Environmental Engineering Reference
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2001]. Crystal structures of several CcOs and of a quinol oxidase have been reported
[Abramson et al., 2000; Ostermeier et al., 1997; Tsukihara et al., 1996]. The O
2
reduction site is buried deep inside subunit I, approximately in the middle of the mem-
brane that hosts the enzyme (Fig. 18.4). The site contains a heme coordinated by a
single axial imidazole moiety at its proximal site (see Fig. 18.4c for the definition
of the proximal and distal sites), and is reminiscent of the O
2
-binding site of myoglo-
bin, the simplest hemoprotein [Frauenfelder et al., 2003]. At the distal site of the heme,
a Cu ion coordinated to three imidazoles is located. The Fe - Cu distance appears to
vary around 5
˚
, depending on the exogenous ligands at the two ions. The 5
˚
dis-
tance is suitable for the formation of a bridging peroxide, although the intermediacy
of such a species in the catalytic cycle remains uncertain. Finally, in most CcOs
and quinol oxidases, one of the Cu-ligating imidazoles is linked covalently to a
phenol residue of a tyrosine. This link is thought to be formed post-translationally,
and is routinely taken as an indication that a phenoxyl radical is formed during the
initial turnover of the enzyme, i.e., reduction of the very first molecule of O
2
after
the enzyme is assembled [Rogers and Dooley, 2001].
In addition to the catalytic heme/Cu site, all heme/Cu oxidases contain a six-
coordinate heme within the catalytic subunit, and all CcOs also contain a binuclear
Cu site (so called Cu
A
to distinguish it from the Cu ion of the O
2
-reduction site,
which is called Cu
B
) in subunit II. Some CcOs have one more redox-active prosthetic
group, a six-coordinate heme, usually located in the same subunit II. On the other
hand, subunit II of quinol oxidases appears to lack redox cofactors. The physiological
functions of these sites are (i) to conduct electrons from the docking site of the external
electron donor (ferrocytochrome c or quinol) on the periphery of the enzyme to the
catalytic site deep within the enzyme; (ii) to control the redox potentials of the catalytic
site (redox cooperativity); (iii) possibly to store reducing equivalents during the turn-
over; and (iv) potentially in the proton pump [Brzezinski, 2006]. The presence of the
additional electron relay sites in CcOs compared with quinol oxidases may be due to
the different physicochemical properties of the electron donor that these two types of
terminal oxidases utilize. Both quinol and quinone are lipophilic, water-insoluble mol-
ecules that are confined to the membrane within which quinol oxidase is embedded.
Quinol binds the enzyme fairly close to the O
2
reducing site. In contrast, cytochrome
c of CcO is a water soluble protein that binds CcO at a site exposed to the aqueous
intermembrane space. Its docking site is at least several ˚ngstr¨ ms farther from the cat-
alytic site, making direct electron tunneling from the ferrocytochrome c to the six-
coordinate heme site too slow and requiring an intermediate electron relay site in
the form of Cu
A
.
In the thermodynamically redox-stable “resting state,” CcOs all Cu ions are in the
Cu
II
state and all hemes are Fe
III
. From this state, CcOs can be reduced by one to four
electrons. One-electron reduced CcOs are aerobically stable with the electron deloca-
lized over the Cu
A
and heme a sites. The more reduced forms—mixed-valence (two-
electron reduced), three-electron reduced, and fully (four-electron) reduced—bind O
2
rapidly and reduce it to the redox level of oxide (22 oxidation state) within ,200 ms
[Wikstrom, 2004; Michel, 1999]. This rate is up to 100 times faster than the average
rate of electron transfer through the mammalian respiratory chain under normal
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