close to the bound O 2 at the catalytic site.^141
Both proposed catalytic cycles indicate formation of the ferryl – oxo species,
Fe(IV)=O in heme a 3 , similar to that found in the myoglobin and hemoglobin
cycles. One of the enigmas of CcO ’ s O 2 reducing ability is the enzyme ’ s capa-
bility to reduce O 2 to the level of water on a much shorter time scale ( < 0.2 ms
in the mixed - valence case and 0.03 ms for the fully reduced enzyme) than the
time it takes CcO to receive an electron from cytochrome c (5 – 20 ms). This
difference is benefi cial because it prevents the escape of partially reduced
oxygen - containing species such as superoxide ( O 2 i−), HO • radicals or peroxide
anions ( O 22 −). However, the rapid reduction of O 2 by CcO has also made it
diffi cult to spectroscopically identify any dioxygen intermediates during the
catalytic cycle.
Researchers have put forward many proposals concerning proton translo-
cation carried out by cytochrome oxidase. First, two types of protons must be
distinguished: (1) “ substrate ” protons used to produce water molecules and
(2) “ pumped ” protons that reach the intermembrane space and provide the
electrochemical energy for the ADP → ATP conversion. In studies of the
bovine heart cytochrome c oxidase, the reference 137 researchers determined
pathways involving hydrogen - bonding networks through water molecules and
amino acid side chains. Figure 10 of reference 137b illustrates possible proton
pumping and substrate proton paths traveling from the matrix side through
the membrane and into the intermembrane (cytosolic) space. Amino acid side
chains, fi xed water molecules, heme a and heme a 3 substituents — especially the
hydroxyl farnesylethyl group (see Figure 7.38 ) — and the magnesium ion are
involved. For instance, protons to be used at the heme a 3 – Cu B dioxygen reduc-
tion site — substrate protons — arrive from the matrix in a hydrogen - bonding
network beginning at lys265, passing through several water molecules with
hydrogen bonding to amino acid side chains thr489, thr490, asn491, his256,
ser255, lys319, tyr244, and Cu B ligand his240. The hemes, Cu B , Cu A , and some
of the hydrogen - bonding network for PDB: 1V54 and 1V55 are visualized in
Figure 7.41A. In this fi gure, the PDB: 1V54 structure is visualized in green
stick format and PDB: 1V55 in cyan.
More recently, Yoshikawa, Tsukihara, and co - workers published a study of
fully oxidized (PDB: 1V54) and fully reduced (PDB: 1V55) bovine heart cyto-
chrome c oxidase structures.^142 In this study, they identifi ed an aspartate
residue, asp51, which undergoes a substantial change in position between the
oxidized and reduced structures (see inset in Figure 7.41A ).
An asp51 – asn51 mutation of the bovine enzyme abolished its proton
pumping capability but showed little change in dioxygen reduction activity.
The asp51 residue resides near the enzyme surface at the interface with the
cytosolic (intermembrane) side. The oxidized and reduced forms of the enzyme
show substantial changes in the hydrogen - bonding interactions near the
membrane surface (see Figure 2 of reference 142 ). The major changes in going
from the oxidized to the reduced structures are to deprotonate asp51 and to