At the end of the electron transport chain, oxygen receives the energy-spent electrons, resulting in the production of water.

½ O2 + 2 e- + 2 H+ → H2O

Oxygen is the final electron acceptor.Almost immediately oxidized into H2O

How is this coupling accomplished?

was originally thought that ATP generation was somehow directly done at Complexes I, III and IV. We now know that the coupling is indirect in that a proton gradient is generated across the inner mitochondrial membrane which drives ATP synthesis.

Mitochondrial respiratory chain: Complex I:

- Transfers e- from NADH to quinone pool & pumps H+. Complex II:- Transfers e- from succinate to quinone pool & H+ released.

Complex III:- Transfers e- from quinol to cytochrome c & pumps H+. Complex IV:- Accepts e- from cytochrome c, reduces O2 to H2O & pumps H+.

Complex V:- Harvests H+ gradient & regenerates ATP.

A total of 10 -12 H+ are ejected from the mitochondrial matrix per 2 e- transferred from NADH to oxygen via the respiratory chain.

Matrix

H+ + NADH NAD+

+ 2H+ 2H+ + ½ O2 H2O

2 e – –

I Q III IV

+ +

4H+ 4H+ 2H+ Intermembrane Space

cyt c

Spontaneous electron flow through each of complexes I, III, & IV is coupled to H+ ejection from the matrix. 

4

Complex I (NADH Dehydrogenase) transports 4H+ out of the mitochondrial matrix per 2e- transferred from NADH to CoQ.

Matrix

H+ + NADH NAD+

+ 2H+ 2H+ + ½ O2 H2O

2 e – –

I Q III IV

+ +

4H+ 4H+ 2H+ Intermembrane Space

cyt c 4

Complex III (bc1 complex):

H+ transport in complex III involves coenzyme Q (CoQ).

Matrix

H+ + NADH NAD+

+ 2H+ 2H+ + ½ O2 H2O

2 e – –

I Q III IV

+ +

4H+ 4H+ 2H+ Intermembrane Space

cyt c 4

A very strong electrochemical gradient is established with few H+ in the matrix and many in the intermembrane space.

The cristae also contain an ATP synthase complex through which hydrogen ions flow down their gradient from the intermembrane space into the matrix.

The flow of three H+ through an ATP synthase complex causes a conformational change, which causes the ATP synthase to synthesize ATP from ADP + P.

High [H+]

Low [H+]

---

+ + +++

+

__

+++++

Matrix

InnerMembrane

H+

H+

H+

OuterMembrane

IntermembraneSpace

Cytoplasm ++ + + + +

+ + ++ + +

H+

High [H+]

H+

H+ H+

H+ H+

H+

H+

---

---

Cristae

Generation of a pH gradient ([H+]) and charge difference (negative in the matrix) across the inner membrane constitute the protonmotive force that can be used to drive ATP synthesis and transport processes.

Mitochondria produce ATP by chemiosmosis, so called because ATP production is tied to an electrochemical gradient, namely an H+ gradient.

Once formed, ATP molecules are transported out of the mitochondrial matrix.

Chemiosmotic Theory --Peter Mitchell A proton gradient isgenerated with energyfrom electron transportby proton pumping by Complexes I,III, IV from the matrix tointermembrane spaceof the mitochondrion.

The protons have a thermodynamic tendency to return to the matrix = Proton-motive forceThe protons diffuse back into the matrix through theFoF1ATP synthase complex. The free energy release drives

ATP synthesis.

The proton pumps are Complexes I, III and IV.

Protons return thru ATP synthase

The Domains Hydrophobic F0 domain sits in the membrane - performs proton

translocation

Hydrophillic F1 portion protrudes from membrane - performs ATP synthesis/hydrolysis 3 alternating alpha and beta subunits

http://nobelprize.org/chemistry/laureates/1997/illpres/boyer-walker.html

ATP synthase: a rotating molecular motor.

a, b, , , and subunits constitute the stator of the motor, and the c, , and subunits form the rotor. Flow of protons through the structure turns the rotor and drives the cycle of conformational changes in and that synthesize ATP.

http://www.bioc.aecom.yu.edu/labs/girvlab/ATPase/ATPsynthase.mov

Animations

http://www.stolaf.edu/people/giannini/flashanimat/metabolism/atpsyn1.swf

Protons cross membrane through the ATP synthase enzyme

http://www.stolaf.edu/people/giannini/flashanimat/metabolism/atpsyn2.swf

Rotary motion of ATP synthase powers the synthesis of ATP

The “stalk” rotates in 120°increments

causes the units in the F1 domain tocontract and expand

The structural changes facilitate the

binding of ADP and Pi to make ATPEach subunit goes through 3 stagesOpen State – releases any ATP

Loose State – ADP and Pi moleculesenter the subunitTight State – the subunit contracts tobind molecules and make ATP

ATP synthesis at F1 results from repetitive conformational changes as rotates

rotates 1/3 turn- energy for ATP release

Interesting Facts

Contains 22722 atoms

23211 bonds connected as 2987 amino acid groups

120 helix units and 94 sheet units

Generates over 100 kg of ATP daily (in humans)

One of the oldest enzymes-appeared earlier then photosynthetic or respiratory enzymesSmallest rotary machine known

Uncoupling. The compound 2,4 dinitrophenol (DNP) allows H+ through the membrane and uncouples.

Blocking. The antibiotic oligomycin blocks the flow of H+ through the Fo, directly inhibiting ox-phos.

Regulation of Respiration -> Primarily by Need for ATP

ATPase inhibited by:

Oligomycin and Dicyclohexylcarbodiimide (DCCD)

Chemiosmosis

Production of ATP in Electron Transport

H+ (Protons) generated from NADH

Electrochemical Gradient Formed between membranes

Electrical Force (+) & pH Force (Acid) thus gradient formed

ATPase enzyme that channels H+ from High to Low concentration3 ATP/NADH

2 ATP/NADH

Overall reaction using NADH funnel NADH + H++ 3ADP + 3Pi+ ½O2 NAD+ + 4H2O + 3ATP

 For the flavoprotein-CoQ funnel, Measure only about 2 ATP produced for each FADH2

Quantify P/O ratio Definition: # Pi taken up in phosphorylating ADP per atomoxygen (½O2)

per 2e-. NADH 3 FADH2 2

What about energy and ATP stoichiometry? -- measured

-- 220 kJ/mole from NADH oxidation-- ATP produced: ADP + Pi ATP

G°= +30.5 kJ/mole-- measure and find about 3 ATP produced for each NADH, which enters. (a little less) [3×(30.5)/220]×100 = 41% efficiency

Shuttling ATP, ADP and Pi

 ATP is required in the cytosol  ADP IN OUT ATP

(Exchange)

So for every ATP transported to cytosol, an ADP must be transported into matrix

Mitochondrial transporters

3 similar 100-residue units (A,B,C)6 membrane-spanning segments

Adding Up the ATP from Cellular Respiration

Figure 6.14

Cytosol

Mitochondrion

Glycolysis

Glucose2

Pyruvicacid

2Acetyl-

CoA

KrebsCycle Electron

Transport

bydirectsynthesis

by directsynthesis

byATPsynthase

Maximumper

glucose:

Figure 6.13

Food

Polysaccharides Fats Proteins

Sugars Glycerol Fatty acids Amino acids

Amino groups

Glycolysis Acetyl-CoA

KrebsCycle Electron

Transport