BCH210 - Lecture 19 - oxidative phosphorylation + ETC

what is the 1st law of thermodynamics?

-energy cannot be created or destroyed

mitochondrial oxidative phosphorylation

-NADH and FADH2 carry electrons to the electron transport chain (passe from NADH to oxygen)

- as electrons are passed from carrier to carrier, a change in redox potential generates free energy

-the energy is used to ”power” a conformational change in the protein complexes, pumping protons from the matrix into the intermembrane space (moves against their concentration gradient)
→ Some energy is lost as heat --> maintains body temperature in animals (done through the hypothalamus) (that lost energy becomes functional for us)

-this proton gradient is used by ATP synthase to make ATP in the matrix

Complex I

-known as NADH-Q oxidoreductase

-NADH donates its electrons to the complex

-pulls across 4 H+ from the mitochondrial matrix into the intermembrane space (where there’s high amounts of protons and low pH)

Complex II

-known as succinate-Q reductase (also in the CAC as succinate dehydrogenase)

-uses FADH2

-doesn’t transport protons across the membrane

Complex III

-known as Q-cytochrome c oxidoreductase

-pulls across 4 H+ from the mitochondrial matrix into the intermembrane space (where there’s high amounts of protons and low pH)

Complex IV

-known as cytochrome c oxidase

-pulls across 2 H+ from the mitochondrial matrix into the intermembrane space (where there’s high amounts of protons and low pH)

how many protons can we pull from NADH in oxidative phosphorylation?

-we can pull across 10 H+ all together (from each complex in total)

how many protons can we pull from FADH2 in oxidative phosphorylation?

-we can pull across 6 H+ all together (from each complex in total)

what is the path of electron flow in the ETC?

-goes from higher pH (low [H+]) to relatively lower pH (high [H+])→ which is more stable

-from complex I to IV

standard reduction potential (E´_o)

-a molecule’s tendency to be oxidized or reduced

(-) = blue side, loses electrons more easily (-3,-2,-1) (oxidize) → the donor

(+) = red side, gains electrons more easily (1,2,3) (reduce) → the acceptor

equation:

ΔE´₀ = acceptor – donor

what is the equation for electron transfer potential?

→ ΔG°’ = - n F ΔE´_o

• F = Faraday constant = 96,485 J/Vmol

• n = number of electrons

(- ΔG°’) → favourable reaction

(+ ΔG°’) → unfavourable reaction

**LEO GER

how to calculate the electron transfer potential?

-if you reduce it, you need to flip the reaction

-if factors are on opposite sides of the arrow, they can be cancelled out

how do you predict the flow of electrons?

-we always try to move electrons from negative to positive, so it would flow in that way (-) → (+)

electron transport chain

-electrons are passed from carrier to carrier

-

what is the electron transfer potential of carriers measured by?

-the electron transfer potential of carriers is measured by standard reduction potential, E´₀

→ good reducing agents give up electrons easily and have negative E´_o values

→strong oxidizing agents have a greater affinity for electrons and have positive E´₀ values

-passage of electrons through the ‘chain’ (from –ve to +ve) results in a free energy change that drives conformational changes in the complexes, setting up a proton gradient for ATP synthase to generate ATP

what is Peter Mitchell’s Chemiosmotic hypothesis?

-ATP synthesis arises due to an electrochemical gradient across the mitochondrial inner membrane

-describes the importance of proton-motive force set up by the electron transport chain for ATP synthesis in the matrix

how does ATP synthase ‘make’ ATP from the proton gradient?

-the proton gradient is produced by e- transport using suitable e- donors

-ATP synthesis arises due to an electrochemical gradient across the mitochondrial inner membrane

what is the driving force behind ADP to ATP conversions?

-proton-motive force (pmf)

ATP synthase

-is membrane-bound, reversible, and dependent on the proton gradient

-is a molecular motor

F1 domain

-carries out the catalytic synthesis of ATP in the matrix

-has a rotor shaft and stator

F0

-the integral membrane protein unit and anchors the complex to the membrane

-has a rotor

-have 6 beta subunits

—> Each β subunit functions independently and alternate between 3 states (and all undergo conformational changes)

process of ATP synthase generating ATP

1. binding of H+ in the rotor causing a rotation in the ring of c subunits of F0

2. the rotation of the ring, rotates the γ subunit, inducing a conformational change in the β subunits. H+ are released into the matrix

3. conformational changes in the F1 β-subunits are responsible for ATP synthesis

**as protons move across it spins the rotor

what are the 3 states each β subunit in F1 alternate between?

1. open or empty/exit (ATP leaves)

2. loose (ADP and Pi bound)

3. tight (ATP bound)

what are the 6 oxidative phosphorylation inhibitors?

1. rotenone (insecticide) inhibit electron flow from complex I to CoQ (turns off complex 1)

2. amytal (barbituate) inhibits electron flow from complex I to CoQ

3. antimycin A blocks complex III

4. cyanide inhibits complex IV

5. azide inhibits complex IV

6. CO inhibits complex IV

7. oligomyocin inhibits ATP synthase (complex V) → gives an ETC that just doesn’t do anything (no product)

8. uncouplers also disrupt the H+ gradient, affecting ATP synthesis

** #1 and 2 → these weaken ATP synthesis since we still have complex 2 (just produces less ATP)

** #4,5 and 6 → can’t convert O2 into H20 but we will still have proton movement

uncouplers

-are molecules that are amphipathic

-are hydrophobic and can cross the membrane (don’t need transporters)

-contain acid groups can be protonated and deprotonated, which allows them to bind H+ and move them from high to low concentrations, disrupting the proton gradient and ATP synthesis

how many H+ are needed for ATP synthesis?

-3 H+ are transported to produce 1 ATP

-then 1 extra H+ is needed for ATP export and ADP and Pi import

→ ATP-ADP translocase and Pi carrier protein

→ maintains the charge across the inner mitochondrial membrane

• OVERALL: ~4 H+ required / ATP made in the matrix

what ATP synthesis to occur in the matrix, what must happen?

-as protons flow through the c ring, rotation in the γ subunit results in conformational changes in the β subunits for ATP synthesis to occur in the matrix

how many ATPs are made from the ETC?

-NADH = 10 H+ / (4 H+/ATP) = 2.5 ATP

-FADH2 = 6 H+ / (4 H+/ATP) = 1.5 ATP

**half ATP doesn’t make sense so you round the number down

-NADH and FADH2 each donate a pair of e- to the ETC, resulting H+ being pumped into a pool of protons used by ATP Synthase

what tells how many ATPs are made per O2 reduced to water?

-The P/O ratio tells you how many ATPs (P) are made per Oxygen reduced to water (2 e- from donor)

P/O ratio

-determines the number of ATPs synthesized per molecular oxygen reduced to water

how much water is formed in the ETC?

in complex IV:

→ NADH + H+ + ½ O2 ——> NAD+ + H2O

→ FADH2 + ½ O2 ——> FAD + H2O

so, 1 H2O is formed at the last step in electron transfer (complex IV) from NADH and FADH2

how much water is formed by ATP synthase?

ADP + Pi ——> ATP + H2O

→ 2.5 H2O made when 2.5 ATP are made from NADH

→ 1.5 H2O made when 1.5 ATP are made from FADH2

so, therefore, 3.5 for NADH, 2.5 for FADH2 (or NADH_cyt.) of water is made

starting from glucose, what is the total number of ATPs and H2Os made through glycolysis, PDC, TCA cycle and oxidative phosphorylation?

-the total is 30 ATP and 36 H2O (based on 2 everything, so 2 rounds)

overall equation:

1 Glucose + 30 ADP + 30 Pi + 6 O2 ———> 6 CO2 + 30 ATP + 36 H2O

where is most ATP made?

-NADH and FADH2 generate way more ATP in oxidative phosphorylation