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physiological conditions, which is one electron transfer per 5 - 20 ms [Alberts et al.,
2002]. As a consequence of this mismatch in rates, (i) the predominant redox state
of CcOs that bind O 2 under normal physiological conditions must be mixed-valence,
whereas three-electron and fully reduced forms may be important under hypoxic
conditions and (ii) in vivo oxidation of four molecules of ferrocytochrome c and
reduction of one molecule of O 2 are kinetically uncoupled, i.e., once bound, O 2 is
reduced all the way to H 2 O regardless of the rate at which electrons flow in the rest
of the respiratory chain. Conceivably, this mechanism minimizes the lifetime of
CcO bound to any peroxo-level intermediates; thereby minimizing the probability
that such an intermediate would dissociate from the enzyme into the solution as the
enzyme waits for an electron from ferrocytochrome c.
CcOs accomplish this kinetic decoupling by at least two mechanisms. First, the
redox potential of the catalytic heme/Cu site depends strongly on the redox state of
the electron transfer site(s), the six-coordinate heme a, and/or Cu A cofactors (redox
cooperativity [Rich and Moody, 1997]). The atomic mechanism of this redox coopera-
tivity is not known, but it manifests itself in the fact that in a singly reduced CcO, the
electron is delocalized over the six-coordinate heme and Cu A sites, whereas the O 2
reducing site remains in the aerobically stable Fe III /Cu II state. However, the arrival
of the second electron (i.e., the formation of mixed-valence CcO) results in redistribu-
tion of both external electrons almost exclusively onto the catalytic site. The catalytic
site is now in the Fe II /Cu I redox state, capable of binding O 2 and reducing it by four
electrons. Hence, a major question in the mechanism of O 2 reduction by CcOs and
quinol oxidases, which are generally assumed to follow similar mechanisms, is the
origin of the additional two electrons required for the four-electron reduction of O 2 .
The current consensus mechanism of O 2 reduction by mixed-valence CcO is shown
schematically in Fig. 18.5. It is derived mostly from single-turnover spectroscopic
experiments. Fully oxidized (compound H) and one-electron reduced (compound E)
states of CcO contain an oxidized catalytic heme/Cu site (Fe III /Cu II ), which has no
affinity for O 2 . As a result of the redox cooperativity between the Cu A and heme a
cofactors and the heme/Cu site, reduction of compound E by another molecule of
ferrocytochrome c results in a two-electron reduction of the heme/Cu site (generating
the Fe II /Cu I state), which rapidly binds O 2 . The resulting adduct, called compound A,
contains an oxyheme moiety that is very similar to that found in oxymyoglobin. It is
regarded formally as a superoxo (O 2 2 ) complex of ferriheme, i.e., binding of O 2 to a
ferroheme results in formal one-electron reduction of O 2 . It is the inability of Fe III to
reduce O 2 by one electron that explains the lack of affinity of ferriheme to O 2 . In com-
pound A, this bound O 2 interacts little if at all with Cu B or the phenol residue, as sur-
mised from the fact the O - O vibrational frequency is very similar to that of isolated
O 2 2 . The next spectroscopically observable intermediate is so-called compound
P M . By this stage of the catalytic cycle, both atoms of the O 2 molecule are reduced
to the “oxide” level (O 22 ), with one atom having been released as H 2 O (or bound
to Cu II as hydroxide, OH 2 , according to calculations [Blombers, 2006]) and the
other atom remaining bound to Fe as a terminal oxide. Two electrons for this four-
electron reduction come from the oxidation of Fe from Fe II in compound R to Fe IV
(ferryl) in compound P M ; oxidation of Cu B yields one electron and oxidation of the
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