Bch210 exam L13

Oxidative Phosphorylation

Mitochondrial Oxidative Phosphorylation

• NADH and FADH2 carry e- to ETC

• their energy used to create conformational change in protein complex

  • pump protons from matrix into intermembrane space

    • proton gradient used by ATP synthase to make ATP in matrix

Oxidative phosphorylation

pump h+ into intermembrane space from matrix using the energy (e-) from NADH and FADH2

• o2 will then be the final e- acceptor and take the e- forming 2 h2o molecules

Oxidative phosphorylation: e- passing and H+ pumping

NADH

• complex 1

  • 2 e- to complex Q

  • release 4 protons

FADH2

• complex 2

  • 2 e- to complex Q

Q

• pass e- to complex iii

• 4 protons released

Complex iii passes to cyt c which passes to complex 4 before releaseing 2 protons

• finally O2 picks up e-

Electron Transfer Potential: Standard Reduction Potential (E ́o)

A molecule’s tendency to be oxidized or reduced.

• more -ve = loses e- more easily (negative E ́0 values.)

• more +ve = gains e- more easily

e- flow from more -ve to +ve

Standard Reduction Potential (E ́o) formula

F = Faraday constant = 96,485 J/Vmol n = number of electrons

• ΔG°’ = - n F ΔE ́o

The Electron Transport ‘Chain’ conformational changes

Passage of electrons through the ‘chain’ (from – ve to +ve) results in a free energy change that drives conformational changes in the complexes to set up a proton gradient for ATP synthase.

What is the experimental evidence that Oxidative Phosphorylation exists, and that the electron transport chain is linked to ATP synthesis?

Mitochondrial O2 experiment

• measure amount of O2 consumed

Monitoring Oxidative Phosphorylation

• measure amount of ATP made

Oxidative Phosphorylation Inhibitors: Rotenone and amytal

inhibit electron flow from complex I to CoQ .

Oxidative Phosphorylation Inhibitors: Complex III

Antimycin A

Oxidative Phosphorylation Inhibitors: Complex IV

• Cyanide, azide and CO

Oxidative Phosphorylation Inhibitors: ATP synthase (complex V).

• Oligomycin

• uncouplers disrupt the H+ gradient

Uncouplers

• molecules that have hydrophobic groups that allow them to cross the membrane.

• acidic group can bind to H+ and move them from high - low conc that disrupts H+ gradient and ATP synthesis

How does ATP synthase make ATP from a proton gradient? Peter Mitchell’s Chemiosmotic Hypothesis:

• ATP synthesis arrives due to electrochemical gradient across mitochondrial inner membrane

  • H+ gradient prod by ETC using suitable e- donor

  • conformation change in atp synthase

  • reversible

ATP synthase: F1

• F1: peripheral protein unit that carries out catalytic synthesis of ATP in matrix

• conformational changes in F1 beta subunit responsible for ATP synthase

ATP synthase: F0

• integral membrane protein unit that anchors the enzyme complex in the inner mitochondrial membrane

• H+ flow through rotor that cause rotation in ring of y subunit of F0

Boyer’s Binding Change Mechanism

• beta subunit functions independently and 3 diff rxns occur simultaneously

• The binding of H+ in the rotor rotates the γ subunit and induces a conformational change in the β subunits.

Each β subunit undergoes a conformational change between 3 states:

• Open or Empty/exit (ATP leaves)

• Loose - ADP and Pi bound
• Tight - ATP bound

ATP Synthase is Reversible

• ATP hydrolysis can also be used to reverse the reaction mechanism.

  • form of active transport that can be used to drive proton transport across the membrane in the opposite direction using a similar rotation mechanism.

recombinant fusion proteins and fluorescence microscopy

Imaging techniques can be used to visualize this rotation in the F1 unit

How many ATPs are made?

• NADH and FADH2 each donate 2 e- to etc

• 1 NADH can make 2.5 atp (10h+ pumped)

• 1 fadh2 can make 1.5 atp (6 h+ pumped)

  • round down if needed

Water formation in Oxidative Phosphorylation

IN ETC at complex 4

• 1 H2O at last step

IN ATP SYNTHASE

• Water ratio: 3.5 for NADH, 2.5 for FADH2 (or NADHcyt.)

Overall summary equation for the complete oxidation of glucose

6 co2 +30 ATP + 36 H2O

Why is hypoxia life threatening? (low O2)

mito matrix makes 95% of ATP, glycolysis only makes 2ATP total

Anaerobic Metabolism

• periods of low o2, etc shits down and anaerobic metabolism occurs

• PDC & CAC also slow down

• ONLY glycolysis will generate ATP

Lactate dehydrogenase

uses NADH made in glycolysis in the cytoplasm to replenishing NAD+ for glycolysis to continue.

• Lactate can be used by liver cells in GNG.

Cori Cycle

Lactate, produced by anaerobic glycolysis in muscles, travels through the bloodstream to the liver