Biochem - ETC and oxidative phosphorylation

How are electrons moved through the mitochondria?

• Electrons in the mitochondrial matric are transferred to the electron transport chain on the inner mitochondrial membrane

• Inner membrane has a large surface area to allow for chemical reactions to take place

What is the overview of the electron transport chain?

• Specialised set of protein complexes and electron carriers

• Three complexes span the membrane (I, III, IV)

• One complex is located on the matrix side (II)

• Two highly mobile electron carriers shuttle electrons (Quinones and Cytochrome c)

• Each complex and carrier in the chain have a lower free energy than the previous

• Electrons flow spontaneously - highest energy carrier is easily oxidised, lowest energy carrier is easily reduced

• Generate ATP as electron transport releases energy

• Energy allows certain complexes to pump protons across the membrane - proton motive force

• Proton motive force is converted into phosphoryl transfer potential by ATP synthase (complex V)

What is an overview of oxidative phosphorylation?

• Process in which ATP is formed

• Due to the transfer of electrons from NADH and FADH2 to O2 by a series of electron carriers

What are the different cofactors involved in the ETC and oxidative phosphorylation?

• Coenzyme Q

• Cytochrome c

• Metal ion cofactors

What is the use of coenzyme Q in the ETC and oxidative phosphorylation?

• A ‘ubiquitous quinone’ that carries electrons

• Carries the electrons from NADH and FADH2 - from complex I or II

• Hydrophobic and diffuses rapidly through the inner mitochondrial membrane without leaving it

• Can exist in three oxidation states (easily oxidised and reduced)

What is the use of cytochrome c in the ETC and oxidative phosphorylation?

• Present in all organisms with a mitochondrial respiratory chain - highly conserved structure

• Tertiary structure of 5 a-helices

• Moves through the intermembrane space

• Binds a haem c cofactor

• Conjugate porphyrin ring with a central ion cation

• Carries 1 electron from complex III to complex IV

• Reduced by Q-cytochrome c oxidoreductase

• Oxidised by cytochrome c oxidase

What are the use of metal ion cofactors in the ETC and oxidative phosphorylation?

• Metal cation containing enzymes can shuttle/carry electrons

• Metal ion cofactors are ‘redox active’

• Can cycle between different oxidation states

• Reduced when they accept electrons

• Oxidised when they pass on electrons

What is the first complex in the ETC?

• NADH-Q reductase

• Quaternary structure comprises ~45 proteins encoded by genes in the nucleus and mitochondria

• Membrane-spanning part and a long arm that extends into the matrix

• Requires flavin mononucleotide and iron-sulphur clusters as cofactors

How does complex I operate in the ETC?

• 2 electrons from NADH are transferred to FMN

• FMN is reduced to FMNH2

• FMNH2 transfers 2 electrons to a series of Fe-S clusters

• Two electrons are transferred to coenzyme Q to form QH2

• Flow of 2 electrons from NADH to Q leads to pumping of 4H+ from the matric to the intermembrane space

• H+ cannot diffuse back and generates a proton gradient

What is the second complex in the ETC?

• Succinate dehydrogenase (succinate Q-reductase)

• Bound to the inner mitochondrial matrix and participates in both the citric acid cycle and the ETC

How does complex II operate in the ETC?

• FADH2 is oxidised to FAD

• 2 electrons are transferred to Q via Fe-S clusters forming QH2

• Does not pump H+

• Less ATP from oxidation of FADH2 compared to NADH

What is the third complex in the ETC?

• Q-cytochrome c oxidoreductase (cytochrome bc1 complex)

• Homodimer

• Requires 3x haem and iron-sulphur clusters as cofactors

How does complex III operate in the ETC?

• Flow of 2 electrons from one QH2 to cytochrome c proteins

• 2H+ ions are pumped into the intermembrane space from the matrix

What is the fourth complex in the ETC?

• Cytochrome c oxidase

• Homodimer

• Each monomer is comprised of 13 proteins

• Requires 2 haem and 3 Cu ion cofactors

How does complex IV operate in the ETC?

• Haema3-CuB is the reduction site of O2 to H2O

• Requires 4 protons and 4 electrons

• 4Cyt cred + O2 —> 4Cyt cox + 2H2O

• 4 ‘chemical’ H+ are taken up from the matrix side to reduce 1 molecule of O2 to 2 molecules of H2O

• 4H+ are transported out of the matrix into the intermembrane space during the reaction

How is proton motive force generated in the ETC?

• 3 of the enzyme complexes in the electron transport chain are proton pumps

• Use electrons to pump H+ into the intermembrane space

• Generates a pH gradient across the inner mitochondrial membrane

• pH in the intermembrane space is 1.4 units lower than the matrix and the membrane potential is 0.14V

• Proton motive force corresponds to a free energy of 21.8kJ per mol of protons

What is the protein involved in oxidative phosphorylation?

• ATP synthase

• Enzyme complex of many proteins

• Form a head, stalk and transmembrane pore

• F0 proton-conducting subunit

• F1 catalytic subunit

How do the different subunits of ATP synthase help to produce ATP in oxidative phosphorylation?

• F0 proton-conducting subunit

• Hydrophobic - spans the inner mitochondrial membrane

• Contains the proton channel of the complex

• Channel consists of 8 to 14 c subunits forming a pore and 1 a subunit

• F1 catalytic subunit

• Extends into the matrix

• Five types of polypeptide chain: a3, B3, y, d, e

• Majority of F1 consists of a3 and B3 subunits

• Stalk consists of y and e

• Each 360 rotation of y leads to the synthesis and release of 3 ATP

• Each ATP synthesised requires transport of 3H+

How does oxidative phosphorylation take place?

• Only route for H+ to return to the matrix is through ATP synthase - transmembrane complex

• Oxidation of fuel molecules and phosphorylation of ADP are coupled by H+ movement across the inner mitochondrial membrane

• Flow of 2 electrons from NADH to O2 results in the pumping of 10H+ from the mitochondrial matrix to the intermembrane space

• Each ATP synthesised requires transport of 3H+

• 1H+ is consumed in transporting each synthesised ATP to the cytoplasm

• Each NADH produces 2.5 ATP

What are the counts of ATP produced per molecule at different parts of metabolism?

• 10 NADH and 2 FADH2 are delivered to the electron transport chain

• 2 NADH from glycolysis produce 1.5 ATP each - used ATP initially

• 2 NADH from the ‘link reaction’ produce 2.5 ATP each

• 2 FADH2 from the citric acid cycle produce 1.5 ATP each

• 6 NADH from the citric acid cycle produce 2.5 ATP each

• Total 26 molecules of ATP are produced during oxidative phosphorylation