Pyruvate Carboxylase

Reversing the final steps

O

OH OH

OH

OH

O

P

OO-

O-

O-

H

O

OH OH

OH

OH

OH P

OO-

O-

-O

OH

OH

OH

O

CH2OPO32- O

P

OO-

O-

O-

H

OH

OH

OH

O

CH2OPO32- OH

P

OO-

O-

-O

Inverse regulation of glycolysis and gluconeogenesis

Hexokinase IV in liverHexokinase I in muscle

HxkIV Km = 10 mM

When blood glucose drops below 5 mM, F6P inhibits it. This way liver does not compete with muscle for glucose

Regulation of phosphofructokinase

Electron transport and oxidative phosphorylation

Glucose is completely oxidized to CO2 through the enzymatic reactions of glycolysis and the citric acid cycle. The redox equation for this process is:

C6H12O6 + 6O2 ---> 6CO2 + 6H2O ΔG°’ = -2823 kJ.mol-1

Which can be represented by two half reactions:

C6H12O6 + 6H2O ---> 6CO2 + 24H+ + 24e- glucose is oxidized

6O2 + 24H+ + 24e- ---> 12H2O molecular oxygen is reduced

In living systems the electron transfer process connecting these two half reactions occurs through a multistep pathway that harnesses the liberated free energy to form ATP.

The sites of electron transfer that form NADH and FADH2 in

glycolysis and the citric acid cycle are represented in the figure.

The 12 electron pairs involved in glucose oxidation are not transferred directly to O2.

Rather they are transferred to coenzymes NAD+ and FAD to form NADH and FADH2

10 NADH : 20 e-

2 FADH2 : 4 e-

The electrons are extracted from the cofactors by reoxidation and then join the electron-transport chain, in this process, protons are expelled from the mitochondrion. The free energy stored in the resulting pH gradient drives the synthesis of ATP from ADP and Pi (inorganic phosphate) through oxidative phosphorylation.

Reoxidation of NADH ~ 3 ATPReoxidation of FADH2 ~ 2 ATP

A total of 38 ATP are produced per each molecule of glucose completely oxidized to CO2 and H2O (including the 2 ATP made in glycolysis and the 2 ATP made in the citric acid cycle)

NAD+ and FAD coenzymes are reduced during glucose oxidation

Mitochondria is the site of eukaryotic oxidative metabolism 0.5 m in

diameter and 1 m long (about the size of a

bacterium)

The outer membrane contains porin, a protein that forms pores and

allows free difussion of up to 10 kD molecules

The inner membrane is a lot more dense and is permeable only to O2, CO2 and H2O. Contains numerous

transport proteins that control metabolite passage.

Mitochondrion is not a regular shaped organelle it is a dynamic organelle that is reticulated throughout the cell

Electrons enter the electron transport chain onto Q

Fatty acid metabolism

Glycerol phosphate

shuttle

Succinate dehydrogenase

NADH:QOxidoreductase

Reduced state has more protons than the oxidized state!

Redox loops pumps out four protons!

The glycerophosphate shuttle mainly occurs in rapidly metabolizing tissues.

NADH from TCA cycle are generated in mitochondrial matrix

NADH from glycolysis are generated in cytoplasm

Problem: No way to transport NADH into the mitochondrion to be reoxidized!

Solution: Use the malate-aspartate shuttle

Complex I: NADH:CoQ oxidoreductase

∆E = 0.360 V ∆G = -69.5 kJ/mol

NADH + H+ + CoQ(ox) + 4H+(in) ---> NAD+ + CoQH2(red) + 4H+

(out)

-0.32

-0.30

-0.030

+0.045-0.25

Ubiquinone +0.045

-0.30

-0.32

What is FMN? Flavin mononucleotide

What are FeS clusters?

Complex II: succinate dehydrogenaseSuccinnate:CoQ oxidoreductase

FADH2 + CoQ(ox) ---> FAD + CoQH2(red)

∆E = 0.085 V∆G = -16.4 kJ/mol

-0.031

-0.040

-0.030

-0.245

-0.060

-0.080+0.045

Succinate -0.031

Heme b

Complex III

CoQH2(red) + 2cyt c(ox) + 2H+(in) ---> CoQ(ox) + 2cyt c(red) + 4H+

(out)

∆E = 0.190 V∆G = -36.7 kJ/mol

+0.045

+0.030

-0.030

+0.280+0.215

+0.235

CoQH2 + cyt c1(ox) ---> CoQ•- + cyt c1(red) + 2H+ (out)

CoQH2 + CoQ•- + cyt c1(ox) + 2H+(in) ---> CoQ + CoQH2 + cyt c1(red) + 2H+ (out)

Cycle I

Cycle II

CoQH2 + 2cyt c1(ox) + 2H+(in) ---> CoQ + 2cyt c1(red) + 4H+ (out)

Net Reaction4 protons pumped instead of 2

Ubiquinone = +0.045

Cytochrome c: an electron bottle neck

Complex IV

∆E = 0.580 V∆G = -112 kJ/mol

4 cytochrome c2+  +   O2   +   8H+(in)   =>4 cytochrome c 3+   +   2H2O   +   4H+(out)

+0.235

+0.245

+0.385

+0.340

+0.815

2H+

If 2 electrons enter at complex I4 + 4 + 2 = 10 protons pumped out

If 2 electrons enter at complex II or Glycerol dehydrogenase or fatty

acid metabolism4 + 2 = 6 protons pumped out

Proton concentration gradient

pH is lower in intermembrane space than in the mitochondrial matrix

GA - GA0’ = RT ln [A]

A(out) A(in)

∆GA = GA(in) - GA(out) = RT ln[A]in

[A]out

( )

∆GA = RT ln[A]in

[A]out

( ) + ZAF ∆

If the solute is charged there is another aspect of the equation: electrical potential

Membrane potential = ∆ = (in) - (out)

Free energy is a combination of chemical and electrical potential

∆GA = RT ln[A]in

[A]out

( ) + ZAF ∆

∆G = 2.3RT [pH(in) - pH(out)] + ZF ∆

∆ = 0.168V = 0.168 J•C-1

∆pH = 0.75

∆G = 21.5 kJ•mol-1

F = 96,485 C•mol-1

Z = +1

∆G of ATP synthesis = 40 to 50 kJ•mol-1

[≈ 210,000V•cm-1!!!!]

ATP synthase allows protons to flow back in

Harnesses the free energy in the process

The gamma subunit: the rotor

Per glucose10 NADH : 20 e-

2 FADH2 : 4 e-

If 2 electrons enter at complex I4 + 4 + 2 = 10 protons pumped out

If 2 electrons enter at complex II or Glycerol dehydrogenase4 + 2 = 6 protons pumped out

Per glucose120 protons120/3 = 40

+ 4ATP from glycolysis and

TCA

44 ATP! Why only 38?

H+ transported

-0.3ATP

2.7 ATP/NADH

ATP synthase is nearly 100% efficient: so why do you not get 1ATP per 3 H+?

H+ transported

-0.3ATP

2.4 ATP/NADH

What about fatty acid biosynthesis, succinate dehydrogenase and glycerol

phosphate shuttle?

1.5 ATP per pair of electrons...

How is electron transport regulated?

What is uncoupling?

The c-subunits of F0 ATPase

pH 8

pH 5

Electrons sometimes leak out of the chain onto molecular oxygen. As much 5% leak

out onto oxygen

Incompletely reduced oxygen is toxic

Reactive oxygen species

Superoxide •O2-

Peroxide O22-

Hydroxyl radical •OH

Where does this occur?

O

O

H3CO

H3CO

CH3

R

O2

O

O

H3CO

H3CO

CH3

R

H

B

O2-

Superoxide comes from Q not Complex IV

Why does this happen?

The ox-tox hypothesis

Oxygen is soluble in membranes and it can oxidize lipids, albeit slowly. Too much

oxygen is toxic.

Mitochondria detoxify oxygen by reducing it to water. This is how they

were beneficial

What about superoxide and peroxide?

Maybe these are allowed to leak out as signal molecules that apprise the cell of the energetic state….

Maybe the generation of superoxide, which is not soluble in membrane is a way to

detoxifying oxygen……

Mitochondria and apoptosis

Cytochrome c is released from intermembrane space