Exploring the Structure and Function of
Cytochrome bo3 Ubiquinol Oxidase from
Escherichia coli
Lai Lai Yap
Department of Biochemistry
Heme-Copper Oxidase Superfamily
• catalyze reduction of oxygen to water, and
utilizes free energy produced to pump protons
across membrane
• transmembrane proton and voltage gradient thus
generated is converted to useful energy forms
(eg. ATP by ATP synthase)
Heme-Copper Oxidase Superfamily
• 2 main groups (based on electron-donating substrate) :
cytochrome c oxidase (eg. mitochondrial cytochrome c oxidase)
and ubiquinol oxidase (eg. cytochrome bo3 ubiquinol oxidase)
• membership based on presence of subunit homologous to subunit
I of mammalian cytochrome c oxidase
• subunit I:
– largest subunit
– binuclear center (where O2 binds and is reduced to water)
consisting of a heme and copper (CuB)
– a second heme (which facilitates transfer of electrons to
binuclear center)
Respiratory Chains of E. coli
Substrates
eg. NADH,
succinate
D
e
h
y
d
r
o
g
e
n
a
s
e
s
Cytochrome bo3
O2
Low O2 affinity
Quinone
Cytochrome bd
High O2 affinity
O2
Cytochrome bo3 ubiquinol oxidase
•
•
•
•
a terminal oxidase in the aerobic respiratory chain of Escherichia coli
member of the heme-copper oxidase superfamily
consists of four subunits
catalyzes two-electron oxidation of ubiquinol-8 (Q8H2) at periplasmic
side of cytoplasmic membrane and four-electron reduction of oxygen
to water at cytoplasmic side
• also functions as a proton pump by translocating protons across the
cytoplasmic membrane to establish an electrochemical proton
gradient
• possible mechanism: ubiquinone bound at the high affinity site (QH)
acts as cofactor and mediates electron transfer from ubiquinol
substrate (at the low-affinity QL site) to heme b
• reduced heme b then provides electrons to the binuclear center for the
reduction of oxygen to water
Electron and proton transfer in cytochrome bo3 ubiquinol oxidase
2H+
periplasm
QL
2e-
2e-
QH
2e-
Heme o3
CuB
QH2
Heme b
II
2e-
III
IV
I
cytoplasm
2H+
translocation
H2 O
½ O2 + 2H+
Spherical rendering structure of ubiquinol oxidase
ubiquinol binding site
Subunit I
Subunit II
Subunit III
Subunit IV
P
C
Structure of ubiquinol oxidase
ubiquinol binding site
Subunit I
Subunit II
Subunit III
Subunit IV
P
C
Redox metal centers of ubiquinol oxidase (in subunit I)
His334
His333
His106
His284
His419
CuB
His421
Heme b
Heme o3
Rainbow rendering of ubiquinol oxidase and cytochrome c oxidase
Cytochrome c binding
site
Cytochrome c oxidase
Ubiquinol binding site
Ubiquinol oxidase
Superposition of ubiquinol oxidase and cytochrome c oxidase
Ubiquinol oxidase
Cytochrome c oxidase
Proton transfer pathways
• D- and K-channels in subunit I
• channels form polar cavities that originate on the
cytoplasmic side, leading to the binuclear center for
proton pumping and water formation
• D-channel : uptake of both chemical and pumped
protons
• K-channel : load enzyme with protons at some earlier
catalytic steps
D- and K-channels of ubiquinol oxidase
H+ out
H421
H334
H106
H333
T149
H284
E286
S145
N142
T201
Y288
T204
T359
N124
K362
S299
S315
T211
D135
D-channel
K-channel
D-channel
K-channel
2H+
periplasm
QL
2e-
2e-
QH
2e-
Heme o3
CuB
QH2
Heme b
II
2e-
III
IV
I
cytoplasm
2H+
translocation
H2 O
½ O2 + 2H+
Structure of ubiquinol oxidase
ubiquinol binding site
Subunit I
Subunit II
Subunit III
Subunit IV
P
C
Ubiquinol oxidase with modeled ubiquinone
(at ubiquinol binding site)
Ubiquinone binding site of ubiquinol oxidase with
modeled ubiquinone
H98
M78
I102
M79
D75
Q101
L160
R71
ubiquinone
Electron and proton transfer in cytochrome bo3 ubiquinol oxidase
2H+
periplasm
QL
2e-
2e-
QH
2e-
Heme o3
CuB
QH2
Heme b
II
2e-
III
IV
I
cytoplasm
2H+
translocation
H2 O
½ O2 + 2H+
Membrane normal view of subunit I with modeled ubiquinone
possible electron path
from ubiquinone to
binuclear center
CuB
H419
heme o3
H421
heme b
M79
H106
I102
ubiquinone