BIOCH 200: Slide 8 (Oxidative phosphorylation)

Catabolic pathways…

a. Oxidize metabolites

b. Generate reduced cofactors

c. Both A + B

d. Neither A nor B

C

What 2 purposes do catabolic pathways serve?

1. Break down large molecules into BUILDING UNITS

2. Release and temporary storage of E in High E molecules

   • ATP/NTPs

   • Reduced cofactors (NADH/FADH2)

Are catabolic pathways oxidative or reductive? What happens to metabolites + cofactors.

Oxidative

• metabolites = oxidized

• Cofactors = reduced

What does the Re-oxidation of cofactors after they are reduced do?

Generate ATP

What 2 processes is oxidative phosphorylation made up of?

1. Oxidation of reduced cofactors (NADH/FADH2) by the reduction of O2

2. Phosphorylation of ADP —> ATP

Where does oxidative phosphorylation occur?

A proton gradient across the INNER Mitochondrial membrane

What is the overall flow to generate ATP?

What are the 6 major components of the ETC?

Complex 1, 2, 3 + 4 (integral membrane proteins)

Coenzyme Q (lipid soluble coenzyme)

Cytochrome c (peripheral membrane protein)

What are 4 four electron-carrying cofactors that play a role in re-oxidation of FADH2 and NADH in the ETC

1. FMN

2. Iron-sulfur clusters

3. Cytochromes

4. Coenzyme Q

How many e- does FMN carry?

2

How many e- do Iron -sulfur clusters carry/pass?

1

What switches between oxidized and reduced sates in cytochromes?/ what takes part in the passing of e- in the ETC?

HEME

• cytochromes = hemoproteins

How many e- do cytochromes pass?

1

• Fe3+ + 1e- —> Fe2+

What type of molecule is Coenzyme Q?

Lipid soluble molecule

What does Coenzyme Q do?

Transports e- from Complex 1 and 2 —> Complex 3

• becomes Ubiquinol QH2

How many e- does Coenzyme Q carry/transport?

2

• Q (ubiquinone) + 2e- + 2H+ —> QH2 (ubiquinol)

What is reduction potential?

affinity for e-

What does a higher reduction potential mean?

More negative delta G

True or False: e- move from compounds with higher reduction potential to those with lower reduction potential

False:

e- move from compounds with lower reduction potential to those with higher reduction potential

• low reduction potential = don’t want e- that much = willing to give up e-

• High reduction potential = really wants e-

Why do e- move spontaneously through the ETC?

e- are passed from components with lower Reduction potential to components with higher reduction potential

What is generated by the redox reactions of the ETC (low —> high reduction potential)

Free energy

How can the Free E from redox reactions of the ETC be used?

TRANSPORT H+ across the membrane

What type of transport is using Free E of redox reactions to transport H+?

PRIMARY active transport

How does this Primary active transport work in the ETC?

Electron transport causes CONFORMATIONAL CHANGE which allows the complexes to pump H+

Which complexes of the ETC can pump H+? How many are pumped at each?

Complex 1 = 4 (Oxidation of NADH = high E)

Complex 3 = 4 (Q cycle)

Complex 4 = 2 (Reduction of O2 = High E)

How many H+ are pumped out as a result of NADH deoxidization? what about FADH?

NADH = 10

FADH = 6

How many e- are transported from NADH?

2

How many e- are transported from FADH2

2

What is the terminal e- acceptor of the ETC?

O2

• reduction of O2 = lots of E

• Very high reduction potential

Illustrate the Path of e- from NADH in ETC

Illustrate the Path of e- from FADH2 in ETC

What is complex 2 also known as? Where else can you find it?

Succinate Dehydrogenase

• part of CAC

What prosthetic group does Complex 2 contain?

FAD

What does Complex 2 do as succinate dehydrogenase?

Catalyzes oxidation of Succinate —> Fumarate as part of CAC

Where do the e- from the oxidation of Succinate go?

To coenzyme Q in the membrane

What determines the Rate of O2 consumption?

B

• A and C both refer to thermodynamics not kinetics

• Affinity = thermodynamics

  • Speed / rate = Kinetics

What drives ATP synthesis by ATP synthase?

the proton electrochemical gradient

What is the proton electrochemical gradient?

PROTON MOTIVE FORCE

• difference in proton (H⁺) concentration and charge across the inner mitochondrial membrane

How is ATP formed by the proton electrochemical gradient?

The potential E of H+ gradient = converted to chemical E in the Phosphoanhydride bonds of ATP

Where is the [H+] high and where is it low?

High = Inter-membrane space (b/c ETC complexes)

Low = Mitochondrial matrix

What are the 2 parts of the ATP synthase?

• Fo and F1

What does Fo and F1 do?

• F0 (Transmembrane protein)= Triggers CONFORMATIONAL CHANGE of F1 when protons pass through

• F1 = Catalytic portion. Synthesizes ATP from ADP

What does the O of Fo stand for? (don’t know how important)

O = Oligomycin

• Oligomycin = inhibits the action of Fo portion of ATP synthase

What determines the rate of O2 consumption?

The rate of ATP synthesis = determines how many [H+] is needed thus ultimately O2 consumption as the Final e- acceptor

How many H+ are needed per ATP by ATP synthase?

3

How many ATP are generated by every complete turn of the central shaft of ATP synthase

3 ATP

• 3 active sites make ATP simultaneously

Adenine Nucleotide Translocase?

ANTIPORTER

• transports newly synth ATP —> cytosol

• Transports ADP (after ATP used) —> matrix

Pi-H+ Symporter?

It transports Pi into matrix for the synthesis of ATP by ATP synthase

• Powered by PMF (H+ gradient)

What is the diff in charge and pH inside vs outside the mitochondrial matrix

In = - charge + High pH

Out = + charge + Lower pH

• all bc of H+ gradient

Overall what is the Net # of H+ needed to generate 1 ATP?

4

• 3 for ATP synthase

• 1 for Pi-H+ symporter

What does it mean by oxidation and phosphorylation are coupled?

The rate of O2 consumption is connected to the rate of ATP synthesis

• NADH reoxidation, ETC + O2 consumption = coupled to Rate of Consumption + synthesis of ATP through the Magnitude of H+ electrochemical gradient

What drives the rate of ATP synthesis

the availability of ADP and Pi

What is the P:O ratio?

Amount of ATP made (P: phosphorylation) per O2 atom reduced to water (O)

How many H2O = made per each NADH/FADH2

1

• 2e- from NADH/FADH2

• 2H+ + 2e- + 1/2O2 —> H2O

What is the P:O ratio for each NADH reoxidized? and why?

~ 2.5

• NADH = 10 H+

• 4 H+ = 1ATP

• 10/4 = 2.5

What is the P:O ratio for FADH2?and why?

1.5

• NADH = 10 H+

• 4 H+ = 1ATP

• 6/4 = 2.5

True or false: The rate of oxidative phosphorylation = determined largely by the relative [ADP]

TRUE

• if no ADP = No ATP can be made by ATP synthase = can’t dissipate gradient = gradient doesn’t need to be replenished by the ETC

How does O2 consumption react in response to Increased ADP

consumption increases

What does [ADP] reflect?

The E consumption of a cell

How does the activity of ATP synthase effect the rate of CAC in tissue?

High ATP synthase activity = HIGH NADH/FADH2 usage = low [ ] = activate CAC

Low ATP synthase activity = High [NADH/FADH2] accumulate = Inhibit CAC

How does Low Energy use affect Coupling of oxidative phosphorylation

PDH = pyruvate dehydrogenase

How does High Energy use affect Coupling of oxidative phosphorylation

How does O2 concentration react to the addition of ADP

They decrease

• ADP addition = ATP synthase stimulated = O2 Consumption = decrease [O2]

What is an uncoupler/ Uncoupling protein? + how does it affect the system?

Uncoupler = Allows H+ into matrix without making ATP = Uncoupled system

What do uncoupling proteins generate instead of ATP?

HEAT

What is Brown adipose tissue?

Adipose tissue with lots of MITOCHONDRIA it produces heat by using uncoupling proteins to generate heat

What happens to O2 consumption in the presence of an Uncoupler?

O2 consumption increases

Why does O2 consumption increase in the presence of an uncoupler?

Uncoupler = transport H+ without generating ATP (heat instead)

• Proton gradient = dissipated faster = ETC rate increases

How do uncouplers affect CAC?

Uncoupler = increase rate of ETC = increase rate of NADH/FADH2 oxidation/usage = low [NADH/FADH2] = Activate CAC

a. Increase

b. Decrease

c. Depends on if NADH/FADH2 = being oxidized

d. no change

B: Less ATP per O2 reduced

Effect of Uncoupler on O2 consumption + ATP prod

2,4-dintrophenol = diet pill (uncoupler)

• dissipates H+ gradient

What is the potential consequence of uncoupling mitochondrial oxidative phosphorylation?

C

A.

Time 2 = ATP synthase inhibitor

Time 3 = Uncoupler