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Cellular RespirationCellular RespirationIII. OxidativeIII. OxidativePhosphorylationPhosphorylation

Lecture 15Lecture 15

October 5, 2005 Chapter 92

Lecture OutlineLecture Outline

1. What do we do with NADH + H+ and FADH2reducing equivalents?

2. Electron Transport – the oxidation phase of Oxidative Phosphorylation

3. ATP synthesis – the Phosphorylation Phase of Oxidative Phosphorylation

5. Why believe the Chemiosmotic hypothesis? 6. Indirect Active Transport into the mitochondrion7. Comparison of yield from Fatty Acid Oxidation

and Monosaccharide Oxidation8. Life without oxygen - Fermentation

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Mitochondrial FunctionsMitochondrial Functions

Oxidize compounds to COOxidize compounds to CO22 + H+ H22OO

Generate >90% of Typical Cell’s ATPGenerate >90% of Typical Cell’s ATP

Fatty acid OxidationOxidation of PyruvateTCA Kreb’s Cycle

OxidativeOxidative Phosphorylation“electron transport”ATP synthesis

ProduceProducereduced carriersreduced carriersNADH & FADHNADH & FADH22

OxidizeOxidize reduced carriersreduced carriersto produce ATP or equivto produce ATP or equiv

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2 H 1/2 O2

(from food via NADH)

2 H+ + 2 e–

2 H+

2 e–

H2O

1/2 O2

Controlled release of energy for

synthesis ofATP

ATP

ATP

ATP

Electron transport chain

Free

ene

rgy,

G

(b) Cellular respiration

+

Figure 9.5 B

Electron transport– oxidizes NADH

and FADH2

back to NAD+

and FAD

ElectronsElectronsGiven to Given to

Electron Transport Electron Transport chainchain

ElectronsElectronsEventually Given to Eventually Given to

Oxygen Oxygen To form WaterTo form Water

EnergyEnergyReleasedReleasedUsed To Used To MakeMakeATPATP

How?How?

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Need ONeed O22 asasfinal acceptorfinal acceptorof reducingof reducingequivalentsequivalents

2H2 + O2 = H2O + BOOM!

Electron transportElectron transportOxidative partOxidative part 3 discrete3 discrete

Proton pumpsProton pumpsPowered byPowered byPassage ofPassage ofelectronselectrons

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Electron Transport ChainElectron Transport Chain

OxidizeOxidize NADH + HNADH + H++ to NAD+

2e2e-- and 2H2H++ given to a multiprotein complex (complex I)

energy of oxidation pumps Henergy of oxidation pumps H+ + to to intermembraneintermembrane spacespace2H+ orphaned as electrons transferred2e- get passed on

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2 Fe++

(2e-)

NADH + H+ NAD+

2 Fe+++

2e-

2H+

2H+

CoQ(2e-) (2e-)

2H+CoQ

H H

(reduced)

(oxidized)

pH 6

pH 8

Complex Iclose up

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Electron Transport ChainElectron Transport Chain

Oxidize NADH + H+ to NAD+

2e2e-- and 2H2H++ given to multiprotein complex (complex I)2e- get passed on2H+ orphaned as electrons transferred

need to pick up 2H2H++ (where from?)to form water

energy of oxidation pumps Henergy of oxidation pumps H++ to to intermembraneintermembrane spacespace

2e2e-- and 2H2H++ (where from?) get passed to a third complex (complex IVcomplex IV)orphaning of 2H+ repeated as 2e- passed onfinally to oxygento oxygen

energy of oxidation pumps Henergy of oxidation pumps H+ + to to intermembraneintermembrane spacespace

2e2e-- and 2H2H++ (where from?) get passed to another complex (complex III)complex III)orphaning of 2H+ repeated as 2e- passed on

energy of oxidation pumps Henergy of oxidation pumps H+ + to to intermembraneintermembrane spacespace

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Net Result of Electron Transport:an electrochemicalelectrochemical HH++ gradientgradient

H+

OH-

2

2

pH 6

pH 8

10H2O

O2

NADH

FADHFADH22

FMN

Fe•S Fe•S

Fe•S

O

FAD

Cyt b

Cyt c1

Cyt c

Cyt a

Cyt a3

2 H + + 1⁄2

I

II

III

IV

Multiproteincomplexes

0

10

20

30

40

50

Free

ene

rgy

(G) r

elat

ive

to O

2 (k

cl/m

ol)

Figure 9.13

What happened to What happened to complex II?complex II?

ThreeThree 2H2H++ pumpingpumpingSteps from NADH + HSteps from NADH + H++

oxidationoxidation

Only Only TWOTWO 2H2H++ pumpingpumpingSteps from FADHSteps from FADH22

oxidationoxidation

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Electron transport – oxidizes NADH and FADH2back to NAD+ and FAD

Electron transport – electrons passed in series from one carrier to anothereventually give e- and H+

to oxygen to form water

BUT PUMP H+ in the process acrossthe inner mitochondrial membrane

into intermembrane space

FORMS BIG H+ gradient 12

What good is the HWhat good is the H++ Electrochemical Gradient ?Electrochemical Gradient ?

H2O

HH++

OHOH--

pH 8pH 8

pH 6pH 6

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INTERMEMBRANE SPACE

H+

H+

H+

H+

H+

H+ H+

H+

P i

+

ADP

ATP

A rotor within the membrane spins clockwise whenH+ flows pastit down the H+

gradient.

A stator anchoredin the membraneholds the knobstationary.

A rod (for “stalk”)extending into the knob alsospins, activatingcatalytic sites inthe knob.

Three catalytic sites in the stationary knobjoin inorganic Phosphate to ADPto make ATP.

MITOCHONDRIAL MATRIXFigure 9.14

MitochondrialMitochondrialHH++ ATPaseATPase

ATP synthase

H+ movement drives conformational change

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pH 6pH 6

pH 8pH 8

pH 6pH 6

pH 8pH 8

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HH++

movtmovt

Mitochondrial HMitochondrial H++ ATPaseATPase

ADP ADP + Pi+ Pi

HH++ HH++

OpenOpenATPATP

ATPATP

ADP ADP + Pi+ Pi

OpenOpen

ATPATPexpelledexpelled 16

ElectronElectronTransport Transport

ChainChainis ais a

Proton Proton PumpPump

ReducingReducingequivalentsequivalentspower the pumppower the pump

Gradient powersGradient powersATP SynthesisATP Synthesis

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Uncoupling Oxidation and Phosphorylation

•Must have intact membranes to make ATP

••Agents that allow HAgents that allow H++ passage (dissipate gradient)passage (dissipate gradient)diminish ATP synthesis diminish ATP synthesis –– dinitrophenoldinitrophenol (DNP)(DNP)

•Agents such as cyanide poison electron transportchain

so no H+ gradient produced- but if supply Hif supply H++ gradient can still make ATPgradient can still make ATP

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• Chemiosmosis and the electron transport chain

Oxidativephosphorylation.electron transportand chemiosmosis

Glycolysis

ATP ATP ATP

InnerMitochondrialmembrane

H+

H+H+

H+

H+

ATPP i

Protein complexof electron carners

Cyt c

I

II

III

IV

(Carrying electronsfrom, food)

NADH+

FADH2

NAD+

FAD+ 2 H+ + 1/2 O2

H2O

ADP +

Electron transport chainElectron transport and pumping of protons (H+),

which create an H+ gradient across the membrane

ChemiosmosisATP synthesis powered by the flowOf H+ back across the membrane

ATPsynthase

Q

Oxidative phosphorylation

Intermembranespace

Innermitochondrialmembrane

Mitochondrialmatrix

Figure 9.15

Roughly3 ATP3 ATP for each NADH + HNADH + H++

2 ATP2 ATP for each FADHFADH22

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Can Use H+ gradient to powerother useful work

How about ATPATP------ out of mitochondrion?

How about ADPADP---- and PPii-- into mitochondrion?

How you get pyruvatepyruvate-- (an acid) across the mitochondrion membrane? (not magic!)

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∆∆pHpH drivesdrivesPPii importimport

∆∆pHpH drivesdrivespyruvatepyruvateimport import

∆Ψ ∆Ψ (charge)(charge)drivesdrives

ATP/ADP ATP/ADP antiportantiport

pH 6pH 8

H+

H+

H+

H+H+

H+

H+

H+

H+

-OH

-OH

-OH

-OH

-OH

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Electrochemical Gradient

Can also power

Indirect ACTIVE TRANSPORTIndirect ACTIVE TRANSPORT

of IONS into MATRIX

instead of making ATP

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Electron shuttlesspan membraneCYTOSOL 2 NADH

2 FADH2

2 NADH 6 NADH 2 FADH22 NADH

Glycolysis

Glucose2

Pyruvate

2AcetylCoA

Citricacidcycle

Oxidativephosphorylation:electron transport

andchemiosmosis

MITOCHONDRION

by substrate-levelphosphorylation

by substrate-levelphosphorylation

by oxidative phosphorylation, dependingon which shuttle transports electronsfrom NADH in cytosol

Maximum per glucose:About

36 or 38 ATP

+ 2 ATP + 2 ATP + about 32 or 34 ATP

or

Figure 9.16

So how many ATP from Glucose oxidation?

~36 ATP per 6 carbon sugar~36 ATP per 6 carbon sugar7.3 kcal/mole x 36 = 263 kcal/mole ∆G = -686 kcal/mole

~39% efficient~39% efficient

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Compare to Fatty Acid OxidationCompare to Fatty Acid Oxidation18 carbon fatty acid (9 18 carbon fatty acid (9 two carbontwo carbon units)units)

Each Each acetyl acetyl CoACoA gives in TCA gives in TCA 3 NADH + H3 NADH + H++

1 FADH1 FADH221 GTP1 GTP

9 ATP9 ATP2 ATP2 ATP1 ATP1 ATP

6 ATP per glucose carbonglucose carbonWhy?Why?

Each Each 2 carbon2 carbon unit 1 2 NADH + Hunit 1 2 NADH + H++

1 2 FADH1 2 FADH22

3 6 ATP3 6 ATP2 4 ATP2 4 ATP

XXXX

XXXX

Each 2 carbonsEach 2 carbons 17 22 ATP17 22 ATPXXOxidation of 18 carbon fatty acid yields 198 ATPOxidation of 18 carbon fatty acid yields 198 ATPXX153153

8.5 11 ATP per FA carbonFA carbonXX24

In Metabolism:

Highly reduced fully oxidized

CH3-CH2-CH2-(CH2)x-CH2-C-O + O2 H2O + CO2 + energy Fatty acid O (captured)

Partially reduced fully oxidized

+ O2 H2O + CO2+ energycarbohydrate (captured)

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CH4

R-CH2 -CH 3

R-CH=CH2

R-CH2-CH2 -OH

R-CH2-C=OH

R-CH2-C=OOH

O=C=O

Organic Oxidation SeriesOrganic Oxidation Series

Fatty AcidsFatty AcidsStart HereStart Here

MonosaccharidesMonosaccharidesStart HereStart Here

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• Chemiosmosis and the electron transport chain

Oxidativephosphorylation.electron transportand chemiosmosis

Glycolysis

ATP ATP ATP

InnerMitochondrialmembrane

H+

H+H+

H+

H+

ATPP i

Protein complexof electron carners

Cyt c

I

II

III

IV

(Carrying electronsfrom, food)

NADH+

FADH2

NAD+

FAD+ 2 H+ + 1/2 O2

H2O

ADP +

Electron transport chainElectron transport and pumping of protons (H+),

which create an H+ gradient across the membrane

ChemiosmosisATP synthesis powered by the flowOf H+ back across the membrane

ATPsynthase

Q

Oxidative phosphorylation

Intermembranespace

Innermitochondrialmembrane

Mitochondrialmatrix

Figure 9.15

Life Without OxygenLife Without Oxygen

XXX

X

XNADH+ H+ X

FADH2 X

27Figure 9.6

Electronscarried

via NADH

GlycolsisGlucose Pyruvate

ATP

Substrate-levelphosphorylation

Electrons carried via NADH and

FADH2

Citric acid cycle

Oxidativephosphorylation:

electron transport andchemiosmosis

ATPATP

Substrate-levelphosphorylation

Oxidativephosphorylation

MitochondrionCytosol

Life Without Oxygen

XX

XX

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The Regeneration Energy CarriersThe Regeneration Energy Carriers

2e2e--

2H2H++

Captured in catabolismCaptured in catabolism

NADH + H+

Energy for cellular work(endergonic, energy-consuming processes)

Energy from catabolism(exergonic, energy yieldingprocesses)

NAD+

2e2e--

2H2H++

Cashed inCashed in

Energy carriers (ATP, NAD+, FAD) present in only minute amounts

X

X

Run outOf

NAD+

X

Can’t

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Glucose

CYTOSOL

Pyruvate

No O2 presentFermentation

O2 presentCellular respiration

Ethanolor

lactate

Acetyl CoA

MITOCHONDRION

Citricacidcycle

Figure 9.18

Life Without Oxygen - FERMENTATIONFERMENTATION

XYeastYeastWine/beerWine/beer

MusclesMuscles

MethodMethodTo RecycleTo RecycleNADH + HNADH + H++

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2 ADP + 2 P1 2 ATP

GlycolysisGlucose

2 NAD+ 2 NADH

2 Lactate

(b) Lactic acid fermentation

O–

C O

C O

CH3O

C O

C OHH

CH3

Figure 9.17

RunningRunningMusclesMuscles

twopyruvate

twolactate

2e2e--

2H2H++

RecyclesRecyclesNADHNADH

Keeps the Motor running

Run in reverseWhen oxygen

returns

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2 ADP + 2 P1 2 ATP

GlycolysisGlucose

2 NAD+ 2 NADH

2 Pyruvate

2 Acetaldehyde���2 Ethanol

(a) Alcohol fermentation

H

H OH

CH3

C

O –

OC

C O

CH3

H

C O

CH3

CO22

Figure 9.17

twopyruvate

twoethanol

2e2e--

2H2H++

RecyclesRecyclesNADHNADH

Gives theFizzTo

ChampagneBeer

Hard cider

alcohol

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Fermentation and cellular respiration– Differ in their final electron acceptorfinal electron acceptor

-- oxygenoxygen-- lactatelactate-- or ethanolor ethanol

Cellular aerobic respiration–– Produces tons more ATPProduces tons more ATP

»» 2 ATP in fermentation2 ATP in fermentation»» 36 ATP in aerobic oxidation of glucose36 ATP in aerobic oxidation of glucose

Fermentation allows organismsFermentation allows organismsto eek out a living in low or no Oto eek out a living in low or no O22

Ancient system Ancient system –– pre oxygen pre oxygen

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Light energy

ECOSYSTEM

CO2 + H2O

Photosynthesisin chloroplasts

Cellular respiration

in mitochondria

Organicmolecules+ O2

ATP

powers most cellular work

HeatenergyFigure 9.2

Where does oxygen come from?

Next time:

photosynthesisphotosynthesis

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Summary:Summary:

1. Oxidative Phosphorylation is comprised of two distinct processesElectron Transport (oxidative) makes H+ gradientATP synthesis (phosphorylation) uses H+ gradient coupled by H+ gradient

2. Processes are separable:cyanide (poisons electron transport)uncouplers (DNP) (dissipate H+ gradient)

3. Electrochemical gradient powersmitochondrial H+ ATPaseindirect active transport

4. Oxidation of fatty acids yields more ATPthan oxidation of glucose

5. Can ferment sugars in absence of oxygenstill produce ATP by substrate level phosphorylationbut no reducing equivalents captured

Why not? Why not?

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• The catabolism of various molecules from food

Amino acids

Sugars Glycerol Fattyacids

GlycolysisGlucose

Glyceraldehyde-3- P

Pyruvate

Acetyl CoA

NH3

Citricacidcycle

Oxidativephosphorylation

FatsProteins Carbohydrates

Figure 9.19