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Lecture 6B – outlineMitochondrial function (e.g. hepatocytes)

1) citric acid cycle as an energy source a) pyruvate or -ketoglutarate dehydrogenase b) lipoic acid therapy

2) the respiratory chain as an energy source

3) oxidative phosphorylation and uncouplers

4) membrane transporters and shuttles a) cytosolic NADH oxidation b) acetyl CoA (NADPH export) c) transport systems in the mitochondria d) gluconeogenesis and glucose transport

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Compartmentalization of the major pathways of metabolism

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Stryer

An overview of the citric acid cycle

1. CITRIC ACID CYCLE AS AN ENERGY SOURCE1. CITRIC ACID CYCLE AS AN ENERGY SOURCE

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Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O 2 CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA

120uM plasma citrate120uM plasma citratecomplexes Fecomplexes Fe

toxic!toxic!

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The citric acid cycle is a source of biosynthetic precursors

Stryer Fig. 20-17.Biosynthetic roles of thecitric acid cycle. Intermediates drawn offfor biosyntheses are replenished by the formation of oxaloacetatefrom pyruvate.

Pyruvate

Acetyl CoA

Citrate

keto-glutarate

Amino acids

SuccinylCoA

Porphyrins

Oxaloacetate

Aminoacids

ADP, Pi

ATP, CO2

Phosphoenolpyruvate

Glucose

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Control of the citric acid cycle

Stryer Fig. 20-22.Control of the citric acid cycle andthe oxidative decarboxylation ofpyruvate: * indicatessteps that require anelectron acceptor (NAD+ or FAD) that is regenerated by therespiratory chain.

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2. THE MITOCHONRIAL RESPIRATORY CHAIN AS AN

ENERGY SOURCE

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The mitochondrial respiratory chain

Sequence of electroncarriers in therespiratory chain

Chemiosmotic theory of oxidative phosphorylation

Diagram of a mitochondrionNADH

NADH-Q reductase

Q

Cytochrome reductase

cyt c

Cytochrome oxidase

O2

complex I

complex III

complex IV

FADH2in flavoproteinssuccinate:Q reductase (complex II)

FMNH2

2Fe-2S

4Fe-4S

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Origin of mitochondria: the endosymbiont hypothesis

The endosymbiont hypothesis suggests that mitochondria have evolvedfrom anaerobic bacteria which were phagocytosed by eukaryote cells at the time oxygen appeared on earth,

Similarities between mitochondria and bacteria include the presence of:• cardiolipin •transporters• ribosomes• circular RNA and DNA

Therefore mitochondria protein synthesis should be inhibited by:• TETRACYCLINE• CHLORAMPHENICOL.E.g. The extensive use of these drugs can inhibit 1. Bone marrow mitochondrial protein synthesis leading to a decline in the production of white or red cells. 2. Intestinal epithelial cells causing them to cease dividing.

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NADH coenzyme Q reductase: complex I

The reduction of ubiquinone to ubiquinol proceeds through a semiquinoneanion intermediate.

NADH

NAD+

FMN

FMNH2

Reduced Fe-S

Oxidised Fe-S

Q

QH2

NADH-Q reductase

C

CC

C

CC

O

O

H3CO

H3CO

CH3

(CH2CH

C

CH3

CH2)10 HC

CC

C

CC

O

OH

H3CO

H3CO

CH3

R

C

CC

C

CC

OH

OH

H3CO

H3CO

CH3

R

e- + H+ e- + H+

Coenzyme Q10(UBIQUINONE

Semiquinone Intermediate (Q )

Reduced Coenzyme Q10 ( UBIQUINOL)

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Q:Cytochrome c reductase (Complex III)

Q

QH

cyt b (+2)

cyt b (+3)

QH

QH2

Fe-S(+2)

Fe-S(+3)

cyt c1(+3)

cyt c1(+2)

cyt c(Fe+2)

cyt c(Fe+3)

cytochrome c reductase

Stryer Fig. 21-11Model of a portion ofQ: cytochrome c reductase

Stryer p. 537

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Cytochrome oxidase (Complex IV)

Lodish Fig. 17-30

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Electron transport can be blocked by specific inhibitorpoisons

NADH

NADH-QReductase

QH2

Cytochrome b

Cytochrome c1

Cytochrome c

Cytochrome Oxidase

O2

Blocked by rotenone and amytal

Blocked by antimycin

Blocked by

CN-, N3-, and CO

Sites of action of someinhibitors of electron transport

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Cytochrome C - catalytic site

The heme in cytochromes c and c1 is covalently attached to 2 cysteine side chainsby thioether linkages

The iron atom of the heme group incytochrome c is bonded to a methioninesulfur atom and a histidine nitrogen atom

R CH

CH2

HSCH2 R'R C

H

CH3

CH2

R'S+

Vinyl groupof the heme

Cysteine residueof the protein Thioether linkage

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Cytochrome C - soluble NOT membrane bound

1. 26/104 amino acids residues have been invariant for > 1.5 x 109 years.

2. Met 80 and His 18 - coordinate Fe.

3. 11 residues from number 70 - 80 lining a hydrophobic crevice have remained virtually unchanged throughout all cytochrome c regardless of species or even kingdom.

4. A number of invariant arginine and lysine clusters can be found on the surface of the molecule.

Cytochrome c has a dual function in the cell. Electron transport for ATP production AND the major cause of most programmed cell death (apoptosis) is initiated by the release of cytochrome c into the cytosol!

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3. OXIDATIVE PHOSPHORYLATION AND

UNCOUPLERS

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Oxidative phosphorylation

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4. Mitochondrial MEMBRANE TRANSPORTERS

A) Cytosolic NADH oxidation

B) Acetyl CoA (NADPH export)

C) Transport systems in the mitochondria

D) Gluconeogenesis and glucose transport

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a) Cytosolic NADH oxidation: membrane transporters glycerol phosphate shuttle (Bucher shuttle)

Glucose

Glyceraldehyde - 3 - phosphate

1,3 - Bisphosphoglycerate

NAD+

NADH, H+Dihydroxyacetone phosphate

Glycerol - 3 - phosphate

Glycerol - 3 - phosphate

Dihydroxyacetone phosphate

Cytosol Outer membrane Inner Membrane

FAD

FADH2

RE

SP

IRA

TO

RY

C

HA

IN

Q

QH2

b

c1

c

a, a3

Glycolysis

1

2

1. Glycerol phosphate dehydrogenase2. Glycerophosphate oxidase

H2C

CHH2C

HO

OH

OP

H2C

CH2CO

OH

OP

See figure 21-30 Stryer 4th Ed.

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b) Acetyl CoA/NADPH export to cytosol for fatty acid synthesis/drug metabolismGlucose

Pyruvate

Acetyl CoA

Oxaloacetate

Pyruvate

Citrate

CitrateSynthase

Citrate

Oxaloacetate

Acetyl CoA fatty acid synthesisor drug metabolism(N-acetylation)

+ATP+CoA

ATP citrate lyaseat high concentration

Malate

Pyruvate

NADH

NAD+

NADP+

NADPH

CO2

malate dehydrogenase

malic enzyme

fatty acid synthesisor P450 catalyzed drug metabolism

Pentose Phosphate Pathway NADPH

Therefore malic enzyme supplies NADPHCitrate Lyase supplies acetyl CoA.

ATP

ADP CO2

Mitochondrial Matrix

Cytosol

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Isocitrate as an NADPH shuttle for drug metabolismGlucose

Pyruvate Acetyl CoA

Citrate

Isocitrate

-ketoglutarate

NAD+

NADH

isocitratedehydrogenase

CO2

Succinyl CoA

NAD+NADH

CO2

Succinate

Fumarate

Malate

Oxaloacetate

NAD+

NADH

Isocitrate

-ketoglutarate

NADP+

NADPH

P450 catalyzedDRUG METABOLISM

isocitratedehydrogenase

CITRICACIDCYCLE

MITOCHONDRIAL MATRIXCYTOSOL

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d) Gluconeogenesis and glucose export by the liver ! 3 irreversible steps

Major antidiabetic drug METFORMINInhibits gluconeogenesisDecr Hepatic Glucose Synth.

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Glucagon 51aa & Insulin 29aa

• Pancreas synthesises both peptide hormones • Insulin required for cells (e.g.liver,muscle,fat) to take up

glucose and synthesise glycogen.• Glucagon hepatocyte receptors signals glycogenolysis

(glycogen breakdown to glucose then increases gluconeogenesis pyruvate -- glucose)

• Drugs. Dipeptidyl peptidase-4 inhibitor (Januvia, new anti type 2 diabetes) increases incretin , a GI hormonal peptide inhibitor of glucagon which lowers plasma glucose.

• Metformin, antidiabetic drug inhibits gluconeogenesis but also can inhibit mitoch.complex I causing lactic acidosis.