Download Document

Bioenergetics
The tiny hummingbirds can store enough
fuel to fly a distance of 500 miles
without resting. This achievement is
possible because of the ability to convert
fuels into the cellular energy currency,
ATP.
• Metabolism - the entire network of chemical
reactions carried out by living cells. Metabolism
also includes coordination, regulation and energy
requirement.
• Metabolites - small molecule intermediates in the
degradation and synthesis of polymers
Most organism use the same general pathway for extraction
and utilization of energy.
All living organisms are divided into two major classes:
Autotrophs – can use atmospheric carbon dioxide as a sole
source of carbon for the synthesis of macromolecules.
Autotrophs use the sun energy for biosynthetic purposes.
Heterotrophs – obtain energy by ingesting complex
carbon-containing compounds.
Heterotrophs are divided into aerobs and anaerobs.
A sequence of reactions that has a specific purpose
(for instance: degradation of glucose, synthesis of
fatty acids) is called metabolic pathway.
Metabolic pathway may be:
(a) Linear
(b) Cyclic
(c) Spiral pathway
(fatty acid
biosynthesis)
Metabolic pathways can be grouped into two paths –
catabolism and anabolism
Catabolic reactions - degrade molecules to create
smaller molecules and energy
Anabolic reactions - synthesize molecules for cell
maintenance, growth and reproduction
Catabolism is characterized by oxidation reactions and
by release of free energy which is transformed to ATP.
Anabolism is characterized by reduction reactions
and by utilization of energy accumulated in ATP
molecules.
Catabolism and anabolism are tightly linked together
by their coordinated energy requirements: catabolic
processes release the energy from food and collect it
in the ATP; anabolic processes use the free energy
stored in ATP to perform work.
Anabolism and catabolism are coupled by energy
Metabolism Proceeds by Discrete Steps
• Multiple-step pathways
permit control of energy
input and output
Single-step vs multi-step
pathways
• Catabolic multi-step
pathways provide energy
in smaller stepwise
amounts
• Each enzyme in a multistep pathway usually
catalyzes only one single
step in the pathway
A multistep
• Control points occur in enzyme
multistep pathways
pathway
Metabolic Pathways Are Regulated
• Metabolism is highly regulated to permit organisms
to respond to changing conditions
• Most pathways are irreversible
• Flux - flow of material through a metabolic pathway
which depends upon:
(1) Supply of substrates
(2) Removal of products
(3) Pathway enzyme activities
Levels of Metabolism Regulation
1. Nervous system.
2. Endocrine system.
3. Interaction between organs.
4. Cell (membrane) level.
5. Molecular level
Feedback inhibition
• Product of a pathway controls the rate of its own
synthesis by inhibiting an early step (usually the first
“committed” step (unique to the pathway)
Feed-forward activation
• Metabolite early in the pathway activates an enzyme
further down the pathway
Stages of metabolism
Catabolism
Stage I. Breakdown of macromolecules (proteins,
carbohydrates and lipids to respective building
blocks.
Stage II. Amino acids, fatty acids and glucose
are oxidized to common metabolite (acetyl CoA)
Stage III. Acetyl CoA is oxidized in citric acid
cycle to CO2 and water. As result reduced
cofactor, NADH2 and FADH2, are formed which
give up their electrons. Electrons are transported
via the tissue respiration chain and released
energy is coupled directly to ATP synthesis.
Fatty Acids
Acetyl Co A
Pyruvate
Glucose
Citric acid
cycle supplies
NADH and
FADH2 to the
electron
transport
chain
Amino Acids
Reduced coenzymes NADH and FADH2 are
formed in matrix from:
(1) Oxidative decarboxilation of pyruvate to
acetyl CoA
(2) Aerobic oxidation of acetyl CoA by the
citric acid cycle
(3) Oxidation of fatty acids and amino acids
The NADH and FADH2 are energy-rich
molecules because each contains a pair of
electrons having a high transfer potential.
The reduced and oxidized forms of NAD
The reduced and oxidized forms of FAD
Electrons of NADH or FADH2 are used to
reduce molecular oxygen to water.
A large amount of free energy is liberated.
The electrons from NADH and FADH2 are not
transported directly to O2 but are transferred
through series of electron carriers that undergo
reversible reduction and oxidation.
The flow of electrons through carriers leads to
the pumping of protons out of the mitochondrial
matrix.
The resulting
distribution of
protons
generates a pH
gradient and a
transmembrane
electrical
potential that
creates a
protonmotive
force.
ATP is synthesized when protons flow back to the
mitochondrial matrix through an enzyme complex
ATP synthase.
The oxidation of fuels and the phosphorylation of
ADP are coupled by a proton gradient across the
inner mitochondrial membrane.
Oxidative
phosphorylation is
the process in which
ATP is formed as a
result of the
transfer of electrons
from NADH or
FADH2 to O2 by a
series of electron
carriers.
OXIDATIVE PHOSPHORYLATION IN
EUKARYOTES TAKES PLACE IN MITOCHONDRIA
Two membranes:
outer membrane
inner membrane (folded into
cristae)
Two compartments:
(1) the intermembrane space
(2) the matrix
The outer membrane
is permeable to small
molecules and ions
because it contains
pore-forming protein
(porin).
The inner membrane
is impermeable to ions
and polar molecules.
Contains transporters
(translocases).
Location of mitochondrial complexes
• Inner mitochondrial membrane:
Electron transport chain
ATP synthase
• Mitochondrial matrix:
Pyruvate dehydrogenase complex
Citric acid cycle
Fatty acid oxidation
THE ELECTRON TRANSPORT CHAIN
Series of enzyme complexes (electron carriers)
embedded in the inner mitochondrial membrane,
which oxidize NADH2 and FADH2 and transport
electrons to oxygen is called respiratory
electron-transport chain (ETC).
The sequence of electron carriers in ETC
NADH
FMN
Fe-S
succinate FAD Fe-S
Co-Q
Fe-S
cyt b
cyt c1 cyt c
cyt a
cyt a3
O2
High-Energy Electrons: Redox Potentials
and Free-Energy Changes
In oxidative phosphorylation, the electron
transfer potential of NADH or FADH2 is
converted into the phosphoryl
transfer
potential of ATP.
Phosphoryl transfer potential is G°' (energy
released during the hydrolysis of activated phosphate compound). G°' for ATP = -7.3 kcal mol-1
Electron transfer potential is expressed as E'o,
the (also called redox potential, reduction
potential, or oxidation-reduction potential).
E'o (reduction potential) is a measure of how easily a
compound can be reduced (how easily it can accept
electron).
All compounds are compared to reduction potential of
hydrogen wich is 0.0 V.
The larger the value of E'o of a carrier in ETC the better
it functions as an electron acceptor (oxidizing factor).
Electrons flow through the ETC components spontaneously
in the direction of increasing reduction potentials.
E'o of NADH = -0.32 volts (strong reducing agent)
E'o of O2 = +0.82 volts (strong oxidizing agent)
NADH
FMN
Fe-S
succinate FAD Fe-S
Co-Q
Fe-S
cyt b
cyt c1 cyt c
cyt a
cyt a3
O2
Important characteristic of ETC is the amount of
energy released upon electron transfer from one
carrier to another.
This energy can be calculated using the formula:
Go’=-nFE’o
n – number of electrons transferred from one carrier
to another;
F – the Faraday constant (23.06 kcal/volt mol);
E’o – the difference
in reduction potential between two carriers.
When two electrons pass from NADH to O2 :
Go’=-2*96,5*(+0,82-(-0,32)) = -52.6 kcal/mol
THE RESPIRATORY CHAIN
CONSISTS OF FOUR
COMPLEXES
Components of electrontransport chain are arranged
in the inner membrane of
mitochondria in packages
called respiratory
assemblies (complexes).
FMN
II
III
IV
III
I
NADH
I
Fe-S
II
succinate FAD Fe-S
Co-Q
Fe-S
cyt b
IV
cyt c1 cyt c
cyt a
cyt a3
O2
Complexes I-IV
• Mobile coenzymes: ubiquinone
(Q) and cytochrome c serve as
links between ETC complexes
• Complex IV reduces O2 to water
Complex I (NADH-ubiquinone oxidoreductase)
Transfers electrons from NADH to Co Q (ubiquinone)
Consist of:
- enzyme NADH dehydrogenase (FMN - prosthetic
group) - iron-sulfur clusters.
NADH reduces FMN to FMNH2.
Electrons from FMNH2 pass to a Fe-S clusters.
Fe-S proteins convey electrons to ubiquinone.
QH2 is formed.
The flow of two electrons from NADH to coenzym Q leads
to the pumping of four hydrogen ions out of the matrix.
Complex II (succinate-ubiquinon oxidoreductase)
Transfers electrons from succinate to Co Q.
Form 1 consist of:
- enzyme succinate dehydrogenase (FAD –
prosthetic group)
- iron-sulfur clusters.
Succinate reduces FAD to FADH 2.
Then electrons pass to Fe-S proteins
which reduce Q to QH
Form 2 and 3 contains2 enzymes acyl-CoA dehydrogenase
(oxidation of fatty acids) and glycerol phosphate dehydrogenase
(oxidation of glycerol) which direct the transfer of electrons
from acyl CoA to Fe-S proteins.
Complex II does not contribute to proton gradient.
All electrons must pass through the ubiquinone (Q)ubiquinole (QH2) pair.
Ubiquinone Q:
- lipid soluble molecule,
- smallest and most hydrophobic of all the
carriers
- diffuses within the lipid bilayer
- accepts electrons from I and II
complexes and passes them to complex III.
Complex III (ubiquinol-cytochrome c oxidoreductase)
Transfers electrons from ubiquinol to cytochrome c.
Consist of: cytochrome b, Fe-S clusters and cytochrome c1.
Cytochromes –
electron transferring proteins containing a heme prosthetic
group (Fe2+  Fe3+).
Oxidation of one QH2 is accompanied by the translocation
of 4 H+ across the inner mitochondrial membrane. Two H+
are from the matrix, two from QH2
Complex IV (cytochrome c oxidase)
Transfers electrons from cytochrome c to O2.
Composed of: cytochromes a and a3.
Catalyzes a four-electron reduction of molecular oxygen (O2) to
water (H2O):
O2 + 4e- + 4H+  2H2O
Translocates 2H+ into the intermembrane space
The four protons used for the
production of two molecules of
water come from the matrix.
The consumption of these four
protons contributes to the
proton gradient.
Cytochrome c oxidase pumps
four additional protons from
the matrix to the cytoplasmic
side of the membrane in the
course of each reaction cycle
(mechanism under study).
Totally eight protons are
removed from the matrix in
one reaction cycle (4 electrons)
Cellular Defense Against Reactive
Oxygen Species
If oxygen accepts four electrons - two molecules of H2O are
produced
single electron - superoxide anion (O2.-)
two electrons – peroxide (O22-).
O2.-, O22- and, particularly, their reaction products are harmful to
cell components - reactive oxygen species or ROS.
DEFENSE
superoxide dismutase (manganese-containing version in
mitochondria and a copper-zinc-dependent in cytosol)
O2.- + O2.- + 2H+ = H2O2 + O2
catalase
H2O2 + H2O2 = O2 + 2 H2O
A PROTON GRADIENT POWERS
THE SYNTHESIS OF ATP
The transport of electrons from NADH or FADH2
to O2 via the electron-transport chain is exergonic
process:
NADH + ½O2 + H+  H2O + NAD+
FADH2 + ½O2  H2O + FAD+
Go’ = -52.6 kcal/mol for NADH
-36.3 kcal/mol for FADH2
How this process is coupled to the synthesis of ATP
The Chemiosmotic Theory
• Proposed by Peter Mitchell in the
1960’s (Nobel Prize, 1978)
• Chemiosmotic theory: electron
transport and ATP synthesis
are coupled by a proton
gradient across the inner
mitochondrial membrane
Mitchell’s postulates for chemiosmotic theory
1. Intact inner mitochondrial membrane is required
2. Electron transport through the ETC generates a proton
gradient
3. ATP synthase catalyzes the phosphorylation of ADP in a
reaction driven by movement of H+ across the inner
membrane into the matrix
Overview of oxidative phosphorylation
+
+
+
-
-
-
+
+
+
-
As electrons flow through complexes of ETC, protons are
translocated from matrix into the intermembrane space.
The free energy stored in the proton concentration gradient is
tapped as protons reenter the matrix via ATP synthase.
As result ATP is formed from ADP and Pi.
REGULATION OF OXIDATIVE
PHOSPHORYLATION
Coupling of Electron Transport with ATP Synthesis
Electron transport is tightly coupled to phosphorylation.
ATP can not be synthesized by oxidative phosphorylation
unless there is energy from electron transport.
Electrons do not flow through the electron-transport chain
to O2 unless ADP is phosphorylated to ATP.
Important substrates: NADH, O2, ADP
Intramitochondrial ratio ATP/ADP is a control mechanism
High ratio inhibits oxidative phosphorylation as ATP
allosterically binds to a subunit of Complex IV
Respiratory control
The most important factor in determining the rate of
oxidative phosphorylation is the level of ADP.
The regulation of the rate of oxidative
phosphorylation by the ADP level is called respiratory
control
Uncoupling of Electron Transport with ATP Synthesis
Uncoupling of oxidative phosphorylation generates heat to maintain
body temperature in hibernating animals, in newborns, and in mammals
adapted to cold.
Brown adipose tissues is specialized for thermogenesis.
Inner mitochondrial membrane contains uncoupling protein (UCP), or
thermogenin.
UCP forms a pathway for the flow of protons from the cytosol to the
matrix.
Uncouplers
• Uncouplers are lipid-soluble aromatic weak acids
• Uncouplers deplete proton gradient by transporting
protons across the membrane
2,4-Dinitrophenol: an uncoupler
• Because the negative charge is delocalized over the ring,
both the acid and base forms of DNP are hydrophobic
enough to dissolve in the membrane.
Specific inhibitors of electron
transport chain and ATP-synthase
Specific inhibitors of electron
transport are invaluable in revealing
the sequence of electron carriers.
Rotenone and amytal block electron
transfer in Complex I.
Antimycin A interferes with electron
flow thhrough Complex III.
Cyanide, azide, and carbon monoxide
block electron flow in Complex IV.
ATP synthase is inhibited by
oligomycin which prevent the influx of
protons through ATP synthase.
ATP Yield
Ten protons are pumped out of the matrix during
the two electrons flowing from NADH to O2
(Complex I, III and IV).
3
Six protons are pumped out of the matrix during
the two electrons
flowing from FADH2 to O2
4
(Complex 4III and IV).
2
Translocation of 3H+ required by ATP synthase for each ATP produced
1 H+ needed for transport of Pi.
Net: 4 H+ transported for each ATP synthesized
For NADH: 10 H+/ 4H+) = 2.5 ATP
For FADH2: 6 H+/ 4 H+ = 1.5 ATP