Oxidative Phosphorylation-- ETC/ATP Synthase :p

role of transporters in chemiosmotic theory

couple downhill flow of electrons through ETC with the uphill flow of protons across the membrane

energy of electron flow is stored as:

electrochemical potential

chemiosmotic theory

transmembrane differences in proton concentration are the reservoir for the energy extracted from biological oxidation reactions

permeability of outer mitochondrial membrane

porous, allows for passage of metabolites, small molecules

4 distinct compartments of mitochondria

1. outer membrane

2. intermembrane space

3. inner membrane

4. matrix

describe pH of intermembrane space compared to cytosol

lower (high [H+])

describe permeability of inner mitochondrial membrane

impermeable; molecules can only cross through specific transporters

where are the ETC complexes located?

inner mitochondrial membrane

purpose of cristae in inner mitochondrial membrane

increase surface area

what increases the surface area of the inner mitochondrial membrane?

cristae

mitochondrial matrix

location of CAC and parts of lipid and amino acid metabolism, contains PDC

describe pH of mitochondrial matrix

higher (lower [H+])

electron carriers

integral proteins with prosthetic groups capable of accepting/donating 1-2 electrons

function of dehydrogenases

collect electrons from catabolic pathways and funnel them into universal electron acceptors (FAD or NAD)

electron carriers required for oxidative phosphorylation

NADH/NAD+, coenzyme Q, flavoproteins (FADH2/FAD, FMN), cytochromes (cytochrome 2), iron-sulfur clusters

3 types of electron transfers

1. direct transfer of electrons

2. transfer as a H + atom

3. transfer of hydride ion (H-)

function of NADH/NAD+

carries electrons from catabolic reactions to their point of entry into the respiratory chain

what complexes use flavoproteins?

I and II

why does electron transfer occur with flavoproteins?

flavoprotein has higher reduction potential than compound oxidized

how many electrons does coenzyme Q carry?

2

where does coenzyme Q carry electrons?

complex I —> III or II —> III

describe the properties of coenzyme Q

small, hydrophobic, freely diffusible within lipid bilayer of inner mitochondrial membrane

how many electrons do cytochromes carry?

1

reduction potential

quantitative measure of relative tendency of chemical species to accept electrons in redox reaction

electrons are transferred from ________ to ________ reduction potential

lower/higher

free energy released in electron transfer is used to …

transport protons from matrix to intermembrane space, storing this energy in an electrochemical gradient

for each NADH ____ H+ are pumped from the matrix to the intermembrane space

10

for each FADH2 ____ H+ are pumped from the matrix to the intermembrane space

6

at what complex does FADH2 start?

II

complex I: NADH:Ubiquinone oxidoreductase

2 e- transferred from NADH to ubiquinone, drives transfer of 4 H+ to IMS (against gradient)

complex II: succinate dehydrogenase

FAD accepts 2 e- from succinate, transfers electrons to ubiquinone; NO H+ PUMPING

which complex does not pump H+?

II

final e- acceptor in complex II reaction

Q

complex III: ubiquinone: cytochrome c oxidoreductase

transfers e- from QH2 to 2 molecules of cytochrome c (each cytochrome c carries 1 e-), pumps 4 H+ to intermembrane space

how many electrons does each cytochrome c carry?

1

how are protons delivered to complex III?

Q cycle

complex IV: cytochrome c oxidase

carries 2e- from 2 cytochrome c to 0.5 molecular oxygen (O2), reducing it to 1 H2O, 2 H+ are picked up from matrix, 2 additional H+ are pumped to intermembrane space

what happens when incompletely reduced oxygen intermediates escape from complex IV?

oxygen radicals can damage cells

final electron acceptor

O2

how does the ETC create an electrochemical proton gradient?

1. actively transport protons across the membrane (complex I and IV)

2. chemically remove protons from the matrix (reduction of CoQ and reduction of oxygen)

3. release protons into the intermembrane space (oxidation of QH2)

which complexes actively transport protons across the membrane?

I and IV

how are protons chemically removed from the matrix?

reduction of CoQ and reduction of O2 to H2O

how are protons released into the intermembrane space?

oxidation of QH2

first electron donor

NADH

chemiosmotic model for ATP synthesis

• electron transport sets up a proton-motive force

• energy of proton-motive force drives synthesis of ATP

• chemical reaction and transport process; energy coupling

2 entry points into electron transport

• for NADH, complex I

• for FAD, complex II

how many H+ does NADH pump total?

12

how many H+ does FAD pump total?

8

why does NADH pump more protons than FAD?

FAD enters ETC at complex II

proton motive force

difference in H+ concentration and separation of charge across inner mitochondrial membrane

does FAD or NADH produce more energy?

NADH (pumps more protons)

effect of inhibitors of ETC on ATP synthesis

block ATP synthesis

effect of inhibition of ATP synthesis on electron transfer

blocks electron transfer

ATP synthesis is driven by:

substrate oxidation

oligomycin

blocks flow of H+ into matrix through H+ channel of ATP synthase

uncoupling reagent/ionophore

weak, hydrophobic, & can diffuse across mitochondrial membranes; can accept or donate H+, relieving H+ gradient by transporting H+; uncouples oxidation from phosphorylation; example DNP

2 functional units of mitochondrial ATP synthase complex

F1 peripheral membrane protein, F0 integral membrane complex

F1 complex

• peripheral (soluble in matrix)

• made of 3 dimers

• when isolated, catalyzes hydrolysis of ATP

F0 complex

• 0 indicates oligomycin sensitivity

• integral membrane complex

• transports protons from IMS to matrix, dissipating proton gradient

• transfers energy to F1 to catalyze phosphorylation of ADP

• has proton pore to leak H+, maintaining H+ gradient

subunits must be _________ to rotate into the membrane

protonated

what causes the gamma subunit to rotate?

attachment of c proteins

what causes the rotary movement of the c ring (F0) to be unidirectional?

large difference in H+ concentration across membrane

what causes the release of formed ATP?

proton gradient

3 active sites of F1 take turns catalyzing:

ATP synthesis

process of ATP synthesis in F1 active site

1. subunit binds ADP + Pi

2. subunit changes conformation to form that tightly binds and stabilizes ATP

3. equilibration of ADP + Pi with ATP on enzyme surface

4. subunit changes to conformation with low ATP affinity (empty)

5. synthesized ATP leaves enzyme surface

F1 dimers can exist in 3 different conformations:

1. open: empty

2. loose: binding ADP and Pi

3. tight: catalyzes ATP formation and binds product

how many H+ are needed to drive the synthesis of 1 ATP molecule?

4

what sets the rate of electron transfer through the respiratory chain?

ATP and ADP concentrations

2 primary mechanisms of oxidative phosphorylation regulation

1. substrate availability (NADH and ADP + Pi)

2. cellular energy needs

effect of low oxygen (hypoxia) on F1 and oxidative phosphorylation

• inhibits F1

• prevents hydrolysis of ATP

• electron transfer to oxygen slows

• pumping of H+ slows

• proton motive force collapses

inhibition of oxidative phosphorylation leads to accumulation of _________

NADH

accumulation of NADH causes __________

feedback inhibition cascade up to PFK-1 in glycolysis