Oxidative Phosphorylation

• NADH from Glycolysis must be transported into the mitochondrion to be oxidized by the respiratory electron transport chain.

• Only the electrons from NADH are transported, these are used to form either NADH or FADH2.

• This is accomplished by one of two shuttle mechanisms.

NADH, H+

Glycerophosphate Shuttle

NAD+

3-Phosphoglycerol Dehydrogenase3-Phosphoglycerol Dehydrogenase

H2C - OH

C = O

CH2OPO32-

DHAPDHAP

H2C - OH

C - H

CH2OPO32-

HO -

3-Phosphoglycerol3-Phosphoglycerol

FAD

FADH2

ETCETC

e -

FlavoproteinD’hase

FlavoproteinD’hase

CytosolCytosol

Inner mitochondrial

membrane

Inner mitochondrial

membrane

(skeletal muscle, brain)

Malate-Aspartate Shuttle(Liver, Kidney, Heart)

Malate

OAA

Aspartate

Malate D’haseMalate D’haseNADHNADH

NAD+NAD+

Aspartate Aminotransferase

Aspartate Aminotransferase

Malate:α-Kgcarrier

Malate

OAA

Aspartate

Aspartate Aminotransferase

Aspartate Aminotransferase

Malate D’haseMalate D’hase

NADHNADH

NAD+NAD+

Glutamate:Aspartate

carrier

Cytosol Matrix

InnerMito.

MembraneGlutamate

Glutamate

a-Kg a-Kg

• Oxidative Phosphorylation: NADH and QH2 are oxidized by the respiratory electron transport chain (ETC).

• ETC is set of membrane-embedded protein complexes that act as electron carriers, passing electrons from NADH and QH2 to molecular oxygen.

• As electrons move through the complexes, protons are transported across the inner mito. membrane from the matrix to the intermembrane space.

• The energy stored in this ion gradient is used to synthesize ATP from ADP and Pi by a membrane bound ATPase.

Chemiosmotic Theory : Formulated by Peter Mitchell in the 1960’s. Nobel Prize for this work awarded in 1978.

Chemiosmotic Theory : Formulated by Peter Mitchell in the 1960’s. Nobel Prize for this work awarded in 1978.

SuccinateSuccinate

ADP + Pi

ATP

Rotenone; Amytal

ADP + Pi

ATP

CN- ; CO

ADP + Pi

ATP

Antimycin AFumarateFumarate

NADH NAD+ (-0.315V)NADH NAD+ (-0.315V)2e-

Complex I

∆Εo’= 0.360V; (∆Go’= -69.5 kJ/mol)

Complex II

Complex III

∆Εo’= 0.190V; (∆Go’= -36.7 kJ/mol)

Complex IV

∆Εo’= 0.580V; (∆Go’= -112 kJ/mol)

FADH2FADH2

CoQCoQ (+0.45V)(+0.45V)

Cytochrome C (+0.235V)Cytochrome C (+0.235V)

2H+ + 1/2 O22H+ + 1/2 O2 H20 (+0.815 V)H20 (+0.815 V)

2e-

2e- Demerol

Meaning of the standard reduction potential:Meaning of the standard reduction potential:When compound loses an e- (serves as a reductant), the structure left behind becomes capable of accepting an e- (serves as an oxidant).

cytochrome b (Fe++)

cytochrome c (Fe+++)+

Reductant X Oxidant Y

cytochrome b (Fe+++)

cytochrome c (Fe++)++

Oxidant X’ Reductant Y’

Reductant X and Oxidant X’ are termed a REDOX PAIR!

Electrons flow from the redox pair with the more negative E0 to the more positive E0.

The Reduction of Q is a 2 Electron Reduction !

The Reduction of Q is a 2 Electron Reduction !

1. Q + 1e- Q.-1. Q + 1e- Q.-

2. Q.- + 1e- + 2H+ QH22. Q.- + 1e- + 2H+ QH2

Electron Transport Through Complex IElectron Transport Through Complex I(NADH Dehydrogenase)(NADH Dehydrogenase)

intermembrane space

matrix

FMN FMNH2

2H+

NADH NAD+

H+

2 one e-

transfers FeS2 one e-

transfers QQH2

2H+

Electron Transport Through Complex IIElectron Transport Through Complex II(Succinate Dehydrogenase)(Succinate Dehydrogenase)

intermembrane space

matrix

FeS2 one e-

transfers QQH2

2H+

2 one e-

transfers b560

FAD2e-

Succinate Fumarate

Complex III (Cytochrome bc1 Complex)Complex III (Cytochrome bc1 Complex)

intermembrane space

matrix

QH2

Q. e-

2H+

Q.

b566

b562

e-

e-

e-Q

Q

e-

Fe-S cyt. c1

Q cycle - first step:Q cycle - first step:

Complex III (Cytochrome bc1 Complex)Complex III (Cytochrome bc1 Complex)

intermembrane space

matrix

QH2

Q. e-

2H+

Qb

566

b562

e-

e-

e-

e-

Q.

Fe-S cyt. c1

Q cycle - second step:Q cycle - second step:

QH2

2H+

Complete Q Cycle:Complete Q Cycle:

matrix2H+

intermembrane space

QH2

Q.e-

2H+

b566

b562

e-

e-

e-

e-

Q.

Fe-S cyt. c1

QH2

Q

Q

e-

2 x 12 x 1

2 x 1

2 x 12

2

2

2 x cyt. c

Electron Transport Through Complex IVElectron Transport Through Complex IV(Cytochrome Oxidase)(Cytochrome Oxidase)

intermembrane space

matrix

2H+

2 one e-

transfers2 one e-

transfersCu-bCu-a

2H+

2H+

1/2 O2 H2OH

2O

cyt. c

Cyt. a Cyt. a3

Complex V (ATP Synthase; FoF1 ATPase)Complex V (ATP Synthase; FoF1 ATPase)A Rotating Molecular MotorA Rotating Molecular Motor

• Consumes the energy in the proton gradient to synthesize ATP from ADP.

• Consumes the energy in the proton gradient to synthesize ATP from ADP.

• Couples the phosphorylation of ADP to the oxidation of substrates in the mitochondrion (hence oxidative phosphorylation).

• Couples the phosphorylation of ADP to the oxidation of substrates in the mitochondrion (hence oxidative phosphorylation).

• Mechanism of ATP synthesis is now known, as well as the x-ray crystal structure. Paul Boyer and John Walker won the Nobel Prize in 1997 for this work.

• Mechanism of ATP synthesis is now known, as well as the x-ray crystal structure. Paul Boyer and John Walker won the Nobel Prize in 1997 for this work.

• F1 contains the catalytic subunits; structure is α3,β3,γ3,δ,ε

• F1 contains the catalytic subunits; structure is α3,β3,γ3,δ,ε

• Fo forms a channel in the membrane that allows the passage of protons; structure is a1;b2;c9-12

• Fo forms a channel in the membrane that allows the passage of protons; structure is a1;b2;c9-12

• Fo is sensitive to oligomycin and DCCD (DCCD reacts with a single glutamate residue on the c subunit to block the channel.

• Fo is sensitive to oligomycin and DCCD (DCCD reacts with a single glutamate residue on the c subunit to block the channel.

Binding Change Mechanism (Boyer):Binding Change Mechanism (Boyer):

• F1 has three interacting and conformationally distinct active sites.

• F1 has three interacting and conformationally distinct active sites.

• Protons bind to aspartate residues in the c subunit rotor and cause it to rotate.

• Protons bind to aspartate residues in the c subunit rotor and cause it to rotate.

• This rotations causes the γ subunit to turn relative to the three β subunit nucleotide sites of F1, changing the conformation of each in sequence, so that ADP is first bound, then phosphorylated, then released.

• This rotations causes the γ subunit to turn relative to the three β subunit nucleotide sites of F1, changing the conformation of each in sequence, so that ADP is first bound, then phosphorylated, then released.

Electron Transport Inhibitors:Electron Transport Inhibitors:

• Rotenone; Amytal and Demerol inhibit Complex I

• Rotenone; Amytal and Demerol inhibit Complex I

• Antimycin A inhibits Complex III• Antimycin A inhibits Complex III

• Cyanide; Azide; Carbon Monoxide inhibit Complex IV

• Cyanide; Azide; Carbon Monoxide inhibit Complex IV

• Electron transport can still proceed in the presence of inhibitors if an electron donor is added that bypasses the site of inhibition.

• Electron transport can still proceed in the presence of inhibitors if an electron donor is added that bypasses the site of inhibition.

Uncouplers:Uncouplers:

• Lipid-soluble weak acids that carry protons from the intermembrane space back into the matrix

• Lipid-soluble weak acids that carry protons from the intermembrane space back into the matrix

• No ATP is produced, but electron transport can proceed.

• No ATP is produced, but electron transport can proceed.

• Uncoupled electron transport generates HEAT. This can be useful to both plants and animals.

• Uncoupled electron transport generates HEAT. This can be useful to both plants and animals.

• Thermogenin (a protein) is a natural uncoupler found in brown adipose tissue.

• Thermogenin (a protein) is a natural uncoupler found in brown adipose tissue.

The Alternative Oxidase (AO):The Alternative Oxidase (AO):• Occurs in plants; bypasses Complexes III and IV (less ATP made)

• Occurs in plants; bypasses Complexes III and IV (less ATP made)

QH2

QH2

AOAOH

2OH

2O

1/2 O2; 2 e-1/2 O2; 2 e-

2H+2H+

• Runs when [ATP] is high. Turned on by wounding; flowering; chemicals. May act as a protective mechanism to alleviate effects of reactive oxygen species that form when the normal chain is backed up

• Runs when [ATP] is high. Turned on by wounding; flowering; chemicals. May act as a protective mechanism to alleviate effects of reactive oxygen species that form when the normal chain is backed up

Control of Ox. Phos.:Control of Ox. Phos.:

Control is tied to the cellular energy demand. This is sensed largely by [ATP].Control is tied to the cellular energy demand. This is sensed largely by [ATP].

As ATP is consumed in the cytosol, ADP is transported into the matrix (in antiport with ATP) by the adenine nucleotide translocase. Electron transport, strictly coupled to [ADP] accelerates.

As ATP is consumed in the cytosol, ADP is transported into the matrix (in antiport with ATP) by the adenine nucleotide translocase. Electron transport, strictly coupled to [ADP] accelerates.

Since ATP carries a charge of -4 and ADP carries a charge of -3, transporting an ATP from the matrix to the cytosol results in addition of one negative charge on the cytoplasmic side of the inner mitochondrial membrane.

Since ATP carries a charge of -4 and ADP carries a charge of -3, transporting an ATP from the matrix to the cytosol results in addition of one negative charge on the cytoplasmic side of the inner mitochondrial membrane.

This is equivalent to moving a proton back into the matrix from the cytosol.This is equivalent to moving a proton back into the matrix from the cytosol.

It takes 3 protons moving through Fo to produce 1 ATP. Taking into account moving this ATP to the cytosol, we can conclude that it takes a total of 4 protons/ATP synthesized.

It takes 3 protons moving through Fo to produce 1 ATP. Taking into account moving this ATP to the cytosol, we can conclude that it takes a total of 4 protons/ATP synthesized.

How many protons are translocatedper 2 e- transferred?

Complex I: 4H+Complex I: 4H+

Complex III: 4H+Complex III: 4H+

Complex IV: 2H+Complex IV: 2H+

10H+10H+ : 2e -: 2e -

1 ATP1 ATP

4 H+4 H+XX

10 H+10 H+

(NADH 1/2 O2)(NADH 1/2 O

2)

2e - 2e -

2e - 2e -

1 ATP1 ATP

4 H+4 H+XX

10 H+10 H+

(NADH 1/2 O2)(NADH 1/2 O

2)

1 ATP1 ATP

4 H+4 H+XX

10 H+10 H+

441010

2.52.5OO

OO P P

Ratio !Ratio !