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

2. 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)

3. 2 main groups (based on electron-donating substrate) : cytochrome c oxidase (eg. mitochondrial cytochrome c oxidase) and ubiquinol oxidase (eg. cytochrome bo 3 ubiquinol oxidase) membership based on presence of subunit homologous to subunit I of mammalian cytochrome c oxidase subunit I: –largest subunit –binuclear center (where O 2 binds and is reduced to water) consisting of a heme and copper (Cu B ) –a second heme (which facilitates transfer of electrons to binuclear center) Heme-Copper Oxidase Superfamily

4. Respiratory Chains of E. coli High O 2 affinity Low O 2 affinity Substrates eg. NADH, succinate DehydrogenasesDehydrogenases Quinone Cytochrome bo 3 Cytochrome bd O2O2 O2O2

5. Cytochrome bo 3 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 (Q 8 H 2 ) 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 (Q H ) acts as cofactor and mediates electron transfer from ubiquinol substrate (at the low-affinity Q L site) to heme b reduced heme b then provides electrons to the binuclear center for the reduction of oxygen to water

6. Electron and proton transfer in cytochrome bo 3 ubiquinol oxidase QLQL QHQH II I III IV ½ O 2 + 2H + H2OH2O 2H + translocation 2H + 2e - periplasm cytoplasm Heme b Heme o 3 Cu B QH 2

7. Subunit I Subunit II Subunit III Subunit IV P C ubiquinol binding site Spherical rendering structure of ubiquinol oxidase

8. P C ubiquinol binding site Subunit I Subunit II Subunit III Subunit IV Structure of ubiquinol oxidase

9. Heme bHeme o 3 Cu B His 106 His 421 His 334 His 333 His 284 His 419 Redox metal centers of ubiquinol oxidase (in subunit I)

10. Rainbow rendering of ubiquinol oxidase and cytochrome c oxidase Cytochrome c oxidaseUbiquinol oxidase Cytochrome c binding site Ubiquinol binding site

11. Superposition of ubiquinol oxidase and cytochrome c oxidase Ubiquinol oxidase Cytochrome c oxidase

12. 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

13. D- and K-channels of ubiquinol oxidase D-channel K-channel D-channel K-channel D135 T211 N142 N124 S145 T204 T149 T201 E286 H106 H421 H333 H334 H284 Y288 T359 K362 S315 S299 H + out

14. QLQL QHQH II I III IV ½ O 2 + 2H + H2OH2O 2H + translocation 2H + 2e - periplasm cytoplasm Heme b Heme o 3 Cu B QH 2

15. P C ubiquinol binding site Subunit I Subunit II Subunit III Subunit IV Structure of ubiquinol oxidase

16. Ubiquinol oxidase with modeled ubiquinone (at ubiquinol binding site)

17. ubiquinone L160 R71 D75 M78 M79 I102 H98 Q101 Ubiquinone binding site of ubiquinol oxidase with modeled ubiquinone

18. Electron and proton transfer in cytochrome bo 3 ubiquinol oxidase QLQL QHQH II I III IV ½ O 2 + 2H + H2OH2O 2H + translocation 2H + 2e - periplasm cytoplasm Heme b Heme o 3 Cu B QH 2

19. ubiquinone heme b heme o 3 Cu B M79 I102 H106 H421 H419 Membrane normal view of subunit I with modeled ubiquinone possible electron path from ubiquinone to binuclear center