Notes #3 - The Stages of Cellular Respiration

Μίτος “mitos” = “thread”, Χονδρίον “chondros” = “granule”. Carl Benda

(1857-1933) coined the term mitochondria in 1898.

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Glycolysis
Where does Glycolysis occur?
- Glycolysis occurs in the cytoplasm where one 6-carbon molecule of glucose is oxidized
to generate two 3-carbon molecules of pyruvate. The fate of pyruvate depends on the
presence or absence of mitochondria and oxygen in the cells.
- It occurs whether or not O2 is present. Glycolysis can be anaerobic.
What happens in Glycolysis?
- Splitting of sugar from glucose to pyruvate.
- Glycolysis (sugar splitting) breaks down glucose into two molecules of pyruvate.
- It has two major phases: The energy investment phase (1st
phase, investment phase, consumes energy, steps 1-5) and
the energy payoff phase (2nd phase, pay off phase,
produces energy, steps 6 – 10).
Net Reaction of Glycolysis
- Glucose + 2 NAD+ + 2 ADP + 2 Pi  2 Pyruvate + 2
NADH + 2 H+ + 2 ATP + 2 H2O.
1st Phase of Glycolysis
- You need to “invest” energy first in order to gain much
more energy later.
- It is endothermic, it consumes energy.
- 2 ATP will be used.
- Step 1: Phosphorylation to Glucose and turns it into
Glucose-6-Phosphate.

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  Glucose enters the cell and is phosphorylated by the enzyme hexokinase, which
transfers a phosphate group from ATP to the sugar. The charge of the phosphate
group traps the sugar in the cell because the plasma membrane is impermeable to
large ions. Phosphorylation also makes glucose more chemically reactive.
 Uses Hexokinase (The word hexo means six. It catalyzes the transfer of a phosphate

group from an ATP to an acceptor molecule).

- Step 2: Glucose 6-phosphate is converted to its isomer, fructose-6-phosphate.

 An isomer is each of two or more compounds with the same formula but a
different arrangement of atoms in the molecule and different properties.
 Since Glucose 6-Phosphate and Fructose-6-phosphate are isomers, it uses
phosphoglucoisomerase.
 Isomerase, any one of a class of enzymes that catalyze reactions involving a
structural rearrangement of a molecule.

- Step 3: Phosphofructokinase transfers a phosphate group from ATP to the sugar,

investing another molecule of ATP in glycolysis. So far, 2 ATP have been used. With
phosphate groups on its opposite ends, the sugar is now ready to be split in half. This is a
key step for the regulation of glycolysis; phosphofructokinase is allosterically regulated
by ATP and its products.
 Fructose-6-phosphate becomes Fructose-1-6-biphosphate

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 Step 4: This is the reaction from which glycolysis gets its name. Fructose bisphosphate
aldolase cleaves the sugar molecule into two different three-carbon sugars:

dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. These two sugars are

isomers of each other.

- Step 5: Isomerase catalyzes the reversible conversion between the two three-carbon
sugars. This reaction never reaches equilibrium in the cell because the next enzyme in
glycolysis uses only glyceraldehyde-3-phosphate as its substrate (and not
dihydroxyacetone phosphate). This pulls the equilibrium in the direction of
glyceraldehyde-3-phosphate, which is removed as fast as it forms. Thus, the net result of

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 steps 4 and 5 is cleavage of a six-carbon sugar into two molecules of glyceraldehyde-3-
phosphate; each will progress through the remaining steps of glycolysis.

2nd Phase of Glycolysis

- It produces 4 ATP and 2 NADH. Which results in a net gain of 2 ATP and 2 NADH
(because 2 ATP were consumed during the 1st phase).
- Step 6: Triose phosphate dehydrogenase catalyzes two sequential reactions while it holds
glyceraldehyde-3-phosphate in its active site. First, the sugar is oxidized by the transfer
of electrons and H+ to NAD+, forming NADH (a redox reaction). This reaction is very
exergonic, and the enzyme uses the released energy to attach a phosphate group to the
oxidized substrate, making a product of very high potential energy. The source of the
phosphates is the pool of inorganic phosphate ions that are always present in the cytosol.
Notice that the coefficient 2 precedes all molecules in the energy payoff phase; these
steps occur after glucose has been split into two-three carbon sugars (step 4).

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- Step 7: Glycolysis produces some ATP by substrate-level phosphorylation. The
phosphate group added in the previous step is transferred to ADP in an exergonic
reaction. For each glucose molecule that began glycolysis, step 7 produces 2 ATP, since
every product after the sugar-splitting step (step 4) is doubled. Recall that 2 ATP were
invested to get sugar ready for splitting; this ATP debt has now been repaid. Glucose has
been converted to two molecules of 3-phosphoglycerate, which is not a sugar. The
carbonyl group that characterizes a sugar has been oxidized to a carboxyl group (---
COO-), the hallmark of an organic acid. The sugar was oxidized in step 6, and now the

energy made available by that oxidation has been used to make ATP.

- Step 8: Phosphoglyceromutase relocates the remaining phosphate group, preparing the

substrate for the next reaction.

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- Step 9: Enolase causes a double bond to form in the substrate by extracting a water
molecule, yielding phosphoenolpyruvate (PEP). The electrons of the substrate are
rearranged in such a way that the resulting phosphorylated compound has a very high
potential energy, allowing step 10 to occur.

- Step 10: The last reaction of glycolysis produces more ATP by transferring the phosphate
group from PEP to ADP, a second instance of substrate-level phosphorylation. Since this
step occurs twice for each glucose molecule, 2 ATP are produced. Overall, glycolysis has
used 2 ATP in the energy investment phase (Steps 1 and 3) and produced 4 ATP in the
energy payoff phase (Steps 7 and 10), for net gain of 2 ATP. Glycolysis has repaid the
ATP investment with 100% interest. Additional energy was stored by step 6 in NADH,
which can be used to make ATP by oxidative phosphorylation if oxygen is present.
Glucose has been broken down and oxidized to two molecules of pyruvate, the end
product of the glycolytic pathway. If oxygen is present, the chemical energy in pyruvate
can be extracted by the citric acid cycle. If oxygen is not present, fermentation may
occur; this will be described later.

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 Note: Magnesium ions (Mg2+) and MgATP2-regulate
the most important glycolytic enzymes, namely
hexokinase, phosphofructokinase, aldolase,
phosphoglycerate kinase, and pyruvate kinase. These
ions are also needed for the Krebs Cycle.

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Citric Acid Cycle/Krebs Cycle/Tricarboxylic Acid Cycle
What happens before Krebs Cycle?
- The pyruvate formed in the cytoplasm (from glycolysis) is brought into the mitochondria
where further reactions take place.
- Pyruvate is first converted to acetyl CoA. Before the citric acid cycle can begin, pyruvate
must be converted to acetyl Coenzyme A (acetyl CoA), which links glycolysis to the
citric acid cycle. This step is carried out by a multienzyme complex that catalyzes three
reactions.

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Krebs cycle Equation/Reaction
- Acetyl CoA + 3 NAD+ + 1 FAD + 1 ADP + 1 Pi → 2 CO2 + 3 NADH + 3 H+ + 1
FADH2 + 1 ATP
What is the Krebs Cycle?
- The citric acid cycle, also called the Krebs cycle, completes the breakdown of pyruvate to
CO2.
- The cycle oxidizes organic fuel derived from pyruvate, generating 1 ATP, 3 NADH, and
1 FADH2 per turn.
- The citric acid cycle has eight steps, each catalyzed by a specific enzyme.
- The acetyl group of acetyl CoA joins the cycle by combining with oxaloacetate, forming
citrate.
- The next seven steps decompose the citrate back to oxaloacetate, making the process a
cycle.
- The NADH and FADH2 produced by the cycle relay electrons extracted from food to the
electron transport chain.

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Where does the Krebs Cycle occur?
The Krebs Cycle occurs in the Mitochondrial Matrix.
The Process of the Krebs Cycle
- One common characteristic in all the enzymes involved in the citric acid cycle is that
nearly all of them require Mg2+.
- Step 1: Condensation of acetyl CoA with oxaloacetate
 The first step of the citric acid cycle is the joining of the four-carbon compound
oxaloacetate (OAA) and a two-carbon compound acetyl CoA.
 The oxaloacetate reacts with the acetyl group of the acetyl CoA and water,
resulting in the formation of a six-carbon compound citric acid, CoA.
 The reaction is catalyzed by the enzyme citrate synthase that condenses the
methyl group of acetyl CoA and the carbonyl group of oxaloacetate resulting in
citryl-CoA which is later cleaved to free coenzyme A and to form citrate.

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- Step 2: Isomerization of citrate into isocitrate.
 Now, for further metabolism, citrate is converted into isocitrate through the
formation of intermediate cis-aconitase.
 This reaction is a reversible reaction catalyzed by the enzyme (aconitase).
 This reaction takes place by a two-step process where the first step involves
dehydration of citrate to cis-aconitase, followed by the second step involving
rehydration of cis-aconitase into isocitrate.

- Step 3: Oxidative decarboxylations of isocitrate

 The third step of the citric acid cycle is the first of the four oxidation-reduction
reactions in this cycle.
 Isocitrate is oxidatively decarboxylated to form a five-carbon compound, α-
ketoglutarate catalyzed by the enzyme isocitrate dehydrogenase.
 This reaction, like the second reaction, is a two-step reaction.
 In the first step, isocitrate is dehydrogenated to oxalosuccinate while the second
step involves the decarboxylation of oxalosuccinate to α-ketoglutarate.
 Both the reactions are irreversible and catalyzed by the same enzyme.
 The first step, however, results in the formation of NADH while the second step
involves the release of CO2.

- Step 4: Oxidative decarboxylation of α-ketoglutarate

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  This step is another one of the oxidation-reduction reactions where α-
ketoglutarate is oxidatively decarboxylated to form a four-carbon compound,
succinyl-CoA, and CO2.
 The reaction irreversible and catalyzed by the enzyme complex α-ketoglutarate
dehydrogenase found in the mitochondrial space.
 This reaction is similar to the oxidative decarboxylation of pyruvate involving the
reduction of NAD+ into NADH.

- Step 5: Conversion of succinyl-CoA into succinate

 In the next step, succinyl-CoA undergoes an energy-conserving reaction in which
succinyl-CoA is cleaved to form succinate.
 This reaction is accompanied by phosphorylation of guanosine diphosphate
(GDP) to guanosine triphosphate (GTP).
 The GTP thus formed then readily transfers its terminal phosphate group to ADP
forming an ATP molecule.
 The reaction is catalyzed by the enzyme, succinyl-CoA synthase.

- Step 6: Dehydration of succinate to fumarate

 Here, the succinate formed from succinyl-CoA is dehydrogenated to fumarate
catalyzed by the enzyme complex succinate dehydrogenase found in the
intramitochondrial space.
 This is the only dehydrogenation step in the citric acid cycle in which NAD+
doesn’t participate.
 Instead, another high-energy electron carrier, flavin adenine dinucleotide (FAD)
acts as the hydrogen acceptor resulting in the formation of FADH2.
 The FADH2 then enters the electron transport chain via the complex II
transferring the electrons to ubiquinone, finally forming 2ATPs.

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- Step 7: Hydration of fumarate to malate
 The fumarate is reversibly hydrated to form L-malate in the presence of the
enzyme fumarate hydratase.
 As it is a reversible reaction, the formation of L-malate involves hydration,
whereas the formation of fumarate involves dehydration.

- Step 8: Dehydrogenation of L-malate to oxaloacetate

 The last step of the citric acid cycle is also an oxidation-reduction reaction where
L-malate is dehydrogenated to oxaloacetate in the presence of L-malate
dehydrogenase, which is present in the mitochondrial matrix.
 This is a reversible reaction involving oxidation of L-malate and reduction of
NAD+ into NADH.
 Oxaloacetate thus formed, allows the repetition of the cycle and NADH formed
participates in the oxidative phosphorylation.
 This reaction completes the cycle.

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A simple flow chart for Krebs Cycle
Oxaloacetate  Citrate  Isocitrate  α-ketoglutarate  Succinyl-CoA  Succinate 
Fumarate  Malate  Repeat the cycle
A list for the enzymes used in the Krebs Cycle
1. Citrate synthase
2. Aconitase
3. Isocitrate dehydrogenase
4. α-ketoglutarate dehydrogenase
5. Succinyl-CoA synthetase
6. Succinate dehydrogenase
7. Fumarase
8. Malate dehydrogenase
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Oxidative Phosphorylation
What happens in Oxidative Phosphorylation?
- This is called oxidative phosphorylation, as the energy to synthesise ATP is derived from
the oxidation of hydrogen carriers.
- Electron Transport Chain and Chemiosmosis occurs here.
- During oxidative phosphorylation, chemiosmosis couples electron transport to ATP
synthesis.
 Following glycolysis and the citric acid cycle, NADH and FADH2 account for
most of the energy extracted from food.
 These two electron carriers donate electrons to the electron transport chain, which
powers ATP synthesis via oxidative phosphorylation.
Where does Oxidative Phosphorylation take place?
- It takes place in the inner-membrane of the mitochondria.
The Pathway of Electron Transport
- The electron transport chain is in the inner membrane (cristae) of the mitochondrion.
- Most of the chainʼs components are proteins, which exist in multiprotein complexes.
- The carriers alternate reduced and oxidized states as they accept and donate electrons.

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 - Electrons drop in free energy as they go down the chain and are finally passed to O2,
forming H2O.
- Electrons are transferred from NADH or FADH2 to the electron transport chain.
- Electrons are passed through a number of proteins including cytochromes (each with an
iron atom) to O2.
- The electron transport chain generates no ATP directly.
- It breaks the large free-energy drop from food to O2 into smaller steps that release energy
in manageable amounts.
- The energy stored in a H+ gradient across a membrane couples the redox reactions of the
electron transport chain to ATP synthesis.
- The H+ gradient is referred to as a protonmotive force, emphasizing its capacity to do
work.
Chemiosmosis: The Energy-Coupling Mechanism
- Electron transfer in the electron transport chain causes proteins to pump H+ from the
mitochondrial matrix to the intermembrane space.
- H+ then moves back across the membrane, passing through the protein complex, ATP
synthase.
- ATP synthase uses the exergonic flow of H+ to drive phosphorylation of ATP.
- This is an example of chemiosmosis, the use of energy in a H+ gradient to drive cellular
work.

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The Simplified Process of the Electron Transport Chain
- Step 1: Generating a Proton Motive Force
 The hydrogen carriers (NADH and FADH2) are oxidised and release high energy
electrons and protons.
 The electrons are transferred to the electron transport chain, which consists of
several transmembrane carrier proteins.
 As electrons pass through the chain, they lose energy – which is used by the chain
to pump protons (H+ ions) from the matrix.
 The accumulation of H+ ions within the intermembrane space creates an
electrochemical gradient (or a proton motive force).

- Step 2: ATP Synthesis via Chemiosmosis

 The proton motive force will cause H+ ions to move down their electrochemical
gradient and diffuse back into matrix.
 This diffusion of protons is called chemiosmosis and is facilitated by the
transmembrane enzyme ATP synthase.
 As the H+ ions move through ATP synthase they trigger the molecular rotation of
the enzyme, synthesizing ATP.

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- Step 3: Reduction of Oxygen
 In order for the electron transport chain to continue functioning, the de-energized
electrons must be removed.
 Oxygen acts as the final electron acceptor, removing the de-energized electrons to
prevent the chain from becoming blocked.
 Oxygen also binds with free protons in the matrix to form water – removing
matrix protons maintains the hydrogen gradient.
 In the absence of oxygen, hydrogen carriers cannot transfer energised electrons to
the chain and ATP production is halted.

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What are the 4 protein complexes for?

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Tables, Net Yield, and Other Terms
An Account of ATP Production by Cellular Respiration
- During cellular respiration, most energy flows in this sequence: glucose → NADH →
electron transport chain → protonmotive force → ATP.
- About 34% of the energy in a glucose molecule is transferred to ATP during cellular
respiration, making about 32 ATP.
- There are several reasons why the number of ATP is not known exactly.
Malate-aspartate Shuttle and Glycerol Phosphate Shuttle
- Malate-aspartate Shuttle – happens in the kidney, liver, and heart.

- Glycerol Phosphate Shuttle – happens in the skeletal, muscle, and brain.

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 - The malate-aspartate shuttle and the glycerol phosphate shuttle act to transfer reducing
equivalents from NADH in the cytosol to the mitochondria since the inner mitochondrial
membrane is impermeable to NADH and NAD+.

Amount of Energy Produced per Glucose

- Phosphorylation of ADP to form ATP stores at least 7.3 kcal mole of ATP.

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Fermentation
- Process for regenerating NAD+ for glycolysis.
- Occurs in organisms that live where oxygen is rare. Ex. Intestines/stomach;
soils/sediments/bogs.
- Fermentation is an anaerobic process in which microorganisms like yeast and bacteria
break down food components (e.g. sugars such as glucose) into other products (e.g.
organic acids, gases or alcohol).
- Two types of Fermentation:
 Lactate Fermentation: Pyruvate converted to lactate (lactic acid).
 Alcoholic Fermentation: Pyruvate converted to ethanol and CO2

The Evolutionary Significance of Glycolysis

- Ancient prokaryotes are thought to have used glycolysis long before there was oxygen in
the atmosphere.
- Very little O2 was available in the atmosphere until about 2.7 billion years ago, so early
prokaryotes likely used only glycolysis to generate ATP.
- Glycolysis is a very ancient process.
Glycolysis and the citric acid cycle connect to many other metabolic pathways
- Glycolysis and the citric acid cycle are major intersections to various catabolic and
anabolic pathways.
The Versatility of Catabolism
- Catabolic pathways funnel electrons from many kinds of organic molecules into cellular
respiration.
- Glycolysis accepts a wide range of
carbohydrates.
- Proteins must be digested to amino acids;
amino groups can feed glycolysis or the citric
acid cycle.
- Fats are digested to glycerol (used in
glycolysis) and fatty acids (used in
generating acetyl CoA).

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 - Fatty acids are broken down by beta-oxidation and yield acetyl CoA.
- An oxidized gram of fat produces more than twice as much ATP as an oxidized gram of
carbohydrate.
Anabolic and Catabolic
- Anabolism is the process by which the body utilizes the energy released by catabolism to
synthesize complex molecules. These complex molecules are then utilized to form
cellular structures that are formed from small and simple precursors that act as building
blocks.
- Catabolism is the break-down of complex molecules. Catabolism is the breakdown of
complex substances to their constituent parts (glucose, amino acids and fatty acids) which
form substrates for metabolic pathways.
How does blood absorb glucose?
Energy Cycle

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