BS MEDICAL LABORATORY
SCIENCE
LESSON NO. 8 | CITRIC ACID CYCLE
TOPIC OUTLINE
1 Biochemical Energy Production
2 Citric Acid Cycle
3 Reactions of the Citric Acid Cycle
4 Regulation of the Citric Acid Cycle
5 Electron Transport Chain
CITRIC ACID CYCLE
BIOCHEMICAL ENERGY PRODUCTION
- The energy needed to run the human body is obtained
from ingested food through a multistep process that
involves several different catabolic pathways.
FOUR GENERAL STAGES IN THE BIOCHEMICAL ENERGY
PRODUCTION PROCESS:
STAGE 1 Digestion
STAGE 2 Acetyl Group Formation
STAGE 3 Citric Acid Cycle
STAGE 4 Electron Transport Chain & Oxidative
Phosphorylation
Each stage also involves numerous reactions
STAGE 1 DIGESTION
- begins in:
Mouth (saliva contains starch digestive enzymes),
Continuous in the stomach (gastric juices),
Completed in the small intestines (the majority of
digestive enzymes and bile salts)
• Results in small molecules than can cross
intestinal membrane into the blood
- End products of digestion:
• Glucose and monosaccharides from carbs
• Amino acid from proteins
• Fatty acids and glycerol from fats and oils
➢ are small enough to pass across intestinal
membranes and into the blood, with the
aid of membrane transport systems
➢ Once in the blood, they are then
distributed to the cells in various parts of
the body
- the digestion products are absorbed into the blood
and transported to body’s cell
STAGE 2 ACETYL GROUP FORMATION
- the small molecules from stage 1 are further oxidized.
- end product of these oxidation is Acetyl CoA
- involves numerous reactions:
• Reaction occur both in cytosol (glucose
metabolism) as well as mitochondria (fatty
acid metabolism) of the cell
TRENT
BIOCHEM: CITRIC ACID CYCLE
Nucum, Thrynt M. 2nd YEAR
STAGE 3 CITRIC ACID CYCLE
- takes place inside the mitochondria
- First intermediate of the cycle is citric acid
• Therefore, designated as Citric acid cycle
- in this stage acetyl groups are oxidized to produce
CO2 and energy
- The carbon oxide we exhale comes primarily from this
stage
- Most energy is trapped in reduced coenzymes NADH
and FADH2
- Some energy produced in this stage is lost in the form
of heat
• Some is carried by the reduced coenzymes
NADH and FADH2 to the fourth stage
STAGE 4 ELECTRON TRANSPORT CHAIN & OXIDATIVE
PHOSPHORYLATION
- Takes place in mitochondria
- NADH and FADH2 are oxidized to release H+ and
electrons
- H+ are transported to the inter-membrane space in
mitochondria
- Electrons are transferred to O2 and O2 is reduced to
H2O
- H+ ions re-enter the mitochondrial matrix and drive
ATP-synthase reaction to produce ATP
- ATP is the primary energy carrier in metabolic
pathways
- NADH and FADH2 supply the “fuel” (hydrogen ions
and electrons) needed for the production of ATP
molecules, the primary energy carriers in metabolic
pathways.
- Molecular O2, inhaled via breathing, is converted to
H2O in this stage
- stage 3 and 4 are the same for all types of foods (carbs,
fats, proteins)
Common Metabolic Pathway
- is the sum total of the biochemical reactions of the citric
acid cycle, the electron transport chain, and oxidative
phosphorylation.
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 CITRIC ACID CYCLE REACTIONS OF THE CITRIC ACID CYCLE
STEP 1 Formation of citrate
- is the series of biochemical reactions in which the acetyl STEP 2 Formation of Isocitrate
portion of acetyl CoA is oxidized to carbon dioxide and STEP 3 Oxidation of Isocitrate and Formation of CO2
the reduced coenzymes FADH2 and NADH are produced. STEP 4 Oxidation of Alpha-Ketoglutarate and
Formation of CO2
- Also know as tricarboxylic acid cycle (TCA) or Krebs STEP 5 Thioester bond cleavage in Succinyl CoA and
cycle Phosphorylation of GDP
• Citric acid is a tricarboxylic acid – TCA cycle STEP 6 Oxidation of Succinate
• Named after Hans Adolf Krebs who elucidated STEP 7 Hydration of Fumarate
this pathway STEP 8 Oxidation of L-Malate to Regenerate
Oxaloacetate
TWO IMPORTANT TYPE OF REACTIONS
OXIDATION DECARBOXYLATION STEP 1 FORMATION OF CITRATE

Oxidation of NAD+ and Decarboxylation of citric - Acetyl CoA, which carries the two-carbon degradation
FAD to produce NADH acid to produce carbon product of carbohydrates, fats, and proteins, enters the
and FADH2 dioxide cycle by combining with the four-carbon keto
dicarboxylate species oxaloacetate.
which produces NADH or -wherein a carbon chain • This results in the transfer of the acetyl group from
FADH2 is shortened by the coenzyme A to oxaloacetate
removal of a carbon • producing the C6 citrate species and free
is encountered in four of atom as a CO2 molecule coenzyme A.
the eight steps
is encountered in two of Two Parts to the Reaction:
the eight steps 1. The condensation of acetyl CoA and
oxaloacetate to form citryl CoA
- a process catalyzed by the enzyme citrate
• The citric acid cycle also produces 2 ATP by synthase
substrate level phosphorylation from GTP 2. Hydrolysis of the thioester bond in citryl CoA
- to produce CoA – SH and citrate, also
catalyzed by the enzyme citrate synthase
SUMMARY OF CITRIC ACID CYCLE REACTIONS
STEP 2 FORMATION OF ISOCITRATE
Acetyl CoA + 3NAD+ + FAD + GDP + Pi + 2H2O →
2CO2 + CoA-SH + 3NADH + 2H+ + FADH2 + GTP - Citrate is converted to its less symmetrical isomer
isocitrate in an isomerization process that involves a
dehydration followed by a hydration
• both catalyzed by the enzyme aconitase.

- The net result of these reactions is that the -OH group

from citrate is moved to a different carbon atom.

CITRATE ISOCITRATE

Is an achiral compound is a chiral compound with

two chiral centers (four
has a tertiary alcohol stereoisomers possible).
group
has a secondary alcohol
group.

Aconitase
- produces only one of the four stereoisomers of
isocitrate

Tertiary alcohols are not readily oxidized

secondary alcohols are easier to oxidize

The next step in the cycle involves oxidation.

TRENT 2
STEP 3 OXIDATION OF ISOCITRATE AND FORMATION OF
CO2
- This step involves oxidation (the first of four oxidation
reactions in the citric acid cycle) and decarboxylation.
- The reactants are a NAD+ molecule and isocitrate.
THE REACTION, CATALYZED BY ISOCITRATE
DEHYDROGENASE, IS COMPLEX:
1. The alcohol group in isocitrate is oxidized to a
ketone (oxalosuccinate) by NAD+, releasing
two hydrogens.
2. One hydrogen and two electrons are
transferred to NAD+ to form NADH; the
remaining hydrogen ion (H+) is released.
3. The oxalosuccinate remains bound to the
enzyme and undergoes decarboxylation
(loses CO2).
- This decarboxylation, which consumes one H+ ion,
converts C6 isocitrate to C5 a-ketoglutarate (a keto
dicarboxylic acid).
- This step yields the first molecules of CO2 and NADH in
the cycle.
STEP 4 OXIDATION OF A-KETOGLUTARATE AND
FORMATION OF CO2
- This second oxidation reaction of the cycle involves one
molecule each of NAD+, CoA – SH, and a-ketoglutarate.
- The catalyst is a three-enzyme system called the a-
ketoglutarate dehydrogenase complex.
The B vitamin thiamin, in the form of TPP, is part of the
enzyme complex, as is Mg2+ ion.
- As in Step 3, both oxidation and decarboxylation occur.
There are three products:
• CO2
• NADH
• C4 species succinyl CoA
STEP 5 THIOESTER BOND CLEAVAGE IN SUCCINYL COA
AND PHOSPHORYLATION OF GDP
- Two reactant molecules are involved in this step a
• Pi (HPO42-)
• GDP (similar to ADP).
- The entire reaction is catalyzed by the enzyme succinyl-
CoA synthetase.
- For purposes of understanding the structural changes
that occur, the reaction can be considered to occur in
two steps.
In the first step, succinyl CoA is converted to succinyl
phosphate (a high-energy phosphate compound)
• CoA–SH is a product of this change.
• The phosphoryl group present in succinyl
phosphate is then transferred to GDP
• the products of this change are GTP and
succinate.
TRENT
Thinking of the two steps as occurring concurrently gives
the following energy analysis:
• When broken, the high-energy thioester bond in
succinyl
• CoA releases energy, which is trapped by
formation of GTP.
• The function of the GTP produced is similar to
that of ATP, which is to store energy in the form of
a high-energy phosphate bond.
Steps 6 through 8 of the citric acid cycle involve a
sequence of functional group changes that have been
encountered several times in the organic sections of the
text.
STEP 7 HYDRATION OF FUMARATE
- The enzyme fumarase catalyzes the addition of water to
the trans double bond of fumarate.
- The enzyme is stereospecific, so only the L-isomer of the
product malate is produced.
STEP 8 OXIDATION OF L-MALATE TO REGENERATE
OXALOACETATE
- In the fourth oxidation reaction of the cycle, a molecule
of NAD+ reacts with malate, picking up two hydrogen
atoms with their associated energy to form NADH + H+
- The needed enzyme is malate dehydrogenase.
- The product of this reaction is regenerated
oxaloacetate, which can combine with another
molecule of acetyl CoA (Step 1)
• and the cycle can begin again.
REGULATION OF THE CITRIC ACID CYCLE
- The rate at which the citric acid cycle operates is
controlled by ATP and NADH levels
- When ATP supply is high, ATP inhibits citrate synthase
(Step 1 of Citric acid cycle)
- When ATP levels are low ADP, ADP activates citrate
synthase
Similarly (in step 3), ADP and NADH control isocitrate
dehydrogenase:
• NADH acts as an inhibitor
• ADP as an activator
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ELECTRON TRANSPORT CHAIN
Electron Transport Chain
- is a series of biochemical reactions in which electrons
and hydrogen ions from NADH and FADH2 are passed to
intermediate carriers and then ultimately react with
molecular oxygen to produce water
- The electron transport chain (ETC) facilitates the
passage of electrons trapped in FADH2 and NADH during
citric cycle
- ETC is a series of biochemical reactions in which
intermediate carriers (protein and non-protein) aid the
transfer of electrons and hydrogen ions from NADH and
FADH2
- The ultimately receiver of electrons is molecular oxygen
- The electron transport (respiratory chain) gets its name
from the fact electrons are transported to oxygen
absorbed via respiration
- The overall ETC reaction: 2 H+ + 2e- + 1/2 O2 → H2O +
energy
- Energy is used to synthesize ATP in oxidative
phosphorylation
- Note that 2 hydrogen ions, 2 electrons, and one half-
oxygen molecule react to form the product water
- This relatively straight forward reaction actually requires
eight or more steps
- The reaction releases energy (exothermic reaction)
TRENT
- The energy released is coupled with the formation of
three ATP molecules per every molecule of NADH
processed through ETC
- The enzymes and electron carriers needed for the ETC
are located along inner mitochondrial membrane
They are organized into four distinct protein complexes
and two mobile carriers
FOUR PROTEIN COMPLEXES TIGHTLY BOUND TO MEMBRANE
COMPLEX I NADH-coenzyme Q reductase
COMPLEX II Succinate-coenzyme Q reductase
COMPLEX III Coenzyme Q - cytochrome C reductase
COMPLEX IV Cytochrome C oxidase
TWO MOBILE ELECTRON CARRIERS ARE
1. Coenzyme Q
2. Cytochrome C
COMPLEX I NADH-COENZYME Q REDUCTASE
- NADH from citric acid cycle is the source of electrons for
this complex
- It contains >40 subunits including flavin mononucleotide
(FMN) and several iron-sulfur protein clusters (FeSP)
- Net result: Facilitates transfer of electrons from NADH to
coenzyme Q
- Several intermediate reactions are involved in this
electron transfer
COMPLEX II SUCCINATE-COENZYME Q REDUCTASE
- Smaller than complex I
- Contains only four subunits including two iron-sulfur
protein clusters (FeSP)
- Succinate is converted to fumarate by this complex
- In the process it generates FADH2
- CoQ is the final recipient of the electrons from FADH2
COMPLEX III COENZYME Q - CYTOCHROME C
REDUCTASE
- Complex III contains 11 different subunits
- Several iron-sulfur proteins and cytochromes are
electron carriers in this complex
- Cytochrome is a heme iron protein in which reversible
oxidation of an iron atom occurs
- Various cytochromes, e.g., cyt a, cyt b, cyt c, differ from
each other by:
• Their protein constituents
• The manner in which the heme is bonded to the
protein
• Attachments to the heme ring
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COMPLEX IV CYTOCHROME C OXIDASE
- Contains 13 subunits including two cytochromes

- The electrons flow from cyt c to cyt a to cyt a3

- In the final stage of electron transfer, the electrons from

cyt a3, and hydrogen ion (H+) combine with oxygen (O2)
to form water

- O2 + 4H+ + 4e- → 2 H2O

- It is estimated that 95 % of the oxygen used by cells

serves as the final electron acceptor for the ETC

TRENT 5