Mitochrondria

Inner membrane	Outer membrane	Matrix	Intermembrane compartment
21% total protein	6% total protein	67% total protein	6% total protein
respiratory chain enzymes, succinate dehydrogenase	monamine oxidase, fatty acid thiokinase	pyruvate dehydrogenase complex, citrate synthase, fumarase	adenylate kinase, nucleoside diphosphokinase

IV. Molecular Organization and Function of the Mitochondria

A. Pathway of Biological Oxidation in Eukaryotes

simplified metabolism

starch, glycogen--> (glycolysis)--> pyruvate--> pyruvate dehydrogenase complex--> acetyl CoA

triacylglycerol--> (lipase)--> fatty acids--> (fatty acid oxidation)--> acetyl CoA

Features of mitochondrial oxidation

1. pyruvate, fatty acids and amino acids

2. acetyl CoA

3. NAD and FAD

4. electron carriers in the inner membrane (ETS)

B. Isolation of the Components of the ETS

simplified version

1. NADH dehydrogenase (16 - 26 polypeptides)

2. Cytochrome b - c₁ (8 polypeptides)

3. Cytochrome oxidase (13 polypeptides)

4. ATPase coupling factor (19 - 22 polypeptides)

C. Two issues to discuss

1. Energetics

Redox potential

What is redox potential? Give an example of a conjugate redox pair. A conjugate redox pair with a negative potential (in volts) is more likely to be an electron donor or an acceptor? Which has the more negative redox potential, NADH/NAD⁺ or H₂O/1/2O₂?

Note that there are large potential drops associated with electron transport in the inner membrane

Where did the energy originally come from that is released from these reactions?

2. Mechanism

Note that the orientation of the three complexes, plus the ATPase coupling factor is essential to the proper functioning of this system?

Why is this asymmetrical orientation a crucial feature to this system?

Where do the electrons that are passed between the complexes come from? Where do these electrons eventually go?

3. Relationship between oxidative phosphorylation and the ETS

- the proton motive force (PMF).

-Calculation of free energy difference associated with PMF

For example, a typical mitochondria involved in ATP synthesis at 37C with a membrane potential difference of -0.16 volts (this means more negative in the matrix than in the intermembrane space because of the outward pumping of protons) and a matrix pH that is 1 unit higher than the surrounding cytoplasm:

(delta)G_outward = 2.303 RT (pH_inside - pH_outside) - nFE_m

= (2.303)(8.314 X 10 -3)(273 + 37)(1) - (1)(96.5)(-0.16)

= +21.4 KJ/mole of protons

This means that the outward pumping of protons against an electropotential gradient is endergonic and requires 21.4 KJ/mole; to bring protons back into the matrix will be exergonic (-21.4 KJ/mole)

The free energy difference is useful to understand features of the energetics of ATP synthesis:

The (delta)G for ATP formation depends on the concentration of ATP, ADP and Pi in the matrix;

ATP + H₂O<--> ADP + P_i

At equilibrium,

(delta)G⁰ = -RT ln K_eq

= -RT ln [ADP][P_i]/[ATP]

(delta)G⁰ (ATP) = -34 KJ/mole (chemist's value!)

In the mitochondria, it takes 3 protons translocated from NADH to make 1 ATP;

that is, (delta)G = -64.5 KJ/mole (biologist's value)

So, if the available energy is greater than -64.5 KJ/mole, ATP will be made. The rate of ATP synthesis is dependent on the concentrations of ATP, ADP and Pi, plus the rate of proton translocation (the electrochemical gradient).

How does the proton gradient get utilized to make ATP?

ATPase is a rotational enzyme

ATPsyn.gif (35994 bytes)
Image from http://rsb.info.nih.gov/NeuroChem/biomach/ATPsyn.html showing ATPase in action (open, loose and tight conformational states)