Oxidative Phosphorylation

Question 1

Describe the [H+] gradient across the mitochondria

Describe the Malate-Aspartate Shuttle in 3 steps.

What does it depend on?

Describe the Glycerol-3-P shuttle in 4 steps.

how is it different to the other shuttle?

Answer 1

High [H+] in inner membrane/cytosol & low [H+] in matrix- electrochemical gradient for moving H+ ions into matrix

1. In cytosol- oxoloacetate converted into malate with malate dehydrogenase & NADH (releasing NAD 2H+ & e-)
2. malate transported from cytosol into matrix down [H+] gradient where mitochondrial malate dehydrogenase & NAD convert it back into oxoloacetate
3. matrix oxoloacetate can then be transported back into cytosol with aspartate

oxidation (O2) of NADH & hence reduction of oxoloacetate into reduced malate (e-)

1. dihydroxyacetone phosphate (glycolysis intermediate) is reduced by NADH and glycerol-3-P dehydrogenase into glycerol-3-P in the cytosol
2. glycerol-3-p transported into transmembrane space
3. reacts with glycerol-3-p dehydrogenase on inner membrane (active site facing inter-membrane space) & reacts with FAD to form dihydroxyacetone phosphate
4. dihydroxyacetone phosphate in intermembrane space then transported back into cytosol

loss of energy (NADH (2.5) used and FADH2 (1.5) made so net loss of 1)
highly expressed in muscle
not efficient
fast- for exercise to produce ATP

Question 2

What is the electron transfer chain?

Which electrode potential is preferred?

Briefly describe in 4 steps how it works.

what 2 things are involved in it?

what are the balanced redox equations?

what are the 2 oxidation reactions of FADH2?

Answer 2

Chain molecules that transfer electrons from donors to acceptors in redox which is coupled with with the transfer of protons across the mitochondrial membrane

reduction: negative gibbs energy & positive Eo means spontaneous, endothermic reaction

1. Complex I reduces coenzyme Q with NADH
2. Complex II reduces coenzyme Q with FADH2
3. complex III helps reduced coenzyme Q passes electrons onto cytochrome C
4. complex IV reduces oxygen with cytochrome C

electron carriers, proteins

reduction: 1/2O2 + 2e- + 2H+ -> H2O
oxidation: NADH -> NAD + 2e- + H+

FADH2 -> FADH + e- + H+
FADH -> FAD + e- + H+

Question 3

what electron carriers are involved?

what 3 steps occur at complex I?

What electron carrier is involved here?

what 2 steps occur at complex II?

where is the entry point?

what electron carriers are involved?

why is complex II different to complex I?

Answer 3

ubiquinone, flavin mononucleotide, iron-sulphur, cytochrome (B & C)

1. NADH is oxidised into NAD+ and H+
2. the H+ reduces coQ into QH
3. 4H+ are pumped out from the inner membrane to intermembrane space

Fe-S

1. FADH2 is oxidised into FAD
2. coQ is reduced into QH2

FADH2 can enter from the citric acid cycle when succinate dehydrogenase catalyses reduction of QH2 (as succinate dehydrogenase is involved in CAC & ETC)

doesn’t pump any protons into the intermembrane space

Fe-S, cytochrome B

Question 4

what 2 steps occurs at complex 3?

what electron carriers involved?

what 2 steps occurs at complex 4?

for 1 NADH molecule reduced, how much H+ is pumped out of the inner membrane & how much oxygen is reduced?

how much is realistically needed?

what happens if you only start from Complex II?

what is the H+ gradient?

Answer 4

1. reduced coQ passes its electrons to 2x cytochrome C (2 electrons) to reduce them
2. 4H+ are pumped out

cytochrome B & FE-S

1. 2x cytochrome C reduce 1/2O2 with 2 electrons producing 1/2 O2
2. This pumps out 2H+ into the matrix & makes H2O

10 H+ (4 from complex I, 4 from III, 2 from IV) 1/2 O2

2NADH - 20H+ so 4x cytochrome C reduces O2 to make 2H2O

Only pump out 6H+ so lose energy

Question 5

What subunits does the ATP synthase complex have? What do they do?

Where is it?

What are the 3 beta conformations?

in 1 full rotation, how much ATP is made? how much H+ needs to be pumped into the matrix for this?

Answer 5

alpha, beta, c subunits & gamma stalk
alpha = holds beta in place
beta = how ADP & Pi binds
c = helps turn
gamma stalk = turns the complex for change beta structural configuration 

embedded in mitochondrial membrane

Loose = irreversibly holds ADP & Pi
tight = active site perfect shape for ATP so makes & holds ATP
open = releases ATP & reversibly binds ADP + Pi

3ATP as 3 subunits - 10H+

Question 6

What happens if there is no H+ gradient?

What happens if there is no ADP + Pi?

What happens when succinate is the substrate instead of malate?

What is state 2 respiration?

state 3?

state 4? what happens if you add more ADP at this stage?

Answer 6

Can’t synthesise ATP- as ATP synthase needs H+ gradient as driven by H+ moving back into matrix from membrane

• no H+ gradient means no ATP
• no ADP means H+ can’t flow back into matrix

H+ can’t flow back into matrix

succinate is oxidised at complex II so 4 less H+ pumped across inner membrane so get less ATP (1.5) per FADH2 oxidised

mitochondria with substrate but no ADP

add ADP until add oxygen is used up- all of the ADP into ATP & oxygen consumption stops
(initial rate of oxygen is proportional to ADP added)

ADP exhausted rate oxygen consumption close to 0- return to stage 3 if add more ADP

Question 7

what are uncouplers?

what is an example?

what is coupled?

what are the 2 requirements in a coupled reaction?

what is the normal coupled reaction?

what happens in an uncoupled reaction?

in a coupled reaction- what is oxidative phosphorylation dependent on?

in a coupled reaction- what is the electron transport chain dependent on?

in an uncoupled reaction- what is oxidative phosphorylation dependent on?

in an uncoupled reaction- what is the electron transport chain dependent on?

in the presence of DNP, lots of oxygen is consumed but why is the P:O ratio wrong?

Answer 7

compounds that carry H+ across membrane for ATP synthase

2,4-dinitrophenol DNP

oxidative phosphorylation and electron transport chain

H+ gradient nd ADP- without, neither reaction will occur

H+ ion doesn’t build up in cytosol as it’s dependent on substrate conc
oxygen reduced no matter ADP conc

H+ is transported across membrane despite substrate concentration so ATP is synthesised
- synthesis of ATP no longer dependent with substrate conc or oxygen
Substrate & oxygen must be present to reduce oxygen

ADP (substrate conc & oxygen levels)

ADP (substrate conc & oxygen levels)

uncoupler will be present, ADP concentration

substrate conc & oxygen levels- not ADP

as oxygen is consumed regardless of ADP concentration, so the P:O will be higher