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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

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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 - PowerPoint PPT Presentation

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

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

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

• 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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