1

MCB 3020, Spring 2005

Chapter 5: Nutrition and Metabolism I

2The Generation of Energy:I. Metabolism (metabolic reactions)II. NutrientsIII. EnergyIV. Review of free energyV. EnzymesVI. Energy generation: oxidation and reduction reactions

3I. Metabolism (metabolic reactions)

• all of the biochemical reactions in a cell

• includes catabolic (degradative) and anabolic (biosynthetic) reactions

4

2. Anabolism• the biosynthesis of complex molecules from simpler compounds with the input of energy

1. Catabolism• the breakdown of complex molecules into simpler compounds with the release of energy

ENERG

Y

5

Catabolismenergysource

wasteproducts

ATP,reductant

small molecules

Anabolism macromolecules(polymers)

B. Catabolic reactions generate ATP. ATP is used for biosynthesis and cell maintenance.

6C. ATP is called the energy currency of the cell.

• anabolic (biosynthetic) reactions require energy in the form of ATP.

• catabolic reactions release energy and store it as ATP.

7II. Nutrition

1. macronutrients2. micronutrients3. growth factors

chemicals taken up from environment and used for cellular reactions

A. Nutrients

81. Macronutrients: chemicals taken up and required in relatively large amounts

CHONPS

K+

Mg2+

Na+

Ca2+

Fe2+/Fe3+

9

CHONPSFe

nucleic acids, phospholipids

cysteine, methionine, vitamins like CoA

amino acids, nucleic acids, cell walls, etc.

many organic molecules

Where do macronutrients occur in cells?

Electron transport proteins

102. Micronutrients: inorganic chemicals required in small amounts

• also called trace elements• usually metals in metabolic enzymes

Co (the metal center of vitamin B12)Cu (found in electron transport proteins)Se (found in selenocysteine)Ni, Zn, Mn, V, W

• examples

113. Growth factors: organic chemicals required in small amounts by some (but not all) cells

a. Examples:vitamins, like B1, B6, B12, biotinsome amino acidspurines, pyrimidines

12b. Many vitamins are precursors of coenzymes used in metabolism. Vitamin CoenzymeB2 (riboflavin) FAD, FMNniacin (nicotinic acid) NAD, NADPB12 cobalaminfolate tetrahydrofolate

Coenzymes are molecules that work together with enzymes to catalyze chemical reactions.

13B. Cells can be grown in laboratory cultures.

Two classes of culture media1. Chemically defined medium

exact chemical composition is known;contains precise amounts of pure chemicals added to distilled water

2. Complex (undefined) mediumexact chemical composition is not known;contains digests of milk proteins, yeast, soybeans, etc. that have growth factors?

14Different organisms can have vastly different nutritional requirements.

Escherichia coli can grow on a simple defined medium. It can synthesize most of the organic molecules required for biosynthesis.

Leuconostoc mesenteroides needs added amino acids, purines, pyrimidines, and vitamins for growth because it cannot synthesize these molecules by itself.

15Laboratory growth medium for E. coliK2HPO4

KH2PO4

(NH4)2SO4

MgSO4

CaCl2Glucose

mineralsH2O

Glucose, H2O, K2HPO4, KH2PO4, NH4Cl, MgSO4, Na acetate, alanine arginine asparagine, aspartate, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, adenine, guanine, uracil, xanthine biotin, folate, nicotinic acid, pyridoxal, pyridoxaminepyridoxine, riboflavin, thiamine, pantothenate, para-aminobenzoic acid, trace elements

Growth medium for L. mesenteroides

(don't memorize)

16

ENERGY

Why do cells need energy?

Where do organisms get energy?

How do cells use energy sources?

the ability to do work

III. Energy

17A. Why do cells need energy?

• growth and biosynthesis• motility • nutrient uptake• reproduction• maintenance, etc.

polysaccharidesnutrients

18B. Where do organisms get energy?

Chemotrophschemicals

Phototrophslight

Chemoorganotrophsorganic chemicals

(eg. sugars)

Chemolithotrophsinorganic chemicals (eg. H2, NH3, H2S)

19C. How do chemotrophs derive energy from energy sources?

Remember: oxidation is the loss of electrons

Organisms capture energy that is released when an organic or inorganic chemical is oxidized.

glucose + 6 O2 6 CO2 + 6 H2O G°’ = - 686 kcal/mol

20

kcal (kilocalorie)• a unit of energy

D. Units of energy

• amount of heat energy required to raise the temperature of 1 kg

of water 1°C• 1 kcal = 4.184 kilojoules (kJ)• 1 kcal = 1 “nutritional” calorie

21

• energy that is available to do useful work

IV. Review of free energy (G)

change in free energychange in enthalpy (total energy)

change in entropy

Review from General Chemistry: G = H - T S

22For biological reactions, the standard conditions for measuring the change in free energy (G°’ ) are

• 25°C• pH 7• reactants and products initially present at 1 M concentration

23A. The G°’ can tell us about the direction a reaction tends to occur.

A + B C + D

FreeEnergy

Progress of reaction

A + B

C + D G°’ is negative

If G°’ is (-)products have lower free energy than substrates

24 1. If G°’ is negative• free energy is released• the reaction is exergonic• the reaction tends to occur in the direction written

Examples: H2 + 1/2 O2 H2O - 57 kcal/mol glucose + 6 O2 6 CO2 + 6 H2O - 686 kcal/mol ATP + H2O ADP + PO4

- - 7.3 kcal/mol

25 2. If G°’ is positive• energy input is usually required• the reaction is endergonic• the reaction does not tend to occur in the direction written

FreeEnergy

Progress of reaction

A + B

C + D If G°’ is (+)products have higher freeenergy thansubstrates

G°’ is positive

26B. Coupled reactionsExergonic reactions (-G°’) can be used to "drive" endergonic reactions (+G°’) to make the overall "coupled" reaction favorable.

A B Go’ = +20 kJ/mole Reaction 1

Reactions 1 and 2 coupledA + C B + D Go’ = -10 kJ/mole

C D Go’ = -30 kJ/mole Reaction 2

27

A + B C + D

C. Equilibrium

• equilibrium occurs when the rates of the forward and reverse reactions are equal

• usually at equilibrium, the concentrations of the products and reactants are not equal

28C. Equilibrium (contd.)

• if the G°’ is large and negative, equilibrium lies towards product; very little of the reactants remain

A + B C + D

29

H2 + 1/2 O2 H2O G°’ = - 57 kcal/mol

If H2 and O2 are mixed without a catalyst, no detectable amount ofwater is formed in our lifetime. Why?

D. “Activation energy” is required to break bonds.

Because before water is formed, chemical bonds have to be broken.

30Activation energy: energy required to bring molecules to the reactive state

FreeEnergy

Progress of reaction

H2 + 1/2 O2

H2O

Activationenergy

31

Activationenergy of catalyzed reaction

E. Catalystschemicals that increase the reaction rate by lowering the activation energy

FreeEnergy

Progress of reaction

H2 + 1/2 O2

H2OG

32

• increase the rate of the reaction, • but DO NOT change the G, • DO NOT change the equilibrium • many reactions in living organisms are catalyzed by biological molecules called enzymes

Properties of catalysts

33V. Enzymes• biological catalysts

• most enzymes are proteins, a few are nucleic acids (ribozymes or catalytic RNAs)

• most enzymes catalyze specific reactions or sets of reactions

34A. Enzyme catalysis

Substrate(s) first combine with the enzyme to form an enzyme-substrate (E-S) complex.

S

E

Substrates (S): reactants, starting materials

S

Enzyme (E): usually a protein E

Products (P): ending materials P

35B. Typical enzymatic reaction sequence:

E + S E-S E-P E + P

Enzyme-substrate complex

At end of reaction, the enzyme returns to its original form

S

ES

EP

EP

E

36C. Important notes on enzymes• Enzymes DO NOT alter the equilibrium of the reaction. • Enzymes can catalyze exergonic and endergonic

reactions.• Substrates bind at the enzyme active site.• Many enzymes contain nonprotein components:

coenzymes (loosely bound) or prosthetic groups (tightly bound).

37Important notes on enzymes (contd.)

• Enzymes tend to be sensitive to pH and temperature.

• Enzymes are often named after the substrate or the reaction catalyzed, plus the ending “-ase” (eg. cellulase breaks down cellulose, ATP synthase makes ATP).

38D. Sometimes enzymes change shape when substrates bind (“induced fit”)glucose + hexokinase (a protein used in glycolysis)

Active site

39E. Metabolic reactions are catalyzed by enzymes.

glucose

ethanol + CO2

12 enzymes

O

OHHOOH

OH

CH2OH

glucose fermentation (anaerobic)

40Respiration of glucose (aerobic)

glucose + 6 O2

6 CO2 + 6 H2O

~30 enzymes~36 [ADP + Pi]

~36 ATP

41

For chemotrophs, utilization of a chemical energy source involves oxidation and reduction reactions (redox reactions).

VI. Energy generation: A. Oxidation and reduction reactions

42Oxidation and reduction reactions

“LEO says GER”

Gain of Electrons = Reduction1/2 O2 + 2 H+ + 2 e- H2O

Loss of Electrons = Oxidation

Glucose (C6H12O6) 12 H+ + 12 e- + 6 CO2

H2 2 H+ + 2 e-

43B. Complete redox reactions can be divided into oxidative and reductive half reactions.

Oxidative half-reaction: H2 2 H+ + 2 e-

e- donor e- acceptor

H2 and H+ are called a redox couple.

Reductive half-reaction: 1/2 O2 + 2 H+ + 2 e- H2O

Complete reaction: H2 + 1/2 O2 H2O

44C. Because electrons do not typically exist alone in solution, complete redox reactions need

an electron acceptor (eg. O2) an electron donor (eg. H2) and

primary electron donor

terminal e- acceptor

glucose + 6 O2 6 CO2 + 6 H2O

45D. Energy is released when an energy source is oxidized.

H2 + 1/2 O2 H2O - 57 kcal/mol

H2 2 H+ + 2 e-

Oxidative half-reaction

ENERG

Y

glucose + 6 O2 6 CO2 + 6 H2O - 686 kcal/mol

G°’

46E. Cells oxidize energy sources and harness the energy released to make ATP.

Explosive release of energy as heat can't be harnessed to do work

H2 1/2 O2

H2O

Electrontransportsystem

H2

2 H+ 2 e-

Hydrogen atoms separated intoprotons & electrons

H2O

2 e-2 H+ 1/2 O2

Some released energy is harnessed to make ATP

H2 + 1/2 O2 H2O G°’ = - 57 kcal/mol

47Study Objectives1. Understand metabolism, catabolism, anabolism, and the role of ATP in metabolism.2. Know the differences between macronutrients, micronutrients, and growth factors. Know where they occur in biological molecules and the examples presented in class.3. Contrast defined and complex media. Know one reason why nutritional requirements differ among organisms.4. Give examples of energy-requiring processes in the cell.5. Define chemotrophs, phototrophs, chemoorganotrophs, chemolithotrophs. (eg. chemotrophs are organisms that use chemicals as an energy source.) Given an energy source (eg. NH3), be able to identify the type of catabolism being used (eg. chemolithotrophy).6. Understand the terms kcal and free energy. What predictions can be made from the Go' value of a reaction. What is reaction coupling and how can it be used by the cell?

487. Understand equilibrium, activation energy, catalysts and their properties. Understand the effect of catalysts on equilibrium. Can catalysts make a nonspontaneous reaction spontaneous?8. Understand enzymes and all the properties presented in class. What is the function of enzymes in the cell?9. Define oxidation, reduction, half reactions, redox couples, electron donor, electron acceptor.10. Describe how cells derive energy from an energy source. What are the roles of the primary electron donor and the terminal electron acceptor?

49Energy generation and glycolysis

I. Oxidation of the energy sourceII. Reduction of NAD+III. Making ATP through substrate level phosphorylationIV. GlycolysisV. Reoxidation of NADH

50

glucosewasteproducts

ATP

I. Oxidation of the energy source: A. Energy released when an energy source is oxidized can be conserved in the form of high energy chemical bonds.

oxidation

ADP + Pi

chemicals with high energy bonds

ENER

GY

51

energy sourceprimary electron donorglucose

one or more intermediate electron carriers, e.g. NAD+

electrons[carbon]

terminal electron acceptor (the last molecule to accept electrons), e.g. O2

B. Electrons are transferred during catabolism.

52C. Redox terminology1. Oxidation is the loss of electrons

Compounds become oxidized afterlosing electrons.

An oxidant is a compound thataccepts electrons. It canoxidize other compounds. TB

Glucose (C6H12O6) 12 H+ + 12 e- + 6 CO2

53

2. Reduction is the gain of electrons

Compounds become reduced aftergaining electrons.

A reductant is a compound thatdonates electrons. It can reduceother compounds.

TB

54

A(red) A(ox)B(ox) B(red)+ +

electron donor

electron acceptor

D. Redox reactions

TBglucose + 6 O2 6 CO2 + 6 H2Oe.g.

551. Redox couples are substances interconverted by redox reactions

Note: the oxidized substance is written to the left. Two redox couples are needed for a redox reaction.TB

A(red) A(ox)B(ox) B(red)+ +

A(ox)/A(red) is a redox couple (CO2/ glucose)

B(ox)/B(red) is a redox couple (O2/ H2O)

56

pyruvate can be reduced to lactate lactate can be oxidized to pyruvate

Example: pyruvate and lactate are a redox couple.

pyruvate/lactate

Eo' (reduction potential) = -0.19 volts

pyruvate + 2H+ + 2e- lactate

half-reaction (hypothetical)

57

(Eo') is a measure of the tendency of a redoxcouple to donate electrons in a redox reaction.

2. Redox couples have associated standard reduction potentials (Eo').

TB

Eo' values can be summarized in a "table of reduction potentials."

In this table, the REDUCED substance of theredox couple is written on the right.

583. Partial table of reduction potentials

Oxidized form / Reduced form Reduction potentialEo' (Volts)

CO2 / glucose (C6H12O2) (- 0.43)2 H+ / H2 (- 0.42)NAD+ / NADH (- 0.32)

NO3- / NO2- (+ 0.42)

pyruvate / lactate (- 0.19)

O2 / H2O (+ 0.82)

fumarate / succinate (+ 0.03)

59a. In a table of reduction potentials, the reduced compound of redox couple with a more negative Eo'

can give electrons to

the oxidized compound of a redox couple lower in the table

electr

ons

60

Eo’ = -0.19 Vpyruvate/lactate

b. Example

In a redox reaction, NADH can donate electrons to pyruvate.

NADH + pyruvate NAD+ + lactate

Two redox couples

NAD+/NADH Eo’ = –0.32 V

61

Reduction potentialEo' (Volts)

CO2 / glucose (C6H12O2) (- 0.43)

O2 / H2O (+ 0.82)

electr

ons

Eo'

4Eo' is the change in standard reduction potential.

625. A large Eo' corresponds to a large Go'. Go' = -nF Eo' (don't memorize equation)

Reduction potentialEo' (Volts)

CO2 / glucose (- 0.43)

pyruvate / lactate (- 0.19)

O2 / H2O (+ 0.82)

small Eo'= -0.24 V

not much energy

large Eo'= -1.25 V

(lots of energy)

636. Electrons can be transferred to intermediate electron carriers in a series of redox reactions.

A(red) A(ox)

B(red)B(ox)

C(red) C(ox)

A(red) = primary electron donor (energy source)

C (ox) = terminal electron acceptorB = intermediate electron carrier

(glucose) (CO2)

(O2)(H2O)

TB

64II. NAD+ is an intermediate electron carrier.

A(red) A(ox)

NADHNAD+

C(red) C(ox)

"A" and "C" can be many numerous compoundsmany of which are catabolic intermediates.

TB

65A. NAD+ and NADP+

1. NAD+nicotinamide adenine dinucleotidecarries 2 electrons and a proton;usually involved in catabolic rxns

2. NADP+similar to NAD+ with an extra PO4

-;usually involved in biosynthesis

66

O

OH HO

adenine

P-P O

OHHO

N

H

NAD+

O

NH2

+

B. The NAD+/NADH couple (Eo' = –0.32V)

H

N

R

H

NADH + H+

O

NH22e– + 2H+

+ H+

TB(look at but don't memorize structures)

67

NAD+ is made by cells in limitedamounts.

The reduction of NAD+ to NADH depletes NAD+.

NAD+ must be regenerated by theoxidation of NADH to NAD+.

C. NAD+ must be recycled

TB

68III. Making ATP by substrate level phosphorylation (SLP)

TB

A. Substrate Level Phosphorylation:

Example

PEP + ADP pyruvate + ATP

*ATP synthesis driven by a high-energy compound, NOT the proton motive force (PMF).

69

COO-

CO~ P

CH2

OP ~ P OCH2 R

OP ~ P ~ P OCH2 R

PEP + ADP

COO-

C=O

CH3

pyruvate + ATP

Ex. of Substrate level phosphorylation

70B. High energy compounds

Compounds that can release large amounts of energy when they react.

Catabolism conserves energy in theform of high energy compounds which can be used to perform cellular work.

TB

71High energy compounds

phosphoenolpyruvate1,3-bisphosphoglycerateacetyl phosphatesuccinyl CoA, acetyl CoA ATPADP

Go' of hydrolysis(kJ / mol)

-52-52-45

-32-32

72

ATP + H2O ADP + Pi

Go' = - 32 kJ / mol

1. ATP is the most important high energy compound in cells.

TB

2. ADP

ADP + H2O AMP + PiGo' = – 32 kJ/mol

73

PEP + H2O pyruvate + Pi

Go' = - 52 kJ / mol

3. Phosphoenolpyruvate (PEP)

COO-CO~PO3

-

CH2

COO-C=O +CH3

PO43-

TB

744. 1,3-bisphosphoglycerate (BPG)

BPG + H2O 3-phosphoglycerate + Pi

Go' = – 52 kJ/mol

The hydrolysis of the above high energycompounds is coupled to energy-consuming cellular reactions to drive them forward.

TB

75SLP and glycolysis

During glycolysis, the hydrolysis of the high-energy compounds PEP or 1,3-bisphosphoglycerate (BPG)is "coupled" to ATP synthesis. This is an example of SLP.

PEP + ADP pyruvate + ATPTB

76 AMP and glucose-6-phosphate are examples of compounds with low energy bonds.

Go' of hydrolysis -14 kJ / mol

77IV. Glycolysis

• one pathway of making energy from glucose• glucose is partially oxidized to pyruvate• NAD+ is the intermediate electron carrier that accepts the electrons• ATP is made by substrate level phosphorylation (SLP) • glycolysis occurs in the cytoplasm

Glucose 2 pyruvate + 2 NADH + 2 ATP

A. Overall reaction of glycolysis:

78

glucose

2 pyruvate (C3)

ATPATP

2 ATP2 ATP

2 NADHredox step

ATP synthesis by SLP

energy input

hexose splitting

(C6)B. Important steps in glycolysis

79

glucose

glucose-6-phosphate

ATP*

ADP

(1)

(1)

hexokinase

energy input*TB

C. Individual steps of glycolysis

80glucose-6-phosphate(1)

fructose-6- phosphate(1)

TB

81fructose-6- phosphate

fructose-1,6- bisphosphate

ATP*

ADP

(1)

(1)

energy input*TB

82fructose-1,6- bisphosphate

glyceraldehyde3- phosphate

dihydroxyacetone phosphate

(1)

splitting reaction

TB

(C3 molecule) (C3 molecule)

(C6 molecule)

83 glyceraldehyde-3- phosphate

1,3-bisphosphoglycerate

(2) NAD+

(2) NADH + (2) H+

Pi

Redox reaction

(2)

(2)

TB

841,3 bisphosphoglycerate (BPG)

3-phosphoglycerate2 ATP

2 ADP

substrate level phosphorylation

(2)

(2)

TB

852-phosphoglycerate

phosphoenolpyruvate

(2)

(2)

TB

86phosphoenolpyruvate

2 ATP

2 ADP

pyruvate

substrate level phosphorylation

(2)

(2)

TB

87V. Reoxidation of NADH to NAD+

Important: in the cellNAD+ is limited, so NADH must be reoxidized

88 A. The reoxidation of electron carriers

1. Fermentation2. Aerobic respiration3. Anaerobic respiration

All organisms on earth that have beenstudied use one or more of 3 generalmethods to reoxidize electron carriers

Organisms that use all three methodsusually prefer aerobic respiration. TB

89

glucose 2 pyruvate

2 NADH + H+2 NAD+

Glycolysis

re-oxidation1. Fermentation reactions2. Aerobic respiration3. Anaerobic respiration TB

90 1. Fermentation

TB

catabolic process in which NADH is re-oxidized using a compound derived from the growth substrate

•

ATP synthesis is by substrate level phosphorylation (SLP) only.

•

Generally used when O2 is not available

•

91a. Fermentation producing ethanol

2NADH + H+2NAD+

2 CO2

2 Acetaldehyde

2 NADH + H+2 NAD+glucose 2 pyruvate

TB

2 Ethanol

2 CH3CH2OH

92b. Fermentation producing lactate

2NADH + H+

2NAD+

2 lactate

2 NADH + H+2 NAD+glucose 2 pyruvate

TB

COO-C=O CH3

COO-HC-OH

CH3

93Fermentation does not use electron transport chains for the reoxidation of electron carriers. Cytoplasmic enzymescatalyze the reoxidation of NADH.

Some of the products of fermentationare valuable.

Many different fermentations are known.

TB

94Study objectives1. Describe how cells derive energy from an energy source. What are the roles of the primary electron donor and the terminal electron acceptor? 2. Understand redox reactions and the terminology used to talk about them.3. Understand redox couples.4. Be able to use the table of standard reduction potentials to predict the direction of a redox reaction.5. Understand the relationship between Eo' and Go'. (Basically, a large Eo' corresponds to a large Go' ) You will NOT be asked to do a calculation.6. Understand how NAD functions in cells.7. Compare and contrast NAD and NADP.8. Understand high energy compounds. Know the examples of high energy and presented in class. Know that GTP is a high energy compound.9. Describe substrate level phosphorylation. Understand the difference between substrate level phosphorylation and oxidative phosphorylation.10. Understand the process of glycolysis. Know the overall reaction. Memorize all the steps. Know which steps involve energy input, hexose splitting, redox reactions, substrate level phosphorylation, ATP synthesis.

9511. What are the 3 general methods microbes use to reoxidize reduced electron carriers formed during catabolic processes? 12. Why must reduced electron carriers be reoxidixed?13. Understand fermentation and its purpose. Memorize the examples and reactions presented in class.

96Respiration and the TCA cycle:I. Aerobic respiration of glucoseII. TCA cycleIII. Electron carriersIV. Electron transport systemV. Oxidative phosphorylation

97

Growth substrates

Oxidized products

Reoxidation of NADH

Oxidized electron carriers

Reduced electron carriers

re-oxidation1. Fermentation2. Aerobic respiration3. Anaerobic respiration TB

98I. Aerobic respiration of glucose

one way to get more energy out of glucose than by fermentation

glucose + 6 O2 6 CO2 + 6 H2O

Respiration: ~36 to 38 ATP / glucose

Fermentation: ~2 ATP / glucose

99A. Respiration1. Oxidation of an organic energy source in the presence of an external terminal electron acceptor

glucose + 6 O2 6 CO2 + 6 H2O

organic energy source

"external" terminal electron acceptor

1001. terminal electron acceptor:

the last molecule to receive theelectrons during catabolism

2. "external" terminal electron acceptor:

terminal electron acceptor that is NOT derived from the energy source

101B. Aerobic respiration 1. Terminal electron acceptor is O2

Anaerobic respiration "external" terminal electron acceptor is NOT O2 eg. NO3

- (nitrate), Fe3+, SO4

-, CO2, CO3

2-, succinate or another organic molecule

O2

102B. Aerobic respiration (continued) 2. Reoxidation of reduced electron carriers with O2 occurs via intermediate electron carriers arranged as electron transport chains (respiratory chains).

3. ATP synthesis occurs mainly by oxidative phosphorylation

103 C. Aerobic respiration of glucose complete oxidation of glucose to CO2

higher energy yield than fermentation

Respiration: 36 to 38 ATP

Fermentation: 2 ATP

C6H12O6glucose

6 CO2 + 6 H2 O2 C3H6O3

6 O2

lactic acid

104D. Oxidative phosphorylation (electron transport phosphorylation)

ATP synthesis at the expense of a proton gradient (proton motive force) produced across a membrane by an electron transport system

H+

H+

ADP + Pi

ATPH+

H+

H+H+H+

H+

H+

H+

Cytoplasmic membrane in prokaryotesInner mitochondrial membrane in eukaryotes

105

NADH

glucose

pyruvate

protonmotive force

membraneoutsideacCoA

NADH

E. Overview: aerobic glucose respiration

TCA NADH

NADH

NADH

FADH2

GTP

2H + + 1/2O2

H2O

ADP + Pi

ATP

H+

NAD+ 2 H+

2 H+

H+

e-

106

Glucose respiration 1. Glycolysis 2. Conversion of pyruvate (3C) to acetyl CoA (2C) 3. Oxidation of acetyl CoA in TCA cycle 4. Reoxidation the intermediate electron acceptors 5. ATP synthesis by oxidative phosphorylation

II. Glucose respiration to CO2 and the TCA cycle glucose

pyruvate

TCA CO2

CO2

CO2 acCoA2

NADH

NADH

1

NAD+

4

3

107

2 CoA + 2 NAD+ 2 NADH

2 acetyl CoA (2C)2 CO2

2 pyruvate (3C)

glucose

glycolysis

A. Conversion of pyruvate to acetyl CoA• pyruvate oxidation produces NADH• decarboxylation makes CO2

108B. Oxidation of acetyl CoA in the TCA cycle (tricarboxylic acid cycle)

also called the citric acid cycle

• two carbons are oxidized to CO2 per acetyl CoA • 3 NADHs and 1 FADH2 are made per acetyl CoA• one GTP is made by substrate level phosphorylation

TCA

acetyl CoA

CO2

CO2

NADH

FADH2

GTP

NADH

NADH

109

acetyl CoA (2C)

(6C)

(5C)(4C)

CH2COO-

HOCH2COO-

CH2COO-

oxaloacetate citrate COO-

O=CH CH2

COO-

OCH3C~SCoA

1. Acetyl CoA (C2) condenses with oxaloacetate (C4) to form citrate (C6).

110

acetyl CoA (2C)

NAD+

NADHCO2

CO2

NADH

NAD+

pyruvate (3C) CO2

CoA + NAD+ NADH

FADH2

FAD

NAD+NADH

GTP GDP + Pi

CoA*SLP

(6C)

(5C)(4C)

2. Redox reactions, decarboxylations, SLP

citrateoxaloacetate

111

acetyl CoA (2C)

citrate

isocitrate

NADH

NAD+ CO2

succinyl CoA

succinate

fumarate

malate

oxaloacetate

NADHCO2

NAD+

GTP GDP + Pi

CoASLP

FADH2

FAD -ketoglutarate

NAD+NADH

(6C)

(5C)(4C)

3. The TCA Cycle

aconitate

1124. In the TCA cycle, there are 4 redox reactions (3 NADH and 1 FADH2) and two decarboxylations.

Four oxidative steps in the TCA cycle

isocitrate -ketoglutarate -ketoglutarate succinyl CoAsuccinate fumarate (FADH2) malate oxaloacetate2 decarboxylations (CO2 removed)

113d. There is one substrate level phosphorylation in the TCA cycle.

GTP is made and is easily converted to ATP

succinyl CoA succinate

GDP + Pi GTP CoA

114e. Sum of reactions for pyruvate oxidation and TCA cycle

pyruvate 3 CO2

4 NADH1 FADH2

1 GTP by SLP

must be reoxidized

TCA

pyruvate acCoA

NADH

NADH

NADH

NADH

FADH2

GTP

15 ATP equivalents per pyruvate

CO2

CO2

CO2

115III. Reoxidation of NADH and FADH2 with O2 occurs via intermediate electron carriers arranged as electron transport chains in the membrane.

TB

Q2H

NADH + H+

NAD+

2H e– e–

e–

e–

1/2 O2 + 2H+ H2O

116

1. NADH dehydrogenasesProtein complexes that acceptprotons and electrons from NADH.

A. Intermediate electron carriers

TB

2H

NADH + H+

NAD+

[2H] = 2 protons + 2 electrons

1172. Flavoproteins

Proteins with FAD or FMN (flavin adenine dinucleotide or flavin mononucleotide) as a prosthetic group.

Flavoproteins carry protons and electrons.

TB

2H

118FMN and FAD (isoalloxazine ring)

N

N N

NH

O

OH3C

H3C

R

Oxidized form

Flavin couples

FAD / FADH2

FMN / FMNH2

FMN and FAD are functionally equivalent, but have a different R-group TB

119Iron-sulfur center

S

S

FeFe

S-cys-E

S-cys-E

E-cys-S

E-cys-S

E-cys-S = the sulfur of a cys residue ofthe protein is bonded to the iron

2Fe2S

TB

120Iron-sulfur center

S

Fe

S

SS

Fe

FeFe

S-cys-E

E-cys-S

S-cys-E

4Fe4S

E-cys-S

TB

1214. QuinonesSmall molecules (nonprotein)

Quinones can diffuse within the membrane.

Quinones carry both protons andelectrons.

TB

122

O

Quinone

CH3O

CH3OO

R

Q

HOCH3O

CH3OOH

R

QH2

Oxidized Reduced

TB

123Diffusion of quinones within the cell membrane.

cytoplasmTB

1245. CytochromesProteins that contain the heme prosthetic group.

Cytochromes carry electrons only.

TBcytochrome

bc1

cytochrome c

cytochromeaa3

e–

e–e–

125Cytochrome

N NNN

Fe

protein

heme

Fe3+/Fe2+ The iron carries the electronsTB

126B. Electron Transport ChainsA series of electron carriers arranged within a membrane.Many different electron transport chains are known and they all function similarly.

TB

• electron transport chains can oxidize intermediate electron carriers like NADH and FADH2 and create proton gradients (PMF)

127

Q

cytoplasm

NADH dehydrogenase

flavoprotein

iron-sulfurprotein

quinone

cytochromebc1

cytochrome c

cytochromeaa3

1. Electron transport chain of E. coli.

TB

128

Q

cytoplasmNADH + H+

NAD

2H2H 2e– e–

e–

e–

1/2 O2 + 2H+H2O

a. In aerobic respiration, the electron transport chain is used to reoxidize NADH with O2.

2H = 2 protons and 2 electrons

TB

129

Q

cytoplasmNADH + H+

NAD

2H2H 2e– e–

e–

e–

2H+ 2H+

222H+

1/2 O2 + 2H+H2O

b. Oxidation via electron transport allows proton pumping. A proton gradient (PMF) is formed across the membrane.

TB

2H+

130

H+

H+H+

H+

H+H+H+

H+

H+

H+

+ + + + + + + + + +

+ + + + + + + + + +

- - - - - - - - - - - -

- - - - - - - - - - - -

++

++

++

----

-

- OH-

ENERGY

PMF

about -20 kJ/mol

In prokaryotes, H+ are pumped out of the cell.The outside becomes slightly acidic and positively charged relative to the inside.

2. Proton motive force (PMF) an energized state of the membrane created by a proton gradient

1313. Results of the electron transport chain

a. intermediate electron carriers (e.g. NADH and FADH2) are reoxidizedb. electrons are ultimately transferred to O2, making water c. proton motive force (PMF) is created, which can be used for ATP synthesis

132V. Oxidative phosphorylation (electron transport phosphorylation)

ATP synthesis driven by PMF

The F1F0 ATPase synthesizes ATP using the PMF.

TB

A. ChemiosmosisUse of an ion gradient (like PMF) to drive ATP synthesis

(Peter Mitchell, 1961):

133

H+

H+

H+

H+H+H+

H+

H+

Energy is released when the H+ gradient is dissipated. The energy can do work (make ATP, rotate flagella, take up nutrients).

ENERGY

ADP + Pi

ATP2 to 4H+

ATP synthase

B. ATP synthesis using PMF

PMF

-20 kJ/mol

134F1F0 ATPase

ADP + Pi ATP

H+H+

H+

H+H+ H+

cytoplasm

TB

F1: catalyzesATP

synthesis

Fo

135C. How many ATPs can be synthesized when NADH and FADH2 are reoxidized through an electron transport chain (respiratory chain)?

NADH ~ 3 ATPFADH2 ~ 2 ATP

136

glucose respiration (bacteria): 38 ATP per glucose

glucose fermentation: 2 ATP per glucose

D. Comparison of glucose fermentation and respiration in bacteria

glycolysis: 2 ATP (net) 2 ATP

2 acCoA (2) x TCA cycles:(2) x 3 NADH (2 x 3 x 3 ATP) 18 ATP(2) x 1 FADH2 (2 x 1 x 2 ATP) 4 ATP(2) x 1 GTP 2 ATP

Total: 38 ATP

2 NADH (2 x 3 ATP) 6 ATP 2 pyr 2 acCoA: 2 NADH 6 ATP

137Study objectives for lecture 91. Understand respiration. Contrast fermentation and respiration.2. Understand oxidative phosphorylation. Contrast oxidative phosphorylation and substrate level phosphorylation.3. Describe the overall process of glucose respiration and the five steps presented in class.4. Memorize and understand the reaction of pyruvate conversion to acetyl CoA.5. Understand the TCA cycle. Know that the TCA cycles begins with the reaction of acetyl CoA and oxaloacetate to make citrate. How does the TCA cycle help cells produce energy?6. Memorize ALL the steps in the TCA cycle. Know which steps involve redox reactions, substrate level phosphorylation, decarboxylation. You do not need to memorize the structures.7. In respiration, glucose is oxidized completely to CO2. How is this done? Where is CO2 released? What happens to the electrons? What is the role of oxygen in respiration? How is energy conserved as ATP? How do cells derive energy from glucose in respiration? In fermentation?

1388. What are electron transport chains? What is their role in metabolism?9. Compare and contrast the electron carriers used in electron transport chains. Understand the particular features of each electron carrier. You do not need to memorize the structures.10. Which electron carrier is nonprotein?11. Can cytochromes carry protons?12. Describe how electron transport chains are used to synthesize ATP. Understand how electron transport in the membrane generates proton motive force. Recall that proton motive force can be used to produce ATP.

Continued on next slide

1391. Describe oxidative phosphorylation. Define chemiosmosis.2. Know the general structure of the F1/F0 ATPase. What is its function?3. How is the reoxidation of intermediate electron carriers related to ATP synthesis?4. Which method allows production of more ATP: aerobic respiration or fermentation?5. Starting with glucose, describe how ATP is made from glucose in fermentation and respiration. 6. Describe how glycolysis, the TCA cycle, electron transport chains, and ATP synthesis are connected in respiration.