Lecture Connections19 | Oxidative Phosphorylation and

Photophosphorylation

© 2009 Jim-Tong Horng

CHAPTER 19 Oxidative Phosphorylation and

Photophosphorylation

Electron transport chain in mitochondria

Building up the the proton-motive force

Synthesis of ATP in mitochondria

Key topics:

Energy from Reduced Fuels is Used to Synthesize ATP in Animals

• Carbohydrates, lipids, and amino acids are the main reduced fuels for the cell

• Electrons from reduced fuels used to reduce NAD+ to NADH or FAD to FADH2.

• In oxidative phosphorylation, energy from NADH and FADH2 are used to make ATP

Oxidative Phosphorylation

• Electrons from the reduced cofactors NADH and FADH2 are passed to proteins in the respiratory

chain

• In eukaryotes, oxygen is the ultimate electron acceptor for these electrons

• Energy of oxidation is used to phosphorylate ADP

Chemiosmotic Theory

• How to make an unfavorable

ADP + Pi = ATP

possible?• Phosphorylation of ADP is not a result of a direct

reaction between ADP and some high energy phosphate carrier

• Energy needed to phosphorylate ADP is provided by the flow of protons down the electrochemical gradient

• The electrochemical gradient is established by transporting protons against the electrochemical gradient during the electron transport

Chemiosmotic Energy Coupling Requires Membranes

• The proton gradient needed for ATP synthesis can be stably established across a topologically closed membrane– Plasma membrane in bacteria– Cristae membrane in mitochondria– Thylakoid membrane in chloroplasts

• Membrane must contain proteins that couple the “downhill” flow of electrons in the electron transfer chain with the “uphill” flow of protons across the membrane

• Membrane must contain a protein that couples the “downhill” flow of proton to the phosphorylation of ADP

Structure of a Mitochondrion

• The proton gradient is established across the inner membrane

• The cristae are convolutions of the inner membrane and serve to increase the surface area

Permeable to Mr. 5,000Porin

37 genes, 13 encoding ETC proteins(most mitochondrial proteins are encoded by nucleus and then transported into

mitochondria)

Metabolic Sources of Reducing Power

Electrons are funneled to universal electron acceptors

• Flavoproteins---FAD or FMN containing proteins (see below)

• NAD-linked dehydrogenase and NADP-linked dehydrogenase

Reduced substrate+NAD+→Oxidized substrate+NADH + H+

Reduced substrate+NADP+→Oxidized substrate+NADPH + H+

Electrons are funneled to universal electron acceptors

• Flavoprotein--Tightly bound FMN or FAD, one or two e- carrier• Coenzyme Q, also called ubiquinone (abbr. CoQ or Q)--one or

two e- carrier, mobile within membrane• cytochromes--- Fe2+/Fe3+, one e- carrier

– cytochrome a’s: Isoprenoid (15-C) on modified vinyl and formyl in place of methyl

– cytochrome b’s: Iron-protoporphyrin IX– cytochrome c’s: Iron-protoporphyrin IX linked to cysteine

• iron-sulfur proteins--- one e- carrier, several types• coppers--- one e- transfer, Cu+/Cu++

Electron Carriers

Coenzyme Q or Ubiquinone

• Ubiquinone is a lipid-soluble conjugated dicarbonyl compound that readily accepts electrons

• Upon accepting two electrons, it picks up two protons to give an alcohol, ubiquinol

• Ubiquinol can freely diffuse in the membrane, carrying electrons with protons from one side of the membrane to another side

Cytochrome c

• Cytochrome c is a soluble heme-containing protein in the intermembrane space

• Heme iron can be either ferrous(Fe3+, oxidized) or ferric(Fe2+, reduced)

• Cytochrome c carries a single electron from the cytochrome bc1 complex to cytochrome oxidase

Iron-Sulfur Centers

• Found in several proteins of electron transport chain, including NADH:ubiquinone oxidoreductase

• Transfers one electron at a time

The Electron Transport Chain—An Overview

The electron transport chain can be isolated in four complexes----

Complex I: NADH::Ubiquinone oxidoreductase,

Complex II: Succinate dehydrogenase,

Complex III: ubiquinone-cytochrome c oxidoreductase

Complex IV: cytochrome c oxidase

NADH:Ubiquinone Oxidoreductasea.k.a. Complex I

• One of the largest macro-molecular assemblies in the mammalian cell

• Over 40 different polypeptide chains, encoded by both nuclear and mitochondrial genes

• NADH binding site in the matrix side• Non-covalently bound flavin mononucleotide

(FMN) accepts two electrons from NADH• Several iron-sulfur centers pass one electron at

the time toward the ubiquinone binding site

The operation of respiratory complex I

NADH:Ubiquinone Oxidoreducase is a Proton Pump

• Transfer of two electrons from NADH to uniquinone is accompanied by a transfer of protons from the matrix (N) to the inter-membrane space (P)

• Experiments suggest that about four protons are transported per one NADH

NADH + Q + 5H+N = NAD+ + QH2 + 4 H+

P

• Reduced coenzyme Q picks up two protons

NADH:Ubiquinone Oxidoreducase is a Proton Pump

Amytal (a barbiturate drug), rotenone (a plant product commonly used as an insecticide), and piericidin A (an antibiotic) inhibit electron flow from the Fe-S centers of Complex I to CoQ

Succinate Dehydrogenasea.k.a. Complex II

• FAD accepts two electrons from succinate

• Electrons are passed, one at a time, via iron-sulfur centers to ubiquinone that becomes reduced QH2

Complex II: succinate-coenzyme Q reductase (succinate dehydrogenase)

• The only TCA cycle enzyme that is an integral membrane protein, also called flavoprotein (FP2).

• Succinate →fumarate + 2H+ + 2e- UQ + 2H + + 2e- → UQH2• Components: FAD and Fe-S centers• No proton pumped• Similar complexes

– Glycerolphosphate dehydrogenase: Reduces CoQ: No protons pumped

– Fatty acyl-CoA dehydrogenase:Reduces CoQ: No protons pumped

Cytochrome bc1 Complexa.k.a. Complex III

• Uses two electrons from QH2 to reduce two molecules of cytochrome c

• CoQ passes electrons to cyt c (and pumps H+) in a unique redox cycle known as the Q cycle

The Q Cycle

• Experimentally, four protons are transported across the membrane per two electrons that reach cyt. c

• Two of the four protons come from QH2

• The Q cycle provides a good model that explains how two additional protons are picked up from the matrix

Cytochrome Oxidase a.k.a. Complex IV

• Mammalian cytochrome oxidase is a membrane protein with 13 subunits

• Contains two heme groups

• Contains copper ions

– Two ions (CuA) form a binuclear center

– Another ion (CuB) bonded to heme forms Fe-

Cu center

Cytochrome Oxidase Passes Electrons to O2

• Four electrons are used to reduce one oxygen molecule into two water molecules

• Four protons are picked up from the matrix in this process

• Four additional protons are passed from the matrix to the inter-membrane space by an unknown mechanism

Summary of the Electron Flow in the Respiratory Chain

Proton-motive Force

• The proteins in the electron transport chain created the electrochemical proton gradient by one of the three means:

–actively transported protons across the membrane via poorly understood mechanisms

–passed electrons to coenzyme Q that picked up protons from the matrix

–took electrons from QH2 and released the

protons to the inter-membrane side

Chemiosmotic Model for ATP Synthesis

• Electron transport sets up a proton-motive force

• Energy of proton-motive force drives synthesis of

ATP

Mitchell’s chemiosmotic hypothesis

• Proton gradient used to drive ATP synthesis• Protons per electron pair

– From succinate (FADH2) 6– From NADH 10

• Four protons per ATP– ADP uptake: 1 proton– ATP synthesis: 3 protons

• One oxygen atom consumed per electron pair• P/O: the number of ATP produced per atom of

oxygen consumed. Used as an index of oxidative phosphorylation.– From NADH: 2.5– From succinate: 1.5

P/O ratio

• ADP + Pi ATP

• P/O: the number of ATP produced per atom of oxygen consumed. Used as an index of oxidative phosphorylation.

– From NADH: 2.5 ATP

– From succinate (FADH2): 1.5 ATP

ATP synthase

Two parts: F1 and F (latter was originally "F-o" for its inhibition by oligomycin)

---F1: ATP synthesis, ---Fo: Proton channel in inner membrane,

ab2c10-12

Binding change mechanism: the three catalytic b subunits are intrinsically identical but are not functionally equivalently at any particular moment where they cycle through three conformational states.

Proton diffusion through the protein drives ATP synthesis!

Mitochondrial ATP Synthase Complex

• The proton-motive force causes rotation of the central shaft

• This causes a conformational change within all the three pairs

• The conformational change in one of the three pairs promotes condensation of ADP and Pi into

ATP

Adenine nucleotide and phosphate translocase

ATP must be transported out of the mitochondria

• Adenine nucleotide translocase• ATP-ADP antiporter located in inner mitochondrial

membrane • But ATP4- out and ADP3- in is net movement of a negative

charge out - equivalent to a H+ going in • The proton-motive force drives ATP-ADP exchange

• Phosphate translocase• Symport of H2PO4

- and H+ located in inner mitochondrial membrane

• Consume some of the energy of e- transport (H+ moved from P to the N side of the inner membrane)

There is no net flow of charge during symport of H2PO4 and H+

ATP Yield From Glucose

Shuttle Systems for e -

Most NADH used in electron transport is cytosolic and NADH doesn't cross the inner mitochondrial membrane

• What to do?? • "Shuttle systems" effect electron movement without

actually carrying NADH • Glycerol 3-phosphate shuttle stores electrons in

glycerol-3-P, which transfers electrons to FAD • Malate-aspartate shuttle uses malate to carry electrons

across the membrane

Malate aspartate shuttle

Liver, kidney and heart

Glycerol 3-phosphate shuttle

Skeletal muscle and brain

Inhibitors of electron transport and oxidative phosphorylation

Complex IV

Complex III

Complex I or III

Uncouplers: Stimulate e- transport: short circuit proton gradient: Block ATP production

• Carry protons from cytosolic surface of the membrane and then carry them back to matrix.

• Proton ionophores: Lipid soluble substance with dissociable proton, eg., 2,4 dinitrophenol (DNP) and FCCP.

• Thermogenin: uncoupler protein: Generates heat using proton gradient.

Heat generation by uncoupled mitochondria

Brown fat thermogenesis

Brown fat, rich in mitochondria and cytochromes

Thorax near shoulders Hibernating animals

         Produces heat instead of ATP

Protein thermogenin is an “uncoupler” (uncoupling protein)

Creates a H+ pore in inner mitochondrial membrane

 

Chapter 19: Summary

• The reduced cofactors pass electrons into the electron

transport chain in mitochondria

• Stepwise electron transport is accompanied by the

directional transport of protons across the membrane

against their concentration gradient

• The energy in the electrochemical proton gradient drives

synthesis of ATP by coupling the flow of protons via ATP

synthase to conformational changes that favor formation

of ATP in the active site

In this chapter, we learned that:

Net Yield of ATP from Glucose

It depends on which shuttle is used!

Mitochondria are from mother side

Fig. 19-32

Mutations in mitochondria genes cause human disease

• Leber’s hereditary optic neuropathy(LHON)– Central nerve system defect, including optic nerves, causing bilateral loss of vision

in early childhood

– A single base change in the mitochondrial gene in complex I

– A single base change in the mitochondrial gene in cyto. b in complex III

• Myoclonic epilepsy and ragged-red fiber disease

(MERRF)– Leucyl-tRNA defect

– Characterized by uncontroled muscular jerking

– Paracrystalline structure

– Also one of the causes of adult-onset diabetes mellitus

• Aging– DNA damage