Citric acid cycle Electron transport...Overview of the citric acid cycle eight reactions of the...
Transcript of Citric acid cycle Electron transport...Overview of the citric acid cycle eight reactions of the...
Citric acid cycle
Electron transport
Citric acid cycle addendum to glycolysis
it continues to oxidize pyruvate to carbondioxide
The electrons obtained by oxidation of glycolytic substrates are ultimately transferred to oxygen.
central pathway that also serves to oxidize amino and fatty acids
Fatty acids are broken down to acetyl-CoA and are used as a major energy source.
can be considered the „hub“ of metabolic chemistry
Overview of the citric acid cycle
eight reactions of the citric acid cycle serve to convert acetyl-
CoA into two molecules of CO2
energy released during that process is conserved in the:
three molecules of NADH
one FADH2
one „high-energy“ compound (GTP)
first recognized by Hans Krebs in 1937
Krebs cycle or tricarboxylic acid cycle
In eukaryotes - in the mitochondrion
All substrates and enzymes must be made in or transported
to the mitochondrion
intermediates are also intermediates of other pathways
Example: oxaloacetate is used for gluconeogenesis
The Citric Acid Cycle
first intermediate of the cycle is citrate
Synthesis of acetyl-coenzyme A The „fuel“ for the citric acid cycle is acetyl-CoA
„high-energy“ thioester compound
derived from the degradation of fatty acids and some amino acids
end product of glycolysis – pyruvate- source for acetyl CoA
Pyruvate is converted to acetyl-CoA by the pyruvate dehydrogenase multienzyme complex – 3 enzymes:
• pyruvate dehydrogenase (E1)
• dihydrolipoyl transacetylase (E2)
• dihydrolipoyl dehydrogenase (E3)
The pyruvate dehydrogenase multienzyme
complex
In E.coli: The core of the complex is made of 24 E2 proteins
forming a cube surrounded by 24 E1 and 12 E3 proteins
advantage of multienzyme complexes:
• short travelling distance of substrates enhances reaction rates
• substrate channeling minimizes loss of substrates due to side reactions
• coordinated control of reactions
Coenzymes and prosthetic groups involved in the pyruvate
dehydrogenase reaction
Enzymes of the citric acid cycle: 1.
Citrate synthase
Another example of acid-base catalysis
one of the few enzymes that can form a carbon-carbon bond
without the assistance of metal ion catalysis
2. Aconitase
Aconitase catalyzes the reversible isomerization of citrate to
isocitrate
contains a so-called iron-sulfur complex - redox cofactor
used in many biochemical transformations
aconitase-catalyzed reaction is a redoxneutral isomerization
iron-sulfur complex stabilizes a transiently occurring
hydroxyl anion
Loss of an iron inactivates the enzyme
3. Isocitrate dehydrogenase
catalyzes the oxidative decarboxylation of isocitrate to α-
ketoglutarate
produces the first CO2 and NADH in the citric acid cycle
similar to the phosphogluconate dehydrogenase reaction in
the pentose phosphate pathway.
4. α-Ketoglutarate dehydrogenase
catalyzes the oxidative decarboxylation of an α-keto acid
producing the second CO2 and NADH of the citric acid
cycle
the second CO2 leaving the cycle is not derived from the
acetyl-CoA that entered the cycle
reaction is chemically identical to the pyruvate
dehydrogenase reaction which produces acetyl-CoA.
5. Succinyl-CoA synthetase
couples the cleavage of the „high-energy“ succinyl-CoA to the
generation of a „high-energy“ nucleotide triphosphate
Goes in 3 steps
6. Succinate dehydrogenase
The remainder of the citric acid cycle is concerned with the
reformation of oxaloacetate from succinate
First of these „rebuilding“ reactions is the dehydrogenation of
succinate to fumarate
Succinate dehydrogenase is the only membrane-bound
protein of the citric acid
feeds the electrons of the reduced FAD directly into the
electron transport chain
7. Fumarase
Fumarase catalyzes the hydration of fumarate to malate
8. Malate dehydrogenase
The last step of the citric acid cycle
dehydrogenation of malate to recover oxaloacetate
NAD+- dependent reaction
reaction is very similar to the lactate and alcohol
dehydrogenase reaction
Endergonic
Overview of ATP generation oxidation of one acetyl group
releases 8 electrons which are used to reduce 3 NAD+ and 1 FAD molecule
electrons are passed on to the electron transport chain where 3 molecules of ATP are generated per NADH and 2 per FADH2
total of 12 molecules of ATP are generated per turn of the citric acid cycle (24 per one molecule of glucose)
Total - 38 molecules of ATP are produced under aerobic conditions from 1 molecule of glucose
Regulation of the citric acid cycle Exergonic reaction steps are possible regulatory points
citrate synthase, isocitrate dehydrogenase and α- ketoglutarate dehydrogenase are subject to regulation
Factors which regulate the activity of enzymes:
1. substrate availability
2. product inhibition
3. competitive feedback inhibition
Acetyl-CoA, citrate and succinyl-CoA act as product inhibitors
ATP and succinyl-CoA act as competitive feedback regulators
NADH plays a major role as a product inhibitor as well as a negative feedback regulator
Relationships to other pathways
Citric acid metabolites are also raw materials for biosynthetic
reactions
Example 1: oxaloacetate for gluconeogenesis
Dual nature of cycle – described as amphibolic
Example 2: acetyl-CoA which is required for fatty acid
biosynthesis in the cytosol
acetyl-CoA cannot cross the mitochondrial membrane -
generated from citrate by the action of ATP-citrate lyase
The glyoxylate cycle
Plants - enzymes that allow the net conversion of acetyl-CoA
to oxaloacetate
can be used for gluconeogenesis
„derivation“ of the citric acid cycle requires two additional
enzymes: isocitrate lyase and malate synthase
so-called glyoxylate cycle operates in two cellular
compartments: the mitochondrion and the glyoxysome
(specialized plant peroxisome)
Net result - conversion of acetyl-CoA to glyoxylate instead of
two CO2
The glyoxylate cycle is essential to
germinating plant seeds
glyoxysomes in germinating seeds - surrounded by lipid bodies
contain triglycerides which are eventually degraded to acetyl-
CoA
converted to glyoxylate and further to oxaloacetate by means of
malate synthase and malate dehydrogenase
Oxaloacetate - used in the reactions leading to the net synthesis
of glucose (gluconeogenesis)
Summary
Citric acid cycle
Glyoxylate cycle
ELECTRON TRANSPORT AND
OXIDATIVE PHOSPHORYLATION
metabolic oxidation of fuel compounds, such as glucose can be summarized as follows:
C6H12O6 + 6O2 -> 6CO2 + 6H2O
Released electrons are not directly transferred to molecular oxygen
first transferred to produce NADH and FADH2
the process of metabolic fuel oxidation can be broken up in two „half“ reactions:
C6H12O6 + 6 H2O -> 6 CO2 + 24 H+ + 24 e-
6 O2 + 24 H+ + 24 e- -> 12 H2O
NADH and FADH2 pass the electrons on to the electron-transport chain in the mitochondrial inner membrane
Functions of the electron-transport
chain
NADH and FADH2 transfer electrons to an electron
acceptor
become reoxidized
oxidized coenzymes reenter the substrate oxidation reactions
of glycolysis and the citric acid cycle
electrons are passed „down“ in a sequence of redox reactions
(10 different redox centers in 4 protein complexes) -
reduce molecular oxygen to water
During the electron transfer processes - protons are pumped
across the inner membrane and generate a proton gradient
free energy of this electrochemical gradient - for the
synthesis of ATP from ADP and phosphate
through oxidative phosphorylation
The Mitochondrion
Greek: mitos, thread + chondros, granule
Contains:
enzymes of the citric acid cycle (including pyruvate
dehydrogenase),
enzymes required for fatty acid oxidation
enzymes and redox proteins involved in electron transport
and oxidative phosphorylation
referred to as the cell’s „power plant“
The Mitochondrion
has about the size of a bacterium (0.5 x 1.0 μm)
eukaryotic cell contains 2000 mitochondria - take up 20%
of the cell’s volume
has a smooth outer membrane
“clefts” of inner membrane - site of the „respiratory activity“
Electron transport
Most of the electron carriers are located in the inner mitochondrial
membrane
form complex integral membrane proteins
electrons are passed from NADH with a redox potential of
-0.315 V to redox centers of gradually more positive redox potential
until the electrons end up on molecular oxygen
Oxidation of NADH in mitochondria electrons from NADH - passed
through 4 different protein complexes
This breaks the free energy change into three smaller parcels
Each contribute to the synthesis of ATP (oxidative phosphorylation)
the oxidation of NADH yields approx. 3 ATP (under standard biochemical conditions)
efficiency of the process is ~42%.
under physiological conditions the efficiency is ~70%.
Complex I: NADH-Coenzyme Q oxidoreductase
largest protein complex in the mitochondrial inner membrane
consist of 43 polypeptides
contains a flavin mononucleotide (FMN)
Six-seven iron-sulfur clusters („non-heme iron“)
Each iron is coordinated by four sulfur atoms – tetrahedral fashion
The iron can undergo one electron reduction/oxidation (Fe3+/ Fe2+)
FMN and CoQ can undergo one and two electron reduction/oxidation
CoQ can move freely within the membrane (compared to FMN which is tightly bound)
Complex II: Succinate-CoQ oxidoreductase
citric acid cycle enzyme succinate dehydrogenase transfers
the electrons to a covalently bound FAD to generated FADH2
In complex II, the electrons are then passed on to one [4Fe-
4S] cluster, two [2Fe-2S] clusters and one cytochrome b560
redox potential difference between succinate and CoQ is not
sufficient to drive ATP synthesis
However, complex II is important as it allows the entry of
high-potential electrons into the electron-transport chain
Cytochromes are electron-transport
heme proteins
are redox active proteins that contain a heme group
heme-bound iron alternates between the ferric and ferrous
state during electron transport
In reduced states the various cytochromes have
distinguishable absorbance spectra
Complex III: CoQ-cytochrome c oxidoreductase
known as cytochrome bc1
passes the electrons from CoQ to cytochrome c
contains two b-type cytochromes, one cytochrome c1 and one
[2Fe-2S] cluster
known as the Rieske center - bound to the iron-sulfur
protein, ISP
The Q-cycles
Electrons from CoQH2 are transferred to cytochrome c in
two so-called Q-cycles
CoQ serves directly as the carrier of protons from the matrix
to the intermembrane space
two cycles pump two protons each (two reactions)
Complex IV: Cytochrome c oxidase takes up the electrons from four reduced cytochrome c
molecules
Used in the reduction of one dioxygen molecule
contains four redox centers:
heme a, heme a3, a copper atom
(CuB) and a pair of copper atoms
(CuA)
Mechanism of cytochrome c oxidase
Electron transfer in cytochrome c oxidase is linear
Dioxygen binds near cytochrome a3 and is reduced to water
The four protons required to generate two molecules of
water originate in the mitochondrial matrix
Additionally, protons are translocated from matrix to the
intermembrane space
for every pair of electrons, two protons are pumped across
the membrane
Oxidative phosphorylation free energy released by the electron-transport chain - stored in the
electrochemical potential of the inner mitochondrial membrane
potential is used by ATP-synthase (complex V) for the highly
endergonic synthesis of ATP
the electrochemical gradient is discharged by ATP-synthase and
this exergonic reaction drives ATP synthesis (chemiosmotic
theory)
Theory- based on the believe that electron-transport results in the
production of a „high energy intermediate“ whose breakdown
yields ATP
search for such an intermediate was unsuccessful
The chemiosmotic theory Explains why:
Oxidative phosphorylation requires intact inner mitochondrial
membranes
The inner mitochondrial membrane is impermeable to H+, K+,
OH- and Cl- (free diffusion of these ions would undo the
electrochemical potential built up by the electron-transport chain)
An electrochemical potential is measurable across the inner
mitochondrial membrane
Compounds that make the inner mitochondrial membrane -
„uncouple“ electron transport from oxidative phosphorylation
ATP synthase (complex V)
membrane-bound multisubunit protein
composed of two functional units F1 and F0
F0 is a water-insoluble transmembrane proton channel
F1 on the other hand is a peripheral water-soluble protein
composed of five types of subunits
This multisubunit protein can be readily dissociated from the
F0 part and is able of ATP hydrolysis but not of ATP synthesis
The binding change mechanism generation of ATP by pumping of protons - broken into three phases
1. The F0 subunit translocates the protons
2. The F1 subunit carries out the synthesis of ATP from ADP and Pi
3. The physical interaction of the F0 and F1 subunit harnesses the exergonic transport of protons to the synthesis of ATP
In the so-called binding change mechanism the three αβ-subunits of the F1 unit exists in three different conformations:
• the L state binds the substrates loosely
• the T state binds the substrates tightly
• the O state binds the substrates very loosely or not at all
Uncoupling of electron transport and
oxidative phosphorylation
Compounds that increase the membrane permeability of
protons - give rise to uncoupling of electron transport and
oxidative phosphorylation
Uncouplers dissipate the proton gradient and disable ATP
synthesis
Lipophilic molecules that freely move in the membrane and
hence shuttle protons across the membrane
Summary
Electron transport chain
Complex I-IV
Oxidative phosphorylation
Binding-change mechanisms
QUIZ 3
Thursday 19.12.2019
10:00
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Chapters: from midterm until today