Lecture 21

Question 1

What is a reduction potential?

Answer 1

• A measure of the potential energy of electrons.
• Electron transfer potential.

X + 2H+—-> + 2e- XH2
ox form red form

• XH2 and X are a redox pair shown in a “half-reaction”
• “Reduction Potential” reflects how much a molecule prefers to be in the reduced state.
• Positive means it prefers to be reduced
• Negative means it prefers to be oxidized

Question 2

E0

Answer 2

1 M everything, (1 atm for gas)
• Reference half cell: H+
+ e- —-> ½ H2
• E0 for the reference cell = 0
• When E0 is < 0 then X has less need than H+ to be reduced so electrons flow towards the reference cell.
• When E0 is > 0 then X wants electrons more than H2
so electrons flow away from the reference cell.

Question 3

Some Standard Reduction Potentials

Acceptors Donors E’0

Answer 3

acceptors: .5O2, FAD, fumarate, oxaloacetate, NAD+, α-ketoglutarate

Donors: H2O,FADH2, succinate, malate, NADH, isocitrate

Question 4

How to calculate ∆E0’

Answer 4

in other words how do you determine the “driving force” for a reaction?

Acetaldehyde + NADH + H+ —–> ethanol + NAD+

two half rxns
Acetaldehyde + 2H+ + 2e- —-> ethanol -0.197
NAD+ + 2e- + H+ —-> NADH -0.32

∆E0’ = E0’ acceptor - E0’donor

Which is the electron acceptor? Acetaldehyde
Which is the electron donor? NADH
What is ∆E0’ for this reaction? (-0.197) – (-0.32) = +0.123

Question 5

What is the ΔE0’ for reduction of O2 by NADH?

Answer 5

NAD+ + 2e- + H+ ——> NADH Eo’ = -0.32V
½ O2 + 2e- +2H+ ——> H2O Eo’ = +0.82V

½ O2 + NADH + H+ ——> H2O + NAD+’

ΔE0’ = Eacceptor – Edonor = +0.82V - (-0.32V) = +1.14 V

ΔG0’ = -nFΔE0’ = -2(96 kJ mol-1 V-1)(1.14 V) = -219kJ/mol

Question 6

In summary

Answer 6

• If ∆G0’ is <0, ∆E0’ is >0
–the reaction is exergonic.

• If ∆G0’ is >0, ∆E0’ is <0
–the reaction is endergonic.

Question 7

Overall reaction catalyzed by complex 1:

Answer 7

NADH + Q + 5 H+ matrix —->NAD+ + QH2 + 4H+ cytoplasm

Question 8

Complex 1:

Answer 8

“NADH-coenzyme Q Oxidoreductase”

46 subunits, 900 kDal

Question 9

Flavin Mononucleotides

Answer 9

• A derivative of Riboflavin (Vitamin B2)

- Tightly bound to proteins in the respiratory complexes.

Question 10

Electron carriers:

Ubiquinone (Q)

Answer 10

• Soluble in the membrane
• Can accept two electrons
• Pass them on one at a time.
• Forms a radical or semiquinone

Question 11

Type of electron carriers in the respiratory chain

Answer 11

• Flavin mononucleotides
• Iron-sulfur proteins
• Ubiquinones
• Cytochromes

Question 12

“Q cycle”:

Answer 12

“Q pool” means a mix of Q and QH2 floating in the membrane

Question 13

Why is the Q cycle important?

Answer 13

• The Q cycle allows a 2 electron carrier (QH2) to

- transfer electrons one a time to a one electron carrier (Cyt c)

Question 14

Cytochromes: Electron carriers

Answer 14

• All have heme prosthetic groups
• Pass one electron Fe3+ –> Fe2+
• Reduction potentials vary from 0.077 to 0.55 volts
• Different micro-environments in the protein.

Question 15

Cytochrome Oxidase (Complex IV)

Answer 15

2cyt(c red) + 4H+(matrix)+ 1/2O2 —–> 2cyt(c ox) + 1H2O +2H+ (cytoplasm)

Question 16

idk add after lecture

Answer 16

ΔE0’ = Eacceptor – Edonor = +0.82V - (-0.32V) = +1.14 V

ΔG0’ = -nFΔE0’ = -2(96 kJ mol-1 V-1)(1.14 V) = -219 kJ/mol

Phosphorylation of ADP to ATP requires 30.5 kJ/mol so this releases plenty of energy to synthesize many ATP’s

Question 17

Inhibitors of Respiration

Answer 17

• Rotenone- (plant toxin used as an insecticide)
• Amytal
• Cyanide
• Antimycin A (an antibiotic)

Question 18

Where do these inhibitors act?

Answer 18

• Treat mitochondria with amytal

– Find that NADH is reduced.– Q, cyt b, cyt c1, cyt c, cyt a, and cyt a3 are oxidized.

Question 19

Reaction 1: Citrate Synthase

Answer 19

The first reaction of the citric acid cycle is catalyzed by the enzyme citrate synthase. In this step, oxaloacetate is joined with acetyl-CoA to form citric acid. Once the two molecules are joined, a water molecule attacks the acetyl leading to the release of coenzyme A from the complex

Acetyl-CoA + Oxaloacetate + H2O <=> Citrate + CoASH + H+ (Enzyme: <>, DeltaG0’= -32.2 kJ/mol)

Question 20

Reaction 2: Acontinase

Answer 20

The next reaction of the citric acid cycle is catalyzed by the enzyme acontinase. In this reaction, a water molecule is removed from the citric acid and then put back on in another location. The overall effect of this conversion is that the –OH group is moved from the 3’ to the 4’ position on the molecule. This transformation yields the molecule isocitrate.

Citrate <=> cis-Aconitate + H2O <=> Isocitrate (Enzyme: <>, DeltaG0’= +6.3 kJ/mol)

Question 21

Reaction 3: Isocitrate Dehydrogenase

Answer 21

Two events occur in reaction 3 of the citric acid cycle. In the first reaction, we see our first generation of NADH from NAD. The enzyme isocitrate dehydrogenase catalyzes the oxidation of the –OH group at the 4’ position of isocitrate to yield an intermediate which then has a carbon dioxide molecule removed from it to yield alpha-ketoglutarate.

Question 22

Reaction 4: Alpha-ketoglutarate deydrogenase

Answer 22

In reaction 4 of the citric acid cycle, alpha-ketoglutarate loses a carbon dioxide molecule and coenzyme A is added in its place. The decarboxylation occurs with the help of NAD, which is converted to NADH. The enzyme that catalyzes this reaction is alpha-ketoglutarate dehydrogenase. The mechanism of this conversion is very similar to what occurs in the first few steps of pyruvate metabolism. The resulting molecule is called succinyl-CoA.

Question 23

Reaction 5: Succinyl-CoA Synthetase

Answer 23

The enzyme succinyl-CoA synthetase catalyzes the fifth reaction of the citric acid cycle. In this step a molecule of guanosine triphosphate (GTP) is synthesized. GTP is a molecule that is very similar in its structure and energetic properties to ATP and can be used in cells in much the same way. GTP synthesis occurs with the addition of a free phosphate group to a GDP molecule (similar to ATP synthesis from ADP). In this reaction, a free phosphate group first attacks the succinyl-CoA molecule releasing the CoA. After the phosphate is attached to the molecule, it is transferred to the GDP to form GTP. The resulting product is the molecule succinate.

Question 24

Reaction 6: Succinate Dehydrogenase

Answer 24

The enzyme succinate dehydrogenase catalyzes the removal of two hydrogens from succinate in the sixth reaction of the citric acid cycle. In the reaction, a molecule of FAD, a coenzyme similar to NAD, is reduced to FADH2 as it takes the hydrogens from succinate. The product of this reaction is fumarate.

FAD, like NAD, is the oxidized form while FADH2 is the reduced form. Although FAD and NAD perform the same oxidative and reductive roles in reactions, FAD and NAD work on different classes of molecules. FAD oxidizes carbon-carbon double and triple bonds while NAD oxidizes mostly carbon-oxygen bonds.

Question 25

Reaction 7: Fumarase

Answer 25

In this reaction, the enzyme fumarase catalyzes the addition of a water molecule to the fumarate in the form of an –OH group to yield the molecule L- malate.

Question 26

Reaction 8: Malate Dehydrogenase

Answer 26

In the final reaction of the citric acid cycle, we regenerate oxaloacetate by oxidizing L–malate with a molecule of NAD to produce NADH.

Question 27

Conclusion

Answer 27

We have now concluded our discussion of the reactions that compose the citric acid cycle. It is helpful at this point to take a minute to take stock of what the citric acid cycle has generated from one acetyl-CoA molecule.

The acetyl-CoA, has been oxidized to two molecules of carbon dioxide.
Three molecules of NAD were reduced to NADH.
One molecule of FAD was reduced to FADH2.
One molecule of GTP (the equivalent of ATP) was produced.

Question 28

reaction order

Answer 28

Citrate Synthase
Acontinase
Isocitrate Dehydrogenase
Alpha-ketoglutarate deydrogenase
Succinyl-CoA Synthetase
Fumarase
Malate Dehydrogenase

Question 29

Key concepts for electron transport

Answer 29

• NADH is oxidized stepwise through three large protein complexes to ultimately transfer electrons to oxygen.
• Complexes I, III and IV translocate protons from the matrix to the cytoplasmic side of the mitochondrial inner membrane.
• FMN, Iron-sulfur clusters, Ubiquinone, Cu ions and the heme of cytochromes act as electron carriers.
• The energy of oxidation is stored in a proton gradient
• The Q cycle allows a 2 electron carrier to transfer one electron at a time to a 1 electron carrier.
• Q and cytochrome c are “couriers” between complexes.