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Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

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Page 1: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

Exploring the Structure and Function of Cytochrome bo3 Ubiquinol Oxidase from

Escherichia coli

Lai Lai Yap

Department of Biochemistry

Page 2: Exploring the Structure and Function of Cytochrome bo 3 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)

Page 3: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

• 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)

Heme-Copper Oxidase Superfamily

Page 4: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

Respiratory Chains of E. coli

High O2 affinity

Low O2 affinitySubstrates eg. NADH, succinate

Dehydrogenases

Quinone

Cytochrome bo3

Cytochrome bd

O2

O2

Page 5: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

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

Page 6: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

Electron and proton transfer in cytochrome bo3 ubiquinol oxidase

QL

QH

II IIII IV

½ O2 + 2H+H2O 2H+

translocation

2H+

2e-2e-

2e-

2e-

periplasm

cytoplasm

Heme b

Heme o3

CuB

QH2

Page 7: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

Subunit ISubunit IISubunit IIISubunit IV

P

C

ubiquinol binding site

Spherical rendering structure of ubiquinol oxidase

Page 8: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

P

C

ubiquinol binding site

Subunit ISubunit IISubunit IIISubunit IV

Structure of ubiquinol oxidase

Page 9: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

Heme b Heme o3

CuB

His106

His421

His334

His333

His284

His419

Redox metal centers of ubiquinol oxidase (in subunit I)

Page 10: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

Rainbow rendering of ubiquinol oxidase and cytochrome c oxidase

Cytochrome c oxidase Ubiquinol oxidase

Cytochrome c binding site Ubiquinol binding site

Page 11: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

Superposition of ubiquinol oxidase and cytochrome c oxidase

Ubiquinol oxidase

Cytochrome c oxidase

Page 12: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

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

Page 13: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

D- and K-channels of ubiquinol oxidase

D-channel K-channelD-channel K-channel

D135T211

N142

N124

S145

T204

T149

T201

E286

H106

H421

H333

H334

H284

Y288

T359

K362

S315S299

H+ out

Page 14: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

QL

QH

II IIII IV

½ O2 + 2H+H2O 2H+

translocation

2H+

2e-2e-

2e-

2e-

periplasm

cytoplasm

Heme b

Heme o3

CuB

QH2

Page 15: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

P

C

ubiquinol binding site

Subunit ISubunit IISubunit IIISubunit IV

Structure of ubiquinol oxidase

Page 16: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

Ubiquinol oxidase with modeled ubiquinone (at ubiquinol binding site)

Page 17: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

ubiquinone

L160

R71

D75

M78

M79I102

H98

Q101

Ubiquinone binding site of ubiquinol oxidase with modeled ubiquinone

Page 18: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

Electron and proton transfer in cytochrome bo3 ubiquinol oxidase

QL

QH

II IIII IV

½ O2 + 2H+H2O 2H+

translocation

2H+

2e-2e-

2e-

2e-

periplasm

cytoplasm

Heme b

Heme o3

CuB

QH2

Page 19: Exploring the Structure and Function of Cytochrome bo 3 Ubiquinol Oxidase from Escherichia coli Lai Lai Yap Department of Biochemistry

ubiquinone

heme b

heme o3

CuB

M79I102

H106

H421

H419

Membrane normal view of subunit I with modeled ubiquinone

possible electron path from ubiquinone to binuclear center