Transcript Document

Pyruvate Carboxylase
Reversing the final steps
O-
O
OO-
P
H
O
O-
O
P
H
CH2OPO3
O
O-
O
2-
O-
O
OH
OH
OH
OH
OH
OH
OH
O
O
P
O-O
P
OH
-O
OH
CH2OPO32O
OH
OH
OH
OH
O-
O-
O
OH
O-
OH
OH
Inverse regulation of glycolysis and gluconeogenesis
Hexokinase IV in liver
Hexokinase 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 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 e2 FADH2 : 4 e-
The sites of electron transfer that
form NADH and FADH2 in
glycolysis and the citric acid cycle
are represented in the figure.
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 ATP
Reoxidation 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
Glycerol
phosphate
shuttle
NADH:Q
Oxidoreductase
Succinate
dehydrogenase
Fatty acid
metabolism
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
NADH + H+ + CoQ(ox) + 4H+(in) ---> NAD+ + CoQH2(red) + 4H+(out)
∆E = 0.360 V ∆G = -69.5 kJ/mol
-0.030
-0.25
-0.30
-0.32
+0.045
-0.32
-0.30
Ubiquinone
+0.045
What is FMN? Flavin mononucleotide
What are FeS clusters?
Complex II: succinate dehydrogenase
Succinnate: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
+0.235
+0.215
+0.280
-0.030
+0.030
+0.045
CoQH2 + cyt c1(ox) ---> CoQ•- + cyt c1(red) + 2H+ (out)
Cycle I
CoQH2 + CoQ•- + cyt c1(ox) + 2H+(in) ---> CoQ + CoQH2 + cyt c1(red) + 2H+ (out)
Cycle II
CoQH2 + 2cyt c1(ox) + 2H+(in) ---> CoQ + 2cyt c1(red) + 4H+ (out)
Net Reaction
4 protons pumped instead of 2
Ubiquinone = +0.045
Cytochrome c: an electron bottle neck
Complex IV
4 cytochrome c2+ + O2 + 8H+(in) =>
4 cytochrome c 3+ + 2H2O + 4H+(out)
∆E = 0.580 V
∆G = -112 kJ/mol
+0.235
+0.245
+0.815
+0.385
+0.340
2H+
If 2 electrons enter at complex I
4 + 4 + 2 = 10 protons pumped out
If 2 electrons enter at complex II or
Glycerol dehydrogenase or fatty
acid metabolism
4 + 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
If the solute is charged there is another
aspect of the equation: electrical potential
Membrane potential = ∆ = (in) - (out)
∆GA = RT ln (
[A]in
) + ZAF ∆
[A]out
Free energy is a combination of
chemical and electrical potential
∆GA = RT ln (
[A]in
) + ZAF ∆
[A]out
∆G = 2.3RT [pH(in) - pH(out)] + ZF ∆
∆ = 0.168V = 0.168 J•C-1
∆pH = 0.75
[≈ 210,000V•cm-1!!!!]
F = 96,485 C•mol-1
Z = +1
∆G = 21.5 kJ•mol-1
∆G of ATP synthesis = 40 to 50 kJ•mol-1
ATP synthase allows protons to flow back in
Harnesses the free energy in the process
The gamma subunit: the rotor
Per glucose
10 NADH : 20 e2 FADH2 : 4 eIf 2 electrons enter at complex I
4 + 4 + 2 = 10 protons pumped out
If 2 electrons enter at complex II or
Glycerol dehydrogenase
4 + 2 = 6 protons pumped out
Per glucose
120 protons
120/3 = 40
+ 4ATP from
glycolysis and
TCA
44 ATP! Why only 38?
ATP synthase is nearly 100% efficient: so why
do you not get 1ATP per 3 H+?
H+ transported
-0.3ATP
2.7 ATP/
NADH
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?
pH 8
pH 5
The c-subunits of F0 ATPase
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 •O2Peroxide O22Hydroxyl radical •OH
Where does this occur?
O2
O
O
O2-
H3CO
CH3
H3CO
CH3
H3CO
R
H3CO
R
O
O
H
B
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