1. Outline the 4 w’s for the bridging reaction (pyruvate dehydrogenase multienzyme complex), and the Krebs Cycle. What feature of the bridging reaction allows amino acids and sugars to be made into fat, but does not allow fat to be made into amino acids or sugars. Explain.

2. Describe the structure of the mitochondria. 3. Describe the function of each of the following enzymes: the pyruvate

dehydrogenase multienzyme complex, NADH dehydrogenase, CoQ-cyctochrome c oxidoreductase, cytochrome c, and cytochrome oxidase.

4. Describe the 4 w’s for Oxidative Phosphorylation. Explain the difference between oxidative phosphorylation and electron transport. Explain how ATP synthase works and why this depends on the proton gradient. Explain how the connection between electron transport and ATP synthase is broken by the uncoupler protein (UCP), and by 2,4 dinitrophenol.

5. Describe the role of the glycerol phosphate shuttle in muscle Oxidative Phosphorylation. What is the difference between the mitochondrial and cytoplasm forms of glycerol phosphate dehydrogenase and how does this impact energy production.

6. Outline the 4 w’s for all the pathways of metabolic main street.

7. Outline the 4Ws for the pentose phosphate pathway. Describe the importance of the pentose phosphate pathway to fatty acid synthesis and neuron health. Explain the role of NADP+/NADPH to Fatty acid synthesis and in the Wernicke Korsakoff syndrome.

8. Indicate the reaction catalyzed by, the pathway involved, and describe the metabolic role of the following enzymes: Glucose-6-P dehydrogenase, transketolase/tranaldolase, superoxide dismutase, and glutathione reductase.

9. Molecular structures to draw/recognize: ribose-5-phosphate, ROS (superoxide, hydroxide radicals, and hydrogen peroxide).

Glucose

AcetylCoA

Pyruvate

NADH/FADH2

Citric Acid Cycle

C6

C4

C5

oxaloacetate

ATP

Glycolysis

Bridging Rx.

Oxidative Phosphorylation

ADP O2

NAD+/FAD

Metabolic Mainstreet

GluconeogenesisFat↑↓

fatty Acids

amino acids↑↓

Protein

no return

ketone bodies

Oxidative Phosphorylation(Respiratory Chain)

NADH, H+ + 1/2 O2 + 3ADP, Pi

NAD+ + H2O + 3ATP

Electron transport

FADH2 2ADP

FAD 2ATP

Where – mitochondria Why – make ATPWhen – supply (ADP) and demand (ATP)

ATP syn

Oxidative PhosphorylationElecton Transport creates a Proton Gradient

ATP synthase utilizes the gradient to fuel ATP production

IIII

IV

II

H+

H+H+

ADP + Pi → ATP

DG˚′ = -nFDE˚′The Oxidative phosphorylation pathway exchanges the free energy provided by redox reactions into a proton gradient. The electric potential of the gradient drives the spontaneous production of ATP from ADP,Pi.

I

III

IV

Electron Transport Complexes

CoQ-Cytochrome C oxidoreductase 250kCoQH2 + 2 CytC(Fe3+) → 2H+ + 2 CytC(Fe2+) + CoQ

Cytochrome C Oxidase 160k 6 CytC(Fe2+) + O2 → 2 H2O + 6 CytC(Fe3+)

II Succinate-CoQ reductase (FADH2) 140kFADH2 + CoQ → FAD + CoQH2

H+

H+

H+

NADH dehydrogenase

IVIQ

III

c

H+ H+ H+

H+H+ H+

NADH, H+ + CoQ → NAD+ + CoQH2

CoQH2 + 2 CytC(Fe3+) → 2H+ + 2 CytC(Fe2+) + CoQ

4 CytC(Fe2+) + O2 → 2 H2O + 4 CytC(Fe3+)

Coenzyme Q is … a) polar b) nonpolar c) ionic

I NADH dehydrogenase: NADH, H+ + CoQ → NAD+ + CoQH2

II Succinate dehydrogenase: FADH2 + CoQ → FAD + CoQH2

III CoQ:cytochrome C oxidoreductase : CoQH2 + 2 CytC(Fe3+) → 2H+ + 2 CytC(Fe2+) + CoQ

IV Cytochrome Oxidase 4 CytC(Fe2+) + O2 → 2 H2O + 4CytC(Fe3+)

CoQ and cytochrome C (Fe3+/Fe2+) are ‘mobile’ electron carriers in Oxidative Phosphorylation

H+

III

III

ATP syn

I’

H+DpH = -1.4

Oxidative PhosphorylationThe pH of the inner membrane space will be ….

a) > than b) < than c) = to The pH of the mitochondrial matrix?

Transport of ADP and Pi into the Matrix

Proton Gradient = Energy Graient

H+

ADP + Pi

ATPsynthase Fo

F1

ATP

H+

The proton gradient turns the transmembrane portion of ATP synthase (F0) creating a pinwheel effect that leads to ATP generation.

The turning of the transmembrane portion of ATP synthase leads to the release of H+ into the matrix and ATP production. At any time a b subunit can have ADP/Pi, ATP, or nothing bound (1 of each).

a

a

a

bb

b

ADP,Pi

ATP

g

F1-ATP Synthase

ATP

Asp- + H+out

Asp Conformational DAsp Asp- + H+

in

A key asparticacid residue

facilitates the H+ driven‘spinner’

ADP,Pi

b

a

a

a

bb

g

F1-ATP Synthase

ADP,PiATP

ATP

ATP

a

a

a

bb

b

ADP,PiATP

g

F1-ATP Synthase

ATP

Transport of ADP and Pi into the Matrix

Summary of the Electron Flow in the Respiratory Chain

Glucose + 2ADP + 2NAD+

AcetylCoA

2Pyruvate + + 2ATP + 2NADH in cytosol

NADH/FADH2

KrebsCycle

C6

C4

C5

C4

ATP

Glycolysis

Bridging Rx.

OP

ADP O2

NAD+/FAD

Metabolic Mainstreet

How?Glycerol Phosphate or

Malate/Aspartate Shuttles

Muscle ― 2 ATP per Glycolysis NADH

Liver ― 3 ATP per Glycolysis NADH

The Glycerol Phosphate shuttle allows NADH produced in the cytosol toProduce aerobic ATP in the matrix without actually entering the mitochondria.

DHAP Glycerol-3-P Dehydrogenase (cytosol)

Glycerol-3-P

E-FADH2 E-FAD

Glycerol-3-P Dehydrogenase (mt)

NADH,H+ NAD+

QQH2II

Glycerol – Phosphate Shuttle

Pathway Direct OP

Glycolysis

Bridging Rx

Krebs Cycle

2ATP

none

2GTP

2NADH out 2FADH2 in = 4

2NADH = 6

6NADH = 182FADH2 = 4

Gross muscle ATP output – Additional loss of ATP due to ‘overhead’

Glucose

AcetylCoA

Pyruvate

NADH/FADH2

KrebsCycle

C6

C4

C5

ATP

Glycolysis

Bridging Rx.

OP

ADP O2

NAD+/FAD

Metabolic Mainstreet

oxaloacetate

2 ATP ― anaerobic

34 ATP ― aerobic Bridging Rx + Krebs + OP

OP UncouplersWhat would happen if H+ entered mitochondria without going through ATP synthase?

a) ATP would be produced b) heat would be produced c) both of above d) neither of above

1. 2,4 – dinitrophenol a weak nonnpolar acid

2. UCP (uncoupler Protein)

a passive H+ transport

Transport H+ across membrane without generating ATP

ADP + Pi ATP

ATPsynthase Fo

F1

NO2

OHNO2

O-

NO2H+

NO2

pH ~ 5.5

pH ~ 7.0

NO2

O-

NO2

+ H+

H+

ATPsynthase Fo

F1

UCP

ADP + Pi > ATP

Hibernating animals use UCP to stay room in winter in lieu of ATP production for muscle activity.

Free RadicalsMolecules that contain unpaired electrons

Superoxide ion •O2

-Nitric oxide NO•

Hydroxide radical OH•

Reactive Oxygen Species (ROS) – Molecules that are free radicals or readily converted into free radicals. They are strong oxidizing agents.Metabolically generated ROS/RNOS include above and hydrogen peroxide (H2O2).

H2O2 + Fe2+ Fe3+ + OH- + OH•

O2- + H2O2 + H+ O2 + H2O + OH•

ROS generation by Metabolism

Oxidative Phosphorylation & cytochrome oxidase O2 + 4H+ + 4e- → 2H2O

About 3-5% of the O2 metabolized by OP gets released prematurely as an ROS.

1) O2 + e- O2-

2) O2- + e- + 2H+ H2O2

3) H2O2 + H+ + e- H2O + OH•4) OH• + H+ + e- H2O

ROS generation by Metabolism

Toxin + cytP450(Fe2+) + O2 reduced toxin + cytP450(Fe3+) + H2O

Toxins include alcohol, pharmaceutical and recreational drugs, non-nutritive food molecules …. , that induce cytochrome P450 production in Liver… etc.

Monoamine oxidase in neurons –Dopamine is neurotransmitter: ↓Parkinson’s ↑Schizophrenia dopamine degradation H2O2

Neurons are particularly sensitive to environmental insult.

ROS – harmful Reactions

DNA + ROS modified bases or single strand breaks. These can lead to ↑mutations or apoptosis.

Protein + ROS fragmented or cross-linked protein. These can lead to ↓[Pro] or plaque buildup.

Polyunsaturated Lipids + ROS damaged cell membrane or athersclerosis . These can lead to cell death or heart disease ….

Defenses against ROS

Superoxide Dismutase (SOD) 2H+ + O2

- H2O2

Glutathione Peroxidase Glutathione (GSH) is the tripeptide gGlu-Cys-Gly

2GSH + H2O2 2 H2O + GS-SGDegrades lipid peroxides as well as H2O2 to minimize lipid damage

Glutathione Reductase

GS-SG + 2NADPH 2 GSH + 2NADP+

Superoxide ion •O2

-Nitric oxide NO•

Hydroxide radical OH•

Hydrogen peroxide H2O2

AntioxidantsVitamin E and Vitamin CFlavinoids: green tea, red wine, chocolate ….Carotinoids: fruits and veggies …..

What do they do? 1) Directly scavenge free radicals 2) Inhibit enzymes that can generate ROS 3) Combination of above.

Caution: Antioxidants can produce FR themselves and excesses may have pro-oxidant rather than antioxidant activity. Some clinical studies show excessive supplements cause more harm than good.

Resveratrol

Found in skin of red grapes & red wine.May activate sirtuin which is implicated in the epigenetic control of gene expression.May up-regulate SOD expression.

Catechin

Found in cocoa and white/green tea.

Epigallocatechin 3-O-gallate EGCG

AntioxidantsThese molecules taken as are considered beneficial components of fruits and vegetables etc.They may function to supplement the bodies normal defense against ROS. This could happen by serving as targets for ROS oxidation but also may involve enzyme expression or inhibition.Studies using pure forms of these compounds show mixed results.

Pentose Phosphate Pathway

Glucose-6-P + 2 NADP+ + H2O

Ribose-5-P + CO2 + 2 NADPH, H+

Why? Production of NADPH as reducing agent fatty acid synthesis & glutathione recycling

Where? Liver and adipose – neurons (brain)

When? NADP+ stimulates – NADPH inhibits mass action signals need for production

particularly important

3 Enzymes- includes … Glucose-6-P dehydrogenase

Transketolase (TPP cofactor) & Transaldolase

TK 2C5 ↔ C7 + C3

Net 3C5 Û 2C6 + C3 or …. 6C5 Û 5C6

TA C7 + C3 ↔ C6 + C4

TK C5 + C4 ↔ C6 + C3

What? – Reversible exchange of ribose/glucose

Why? – Retain proper balance of ribose/glucose

Where? – Liver, adipose, neurons (brain)

When? – As needed – regulated by mass action (equilibrium) DG = DG°´ + RT ln Q

Glycolysis ---Gluconeogenesis

Glucose ↓↑Glucose-6-P ↓↑C3 Intermediate ↓↑Pyruvate

NADPH + R-5-P (C5) + CO2

PPP

Nucleic Acids

Fat Synthesis & Glutathione Reductase

NADP+

TK

TA

Pentose Phosphate Pathway

Wernicke Korsakoff SyndromeLesions in Wernicke’s area of brain - left posterior of temporal lobe – probably due to neuron death.Causes speech comprehension problems, amnesia, peripheral neuritis.

Genetics (‘nature’)TK binding to TPP 10x weaker more common in “Europeans”

Environment (‘nurture’)Exacerbated by thiamine deficiency – common in alcoholics.

TK mutationv

[TPP] Normal [TPP]Thiamine deficiency