Download Respiration involves the oxidation of glucose and other compounds

Oxidation of glucose and fatty acids to CO2
Respiration involves the oxidation of glucose and other
compounds to produce energy.
C6H12O6 + 6 02 + 36 Pi + 36 ADP + 36 H+
6 CO2 + 36 ATP + 42 H20
Step I: glycolysis
Glucose + 2ADP
2 pyruvate + 2ATP
key enzyme:
1. phosphofructokinase (step 3 in Figure 1).
• stimulated by high ADP levels
• inhibited by high ATP and citrate.
2. glyceraldehyde 3-phosphate dehydrogenase (step 5).
• can be inhibited by NEM.
Step II: Mitochondria in respiration
Transport of pyruvate into the mitochondria and its
subsequent oxidization by O2 to produce CO2, generating 34
of the 36 ATP molecules produced during respiration.
1. Structure and function of mitochondria
(1). Generation of acetyl CoA.
Ethanol, a substrate that you will be testing in today’s lab, can also be
converted to acetyl CoA.
(2). Kreb’s cycle/citric acid cycle.
mitochondria matrix
Acetyl CoA is oxidized to yield CO2
and reduced coenzymes.
Three reactions in the Krebs cycle
reduce the coenzyme NAD+ to NADH,
and one reduces the coenzyme FAD to
FADH2.
The reduced coenzymes (NADH and FADH2)
store the energy released in glucose oxidation.
succinate, malate
3. Oxidative phosphorylation
Oxidative phosphorylation is the process by which the energy
stored in NADH and FADH2 is used to produce ATP.
A. Oxidation step----electron transport chain
1
O
2 2
1
FADH + O2
2
NADH + H+ +
B. Phosphorylation step
NAD+ + H2O
FAD + H2O
(1). Generation of acetyl CoA.
Ethanol, a substrate that you will be testing in today’s lab, can also be
converted to acetyl CoA.
Electrons are moved from
molecules with low
reduction potential (low
affinity for electrons) to
molecules with
successively higher
reduction potential (higher
electron affinity).
Electrons are transferred through four large protein complexes and two
smaller proteins, each of which has an electron carrier group.
At complex I, III and IV, the energy released during electron transfer is
used to move H+ ions from the matrix to the inner mitochondrial space,
generating a gradient of protons across the membrane.
B. Phosphorylation step
Peter Mitchell’s chemiosmotic hypothesis:
The energy stored in an electrochemical gradient across the
inner mitochondrial membrane could be coupled to ATP
synthesis.
F0F1 ATP synthase
Movement of protons down the
electrochemical gradient through a channel
between the a and c subunits releases
energy that is coupled to the rotation of the
c,  and  subunits. This rotation causes a
conformational change in the  and 
subunits that promotes synthesis of ATP
from ADP and Pi.
Other factors involved in oxidative phosphorylation
Transporters and shuttles move small molecules across the inner
mitochondrial membrane.
Transporters include the ATP/ADP antiporter, which transports ADP into
the matrix and ATP out of the matrix.
In addition there are separate transporters for Pi and pyruvate.
(1). Generation of acetyl CoA.
Ethanol, a substrate that you will be testing in today’s lab, can also be
converted to acetyl CoA.
Inhibitors and substrates
Inhibitors:
Rotenone (also amytal): inhibits complex I.
Antimycin A: inhibits cytochrome c.
Sodium azide: inhibits complex IV.
Oligomycin: inhibits the F0F1 ATP synthase.
Atractyloside: inactivates the ATP/ADP antiporter.
Substrates:
glutamate malate: reduces NAD+ to NADH
Succinate: reduces FAD to FADH2
ascorbate/TMPD enters the electron transport chain in
complex IV.
• Respiratory Control
The rate of oxidative phosphorylation depends on the levels ADP/ATP in
mitochondria– ie. oxidation of NADH and FADH2 only occurs if there is
a there is ADP and Pi available to generate ATP.
Oxidation of
NADH and FADH2
H+ gradient
ADP/ATP
levels
Examining the phenomenon of respiratory control:
Inhibitors: Atractyloside, Oligomycin
CaCl2: stimulates oxidative phosphorylation and ATP production.
Uncouplers--DNP
DNP acts to shuttle H+ across the inner mitochondrial membrane,
causing a dissipation of the H+ gradient, thus uncouples the process of
oxidation and phosphorylation.
The uncouplers overcome respiratory control, but do not stop
oxidation/the electron transport chain. The energy released from the
oxidation of NADH/FADH2 is dissipated as heat. An uncoupler called
thermogenin occurs naturally in brown-fat tissue and functions to
uncouple oxidation and phosphorylation, enhancing heat generation.
This week’s experiments: measuring O2 consumption
1. Yeast respiration lab:
Goal: learn how to measure O2 consumption.
Compare O2 consumption by normal and starved yeast.
2. Mitochondria respiration lab:
Examine the effects of various inhibitors and substrates on the rate
of respiration.
Determine the identity of your unknown (think what substrates you need
to add and in what order together with the unknown).