Module 11 - Regulation of Citrate Cycle and Chemiosmotic Theory

Question 1

How did the relative concentrations of sucrose and citrate change over days of aspergillus fermentation?

Answer 1

started at 100% sucrose, shift to 100% citrate

Question 2

What is an amphibolic pathway?

Answer 2

the citrate cycle is amphibolic because it functions in catabolic (oxidation of acetyl coA) and anabolic (production of precursors for fatty acid, amino acid, and heme biosynthesis) pathways

Question 3

How is the flux of pyruvate entering the citrate cycle controlled?

Answer 3

pyruvate dehydrogenase is activated by CoA and stimulates acetyl CoA production

pyruvate carboxylase is stimulated by acetyl CoA to maintain oxaloacetate for citrate synthesis

This means that pyruvate carboxylase balances the input of oxaloacetate with acetyl CoA when stored fat is metabolized. If there is a lot of acetyl CoA, that means that there isn’t enough oxaloacetate to convert it, so PC is stimulated to make more.

Question 4

When Aspergillus niger fermentation creates citrate, why does the natural citrate cycle NOT occur instead?

Answer 4

the culture condition inhibit part of the citrate cycle, and instead the citrate formed is exported out of the mitochondria

citrate lyase is inhibited (so it is not turned into acetyl CoA and oxaloacetate) and it continues to be exported out of the cell

Question 5

What is an anaplerotic reaction?

Answer 5

reactions that replenish citrate cycle intermediates that have been shunted to other metabolic pathways

Question 6

What metabolites of the citrate cycle are shared, and to what other processes?

Answer 6

acetyl coA: fatty acids and cholesterol
oxaloacetate: amino acids and glucose
a-ketoglutarate: amino acids
succinyl coA: heme

Question 7

How does pyruvate carboxylate form oxaloacetate?

Answer 7

uses biotin to catalyze an ATP dependent reaction

PC binds carboxybiotin which is decarboxylated to release a CO2, a pyruvate enolate forms with the carboxybiotin, then the CO2 reacts with the pyruvate enolate to form oxaloacetate

Question 8

How does pyruvate carboxylase function as an anaplerotic reaction?

Answer 8

phosphoenolpyruvate is…

• turned into oxloaetate by phosphoenolpyruvate carboxylase
  OR
• turned into malate by pyruvate kinase and malic enzyme

Question 9

What is the chemiosmotic theory statement?

Answer 9

energy from redox reactions is translated into vectorial energy in the form of a proton gradient by coupling the electron transfer to membrane bound proton pumps

Question 10

What are the two types of gradients in a proton circuit?

Answer 10

chemical gradient (delta pH)
membrane potential gradient (delta psi)

Question 11

What is required for the chemiosmotic theory to work?

Answer 11

an enclosed mitochondrial membrane

Question 12

In one sentence, how does the chemiosmotic theory work?

Answer 12

H+ are pumped. out of the matrix by the electron transport system and then flow back down the chemical and electrical gradients through an ATP-synthase complex

Question 13

What do uncoupler proteins and inhibitors do to the proton circuits?

Answer 13

Uncoupler proteins direct energy away from ATP-synthesis and produce heat, while inhibitors stop proton flow and lead to cell death

Question 14

The inner mitochondrial membrane is composed of…

Answer 14

80% protein, mostly ETC complexes and ATP synthase enzyme

Question 15

Oxidative phosphorylation couples…

Answer 15

NADH oxidation with ATP synthesis

Question 16

What are the three “parts” of oxidative phosphorylation?

Answer 16

electron transport system, cytochrome C, and ATP synthase

Question 17

What does Ox Phos accomplish for the cell?

Answer 17

generate 28 ATP (of the 32 from glucose catabolism), and produce heat for thermoregulation via uncoupling protein 1 in brown adipose tissue

Question 18

What is the overall net reaction of NADH oxidation in the OxPhos pathway?

Answer 18

2 NADH + 2 H + + 5 ADP + 5 Pi + O2 → 2 NAD+ + 5 ATP +2 H2O

Question 19

How was Mitchell’s idea (that proton circuits could generate energy for ATP synthesis) proven?

Answer 19

an artificial membrane contained bacteriorhodopsin and light activated ATP synthase

when light energy was present, ATP was synthesized

Question 20

Using 2e- from NADH oxidation, how many H+ are translocated in the different complexes of the electron transport system?

Answer 20

4 H+ from complex I
4 H+ from complex III
2 H+ from complex IV

Question 21

What is the difference between complex 1 and complex 2 starting the electron transport system?

Answer 21

complex 1: NADH 2e- transports 10 H+ total
complex 2: FADH2 2e- transports 6 H+ total

Question 22

How does the dG of chemiosmosis differ in mitochondria and chloroplasts?

Answer 22

mitochondria: charge difference is more significant than H+ difference

chloroplast: H+ difference is more significant than the charge difference

Question 23

What is the difference between a redox loop and a redox driven proton pump?

Answer 23

redox loop: separation of H+ and e- occurs on opposite sides of the membrane
proton pump: conformational changes in the protein complex pumps protons across the membrane

Question 24

What is complex 1 and how does it work?

Answer 24

NADH-ubiquinone oxidoreductase

1. NADH transfers 2 e- to FMN
2. 2 e- transferred through carriers
3. 2 e- and 2 H+ bind to Q forming QH2

Question 25

What is the structure of complex 1?

Answer 25

63 transmembrane alpha helice
Fe-S (iron sulfur centers) that carry one electron at a time from one end of the complex to the other

Question 26

What is another name for Coenzyme 10? What is the structure?

Answer 26

CoQ10 contains 10 isoprenoid units

Question 27

What is complex 2 and how does it work?

Answer 27

succinate dehydrogenase

1. a coupled redox reaction involving FAD turns succinate to fumarate
2. 2 e- go through carriers
3. 2 e- and 2 H+ bind to Q forming QH2

Question 28

What is the structure of cytochrome c and how does it work?

Answer 28

an iron containing heme group transfers 1 e- at a time to complex IV

oxidized contains Fe 3+ (ferric iron)
reduced contains Fe 2+ (ferrous iron)

Question 29

What is complex 3 and how does it work?

Answer 29

ubiquinone-cytochrome C oxidoreductase
(used for the Q cycle)

1. QH2 releases 2 H+ into inter membrane space and 2 e-
2. 2 e- split into single electrons (one to cytochrome C, other forms a semiquinone)
3. another QH2 releases 2 H+ and 2 e- (one to cytochrome C and the other reforms a QH2 from the previous semiquinone)
4. reformed QH2 grabs 2 more H+ to prep for the next round

Question 30

What are the overall transformations done by complex 3?

Answer 30

2 QH2 oxidized, but 1 regenerated
4 H+ translocated
2 cytochrome C molecules each transporting an electron to complex IV

Question 31

Complex 3 functions as a —- and a ——

Answer 31

mobile electron carrier (pushing electrons through the cycle)
transformer (2 e- system to 1 e- system used by cytochrome c)

Question 32

What is the structure of cytochrome C?

Answer 32

cytochrome proteins are classified as a, b, or c based on the type of heme they contain
a: hydrophobic tail
b: normal
c: protein link

the heme is covalently linked to the protein

Question 33

What is complex 4 and how does it work?

Answer 33

cytochrome c oxidase (a kind of oxidoreductase)

1. cytochrome c drops 2 e-, which move through carriers
2. 2 H+ and 1/2 O2 is turned into water
3. 2 other H+ are translocated into the inter membrane space

overall: 4 H+ are involved, but only 2 H+ translocate

Question 34

What is the structure of complex 4?

Answer 34

• homodimer containing two copper centers and two heme groups
• cytochrome c binds near copper A leading to oxidation of the heme in cytochrome c

Question 35

What is the energy requirement to transport 1 H+ across the membrane at 37 C? Where does this energy come from? How much energy is actually generated through redox reactions, and what does this mean for transport potential?

Answer 35

22.4 kJ/mol
reduction potential in NADH and FADH2 made available by oxidation

-220 kJ/mol are generated and 10 H+ are translocated, which is -22 kJ/mol each. This is pretty close to the amount needed for each H+ (22.4)