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

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