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Objectives

1.1. To know about the structural and biochemical To know about the structural and biochemical organizations of a mitochondrionorganizations of a mitochondrion

2.2. To understand the electrochemical reactions To understand the electrochemical reactions through which the chemical energy in food through which the chemical energy in food can be converted to chemical energy in ATPcan be converted to chemical energy in ATP

3.3. To realize how the structural organizations of To realize how the structural organizations of mitochondria have allowed the above mitochondria have allowed the above electrochemical reactions to be carried out electrochemical reactions to be carried out effectivelyeffectively

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Energy Conversion (1): MitochondriaEnergy Conversion (1): Mitochondria

• Cellular respirationCellular respiration

- Flow of electrons from Flow of electrons from reduced coenzymesreduced coenzymes to an to an electron acceptor; generation of ATPelectron acceptor; generation of ATP

- NADH and FADHNADH and FADH22 from glycolysis, TCA cycle, from glycolysis, TCA cycle, --

oxidations, etc.oxidations, etc.

- Ultimate electron acceptor is oxygen; reduced Ultimate electron acceptor is oxygen; reduced form as water form as water (aerobic respiration); (aerobic respiration); takes place takes place with mitochondria in eukaryotic cellswith mitochondria in eukaryotic cells

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The Energy PowerhouseThe Energy Powerhouse• Discrete sausage-shaped structures; the Discrete sausage-shaped structures; the

second largest organelle in most animal cellssecond largest organelle in most animal cells

• A double-membrane organelle; outer A double-membrane organelle; outer membrane separated from inner membrane membrane separated from inner membrane by intermembrane spaceby intermembrane space

A.A. Outer membraneOuter membrane

• Not a significant permeability barrier for Not a significant permeability barrier for ions and small molecules; transmembrane ions and small molecules; transmembrane proteins proteins (porins)(porins)

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B.B. Intermembrane spaceIntermembrane space

• Continuous with the cytosolContinuous with the cytosol

C.C. Inner membraneInner membrane

• A permeability barrier to most solutes A permeability barrier to most solutes

• Locale of the protein complexes of electron traLocale of the protein complexes of electron transport and ATP synthesisnsport and ATP synthesis

• Distinctive foldings Distinctive foldings (cristae); (cristae); increase surface increase surface area to accommodate more the protein complearea to accommodate more the protein complexesxes

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D.D. MatrixMatrix

• Semi-fluid enclosed by Semi-fluid enclosed by inner membrane; inner membrane;

- Enzymes for Enzymes for mitochondrial functionsmitochondrial functions

- A circular DNA A circular DNA molecule; coding for its molecule; coding for its own rRNAs, tRNAs, and own rRNAs, tRNAs, and a number of polypeptide a number of polypeptide subunits of inner-subunits of inner-membrane proteins membrane proteins (genetic competence)(genetic competence)

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Electron Transport System (ETS)Electron Transport System (ETS)

• Transfer of electrons from NADH and Transfer of electrons from NADH and FADHFADH22 is highly exergonic is highly exergonic

• Multistep process;Multistep process; a series of reversibly a series of reversibly oxidizable electron carriers; total free energy oxidizable electron carriers; total free energy difference is released in increments to difference is released in increments to prevent excessive amount being released as prevent excessive amount being released as heat (energy conservation for ATP)heat (energy conservation for ATP)

• 4 different kinds of carriers::4 different kinds of carriers::

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A.A. FlavoproteinsFlavoproteins

• Membrane-bound proteins using either Membrane-bound proteins using either flavin flavin adenine dinucleotide (FAD)adenine dinucleotide (FAD) or or flavin mononuflavin mononucleotide (FMN)cleotide (FMN) as prosthetic group as prosthetic group

• Transfer both electrons and protonsTransfer both electrons and protons

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B.B. Iron-Sulfur ProteinsIron-Sulfur Proteins

• Proteins containing Proteins containing iron-sulfur (Fe/S) centers;iron-sulfur (Fe/S) centers; i iron and sulfur atoms complexed with ron and sulfur atoms complexed with cysteinecysteine g groups of the proteinroups of the protein

• Alternates between the Alternates between the FeFe3+3+(ferric) and Fe(ferric) and Fe2+2+(fer(ferrous)rous)

• Do not pick up and release protonsDo not pick up and release protons

C.C. Cytochromes (Cyt)Cytochromes (Cyt)

• Contain Contain iron;iron; part of a porphyrin prosthetic g part of a porphyrin prosthetic group roup (heme)(heme)

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• One-electron carriers; transfer electrons only:One-electron carriers; transfer electrons only:

1.1. Cyt b, cCyt b, c11, a and a, a and a33 are integral membrane are integral membrane

proteinsproteins

2.2. Cyt cCyt c is relatively hydrophilic; loosely associated is relatively hydrophilic; loosely associated with inner face of membrane; not a part of the with inner face of membrane; not a part of the complexes; mobile electron carrier complexes; mobile electron carrier

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3.3. Cyt a and aCyt a and a33

• Copper - containing - cytochromes Copper - containing - cytochromes (bimetallic iron-copper (Fe/Cu) center)(bimetallic iron-copper (Fe/Cu) center)

• Components of Components of cytochrome c oxidasecytochrome c oxidase

• Keeping an OKeeping an O22 molecule bound to the molecule bound to the

oxidase complex; completely picked up oxidase complex; completely picked up the four electrons and four protonsthe four electrons and four protons

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D.D. Coenzyme Q (CoQ)Coenzyme Q (CoQ)

• UbiquinoneUbiquinone (a benzene (a benzene derivative); the only derivative); the only nonprotein componentnonprotein component

• Carries both protons and Carries both protons and electronselectrons

• Not part of a respiratory Not part of a respiratory complex; a collection point complex; a collection point for electrons from for electrons from FMN- and FMN- and FAD-linked dehydrogenasesFAD-linked dehydrogenases

• Active transport of protons Active transport of protons across inner mitochondrial across inner mitochondrial membranemembrane

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• The electron carriers function in a The electron carriers function in a sequence determined by their relative sequence determined by their relative reducing power reducing power (reduction potentials)(reduction potentials)

- Two interconvertible molecules or ions by Two interconvertible molecules or ions by the loss or gain of electrons the loss or gain of electrons (redox pair)(redox pair)

• With exceptions of With exceptions of CoQ and Cyt c,CoQ and Cyt c, the the electron carriers are organized into four electron carriers are organized into four large multiprotein complexeslarge multiprotein complexes (respiratory (respiratory complexes) complexes)

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A.A. Complex I Complex I

• NADH-coenzyme Q oxidoreductaseNADH-coenzyme Q oxidoreductase

- Transfers electrons from NADH to coenzyme Transfers electrons from NADH to coenzyme QQ

B.B. Complex IIComplex II

• Succinate-coenzyme Q oxidoreductaseSuccinate-coenzyme Q oxidoreductase

- Transfers electrons derived from succinate Transfers electrons derived from succinate oxidation in TCAoxidation in TCA

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C.C. Complex IIIComplex III

• Coenzyme Q – cytochrome c oxidoreductase Coenzyme Q – cytochrome c oxidoreductase

- Accepts electrons from coenzyme Q and passes Accepts electrons from coenzyme Q and passes them to cytochrome cthem to cytochrome c

D.D. Complex IVComplex IV

• Cytochrome c oxidase Cytochrome c oxidase

- A terminal oxidase; capable of direct transfer A terminal oxidase; capable of direct transfer of electrons to oxygenof electrons to oxygen

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Respiratory ComplexRespiratory Complex 　　 　　Electron FlowElectron Flow 　　

NumberNumber NameNameNumber of Number of PolypeptidesPolypeptides

Prosthetic GroupsProsthetic Groups Accepted fromAccepted from Passed toPassed toProton Proton TransportTransport??

IINADH NADH dehydrogenase dehydrogenase (NADH-coenzyme Q (NADH-coenzyme Q oxidoreductase)oxidoreductase)

22-2622-261 FMN 1 FMN 6-9 Fe/S centers6-9 Fe/S centers

NADHNADH Coenzyme QCoenzyme Q YesYes

IIIISuccinate-coenzyme Succinate-coenzyme Q oxidoreductase Q oxidoreductase (succinate (succinate dehydrogenase)dehydrogenase)

4-54-51 FAD 1 FAD 3 Fe/S centers3 Fe/S centers

Succinate Succinate (via enzyme-bound (via enzyme-bound FAD)FAD)

Coenzyme QCoenzyme Q NoNo

IIIIIICoenzyme QCoenzyme Q-cytochrome -cytochrome ccoxidoreductaseoxidoreductase(cytochrome (cytochrome bb--cc11

complex)complex)

8-108-102 cytochrome 2 cytochrome b b 1 cytochrome1 cytochrome c c11

1 Fe/S center1 Fe/S centerCoenzyme QCoenzyme Q Cytochrome Cytochrome cc YesYes

IVIVCytochrome Cytochrome cc oxidaseoxidase

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1 cytochrome 1 cytochrome aa1 cytochrome 1 cytochrome aa33

2 Cu centers 2 Cu centers (as Fe/Cu centers(as Fe/Cu centerswith cytochrome with cytochrome aa33))

Cytochrome Cytochrome cc Oxygen (OOxygen (O22)) YesYes

Properties of the Mitochondrial Respiratory Complexes

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ATP Generation / Electron Transport ATP Generation / Electron Transport

• ATP generation: ADP + Pi ATP generation: ADP + Pi ATPATP

A.A. PhotophosphorylationPhotophosphorylation

B.B. Substrate level phosphorylationSubstrate level phosphorylation

• Glycolysis:Glycolysis: 1,3-bisphosphoglycerate 1,3-bisphosphoglycerate 3-phosp 3-phospho-glycerate; phosphoenolpyruvate ho-glycerate; phosphoenolpyruvate pyruvate pyruvate

• TCA:TCA: succinyl CoA succinyl CoA succinate succinate

- 4 ATP4 ATP molecules/glucose: 2 from glycolysis molecules/glucose: 2 from glycolysis ++ 2 2 from TCAfrom TCA

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C.C. Oxidative phosphorylationOxidative phosphorylation

• 6 different oxidations 6 different oxidations (12 pairs of electrons):(12 pairs of electrons):

1.1. Glycolysis:Glycolysis: glyceraldehyde-3-phosphate glyceraldehyde-3-phosphate 1,3-bi 1,3-bisphosphoglycerate sphosphoglycerate (+NADH)(+NADH)

2.2. Pyruvate Pyruvate acetyl CoA acetyl CoA (+NADH)(+NADH)

3.3. TCA:TCA: isocitrate isocitrate -ketoglutarate -ketoglutarate (+NADH)(+NADH); ; --KG KG succinyl CoA succinyl CoA (+NADH)(+NADH); succinate ; succinate fuma fumarate rate (+FADH(+FADH22)); malate ; malate oxaloacetate oxaloacetate (+NADH) (+NADH)

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Chemiosmotic ModelChemiosmotic Model

• Electrochemical potentialElectrochemical potential across a across a membrane; the link between electron membrane; the link between electron transport and ATP formation transport and ATP formation

- Exergonic transfer of electrons between Exergonic transfer of electrons between and within respiratory complexes; and within respiratory complexes; unidirectional pumping of protonsunidirectional pumping of protons across the membrane where the across the membrane where the transport system is localizedtransport system is localized

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The FThe F00FF11 Complex Complex

• A A F-type ATPase; F-type ATPase; both ATPase and ATP synthase activitiesboth ATPase and ATP synthase activities

• Converts electrochemical energy (proton gradient) into Converts electrochemical energy (proton gradient) into potential chemical energy (ATP)potential chemical energy (ATP)

A.A. FF11 complex complex

• 33 and 3 and 3 polypeptides; polypeptides; 3 3 complexes complexes (catalytic hexagon)(catalytic hexagon)

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subunit:subunit: catalytic site for ATP catalytic site for ATP synthesis/hydrolysis; synthesis/hydrolysis; subunit: subunit: ATP/ADP- ATP/ADP-binding sitebinding site

• Both ATP synthase and ATPase activities Both ATP synthase and ATPase activities

• Proton translocation through FProton translocation through F00 drives ATP drives ATP

synthesis by Fsynthesis by F11

B.B. StalkStalk

• Composes of Composes of , , and and subunits subunits

• Allows rotation of FAllows rotation of F11 complex about F complex about F00

complexcomplex

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C.C. FF00 complex complex

• Consists of Consists of 1a, 2b and 9-12 c subunits1a, 2b and 9-12 c subunits

• c subunits are organized in a circle; c subunits are organized in a circle; proton channelproton channel

• As a As a proton translocator:proton translocator: channel channel through which protons flow through which protons flow (protonation and deprotonation of aspartate)

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Binding Change ModelBinding Change Model

• To explain how To explain how exergonic exergonic flow of protons flow of protons through Fthrough F00 can drive can drive endergonic endergonic

phosphorylation of ADP to ATPphosphorylation of ADP to ATP

• Electrochemical–to–mechanical–to–chemical Electrochemical–to–mechanical–to–chemical transducertransducer

• Each of the three Each of the three subunits subunits exists in 3 exists in 3 different conformations at any point in time:different conformations at any point in time:

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- (O)pen:(O)pen: little affinity; ADP and Pi are free to little affinity; ADP and Pi are free to enter (ATP is free to leave) the catalytic siteenter (ATP is free to leave) the catalytic site

- (L)oose:(L)oose: higher affinity; lose binding of ADP higher affinity; lose binding of ADP and Piand Pi

- (T)ight:(T)ight: Packing the ADP and Pi together tig Packing the ADP and Pi together tightly; facilitating the condensation htly; facilitating the condensation

- O O L L T: T:

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1.1. Flowing of protons flow through a channel in Flowing of protons flow through a channel in the the a subunita subunit of F of F00

2.2. Rotation of the ring of Rotation of the ring of c subunits;c subunits; rotation of rotation of the attached the attached subunit subunit

3.3. AsymmetryAsymmetry of the of the subunit; different subunit; different interactions with the three interactions with the three subunits at any subunits at any point in timepoint in time

4.4. Each Each subunit passes successively through subunit passes successively through the O, L, and T conformations as the the O, L, and T conformations as the subunit rotates 360subunit rotates 360ºº

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