Chapter Two: part five:
1. oxidation of pyruvate and the citric acid cycle
In the next stage of cellular respiration—and in the presence of oxygen—
pyruvate produced in glycolysis is transformed into an acetyl group
attached to a carrier molecule of coenzyme A.
The resulting acetyl CoA is usually delivered from the cytoplasm to the
mitochondria, a process that uses some ATP.
In the mitochondria, acetyl CoA continues to the citric acid cycle. The
citric acid cycle (CAC or TCA- tricarboxylic acid cycle) is also known as
the Krebs cycle.
During the conversion of pyruvate into the acetyl group, a molecule of
CO2 and two high-energy electrons are removed. Remember that
glycolysis produces two molecules of pyruvate, and each can attach to a
molecule of CoA and then enter the citric acid cycle.
The electrons are picked up by NAD+, and NADH carries the electrons to
a later pathway for ATP production.
The glucose molecule that originally entered cellular respiration in
glycolysis has been completely oxidized.
The chemical potential energy stored within the glucose molecules has
been transferred to NADH or has been used to synthesize ATP
molecules.
The citric acid cycle occurs in the mitochondrial matrix and involves a
series of redox and decarboxylation reactions that again remove high-
energy electrons and produce CO2.
These electrons are carried by NADH and FADH2 to the electron
transport chain located in the cristae of the mitochondrion.
During the cycle, ATP is synthesized from ADP and inorganic phosphate
by substrate-level phosphorylation.
NOTE THAT: 1 NADH=3ATP, and 1 FADH2=2 ATP
If oxygen is available, aerobic respiration will go forward.
In eukaryotic cells, the pyruvate molecules produced at the end of glycolysis
are transported into mitochondria.
There, pyruvate will be transformed into an acetyl group that will be picked
up and activated by a carrier compound called coenzyme A (CoA).
The resulting compound is called acetyl CoA. CoA is made from vitamin
B5, pantothenic acid.
Acetyl CoA can be used in a variety of ways by the cell, but its major
function is to deliver the acetyl group derived from pyruvate to the next
stage of the pathway in glucose catabolism.
Breakdown of Pyruvate
For pyruvate, the product of glycolysis, to enter the next pathway, it must undergo
several changes. The conversion is a three-step process.
Step 1.
A carboxyl group is removed from pyruvate, releasing a molecule of carbon
dioxide into the surrounding medium.
The result of this step is a two-carbon hydroxyethyl group bound to the
enzyme (pyruvate dehydrogenase). This is the first of the six carbons from
the original glucose molecule to be removed.
This step proceeds twice for every molecule of glucose metabolized; thus,
two of the six carbons will have been removed at the end of both steps.
Step 2.
The hydroxyethyl group is oxidized to an acetyl group, and the electrons are
picked up by NAD+, forming NADH.
The high-energy electrons from NADH will be used later to generate ATP.
Step 3.
The enzyme-bound acetyl group is transferred to CoA, producing a molecule
of acetyl CoA.
Note that during the second stage of glucose metabolism, whenever a
carbon atom is removed, it is bound to two oxygen atoms, producing
carbon dioxide, one of the major end products of cellular respiration.
Acetyl CoA to CO2
In the presence of oxygen, acetyl CoA delivers its acetyl group to a four-
carbon molecule, oxaloacetate, to form citrate, a six-carbon molecule with
three carboxyl groups; this pathway will harvest the remainder of the
extractable energy from what began as a glucose molecule.
This single pathway is called by different names: the citric acid cycle (for
the first intermediate formed—citric acid, or citrate—when acetate joins to
the oxaloacetate), the TCA cycle (since citric acid or citrate and isocitrate
are tricarboxylic acids), and the Krebs cycle, after Hans Krebs, who first
identified the steps in the pathway.
Citric Acid Cycle
Like the conversion of pyruvate to acetyl CoA, the citric acid cycle takes
place in the matrix of mitochondria.
Almost all the enzymes of the citric acid cycle are soluble, with the single
exception of the enzyme succinate dehydrogenase, which is embedded in the
inner membrane of the mitochondrion.
Unlike glycolysis, the citric acid cycle is a closed loop: The last part of the
pathway regenerates the compound used in the first step.
The eight steps of the cycle are a series of redox, dehydration, hydration, and
decarboxylation reactions that produce two carbon dioxide molecules, one
GTP/ATP, and reduced forms of NADH and FADH2.
This is considered an aerobic pathway because the NADH and
FADH2 produced must transfer their electrons to the next pathway in the
system, which will use oxygen.
If this transfer does not occur, the oxidation steps of the citric acid cycle also
do not occur. Note that the citric acid cycle produces very little ATP directly
and does not directly consume oxygen.
Steps in the Citric Acid Cycle
Step 1.
Prior to the start of the first step, a transitional phase occurs during which
pyruvic acid is converted to acetyl CoA.
Then, the first step of the cycle begins: This is a condensation step,
combining the two-carbon acetyl group with a four-carbon oxaloacetate
molecule to form a six-carbon molecule of citrate.
CoA is bound to a sulfhydryl group (-SH) and diffuses away to eventually
combine with another acetyl group. This step is irreversible because it is
highly exergonic.
The rate of this reaction is controlled by negative feedback and the amount
of ATP available. If ATP levels increase, the rate of this reaction decreases.
If ATP is in short supply, the rate increases.
Step 2.
In step two, citrate loses one water molecule and gains another as citrate is
converted into its isomer, isocitrate.
Step 3.
In step three, isocitrate is oxidized, producing a five-carbon molecule, α-
ketoglutarate, together with a molecule of CO2 and two electrons, which
reduce NAD+ to NADH.
This step is also regulated by negative feedback from ATP and NADH, and
a positive effect of ADP.
Steps 3 and 4.
Steps three and four are both oxidation and decarboxylation steps, which
release electrons that reduce NAD+ to NADH and release carboxyl groups
that form CO2 molecules. α-Ketoglutarate is the product of step three, and a
succinyl group is the product of step four.
CoA binds the succinyl group to form succinyl CoA. The enzyme that
catalyzes step four is regulated by feedback inhibition of ATP, succinyl CoA,
and NADH.
Step 5.
In step five, a carboxyl group is substituted for coenzyme A, and a high-
energy bond is formed.
This energy is used in substrate-level phosphorylation (during the
conversion of the succinyl group to succinate) to form either guanosine
triphosphate (GTP) or ATP.
There are two forms of the enzyme, called isoenzymes, for this step,
depending upon the type of animal tissue in which they are found.
One form is found in tissues that use large amounts of ATP, such as the
heart and skeletal muscle. This form produces ATP.
The second form of the enzyme is found in tissues that have a high number
of anabolic pathways, such as liver tissues. This form produces GTP. GTP is
energetically equivalent to ATP; however, its use is more restricted. In
particular, protein synthesis primarily uses GTP.
Step 6.
Step six is a dehydrogenation process that converts succinate into fumarate.
Two hydrogen atoms are transferred to FAD, producing FADH2.
The energy contained in the electrons of these atoms is insufficient to reduce
NAD+ but adequate to reduce FAD. Unlike NADH, this carrier remains
attached to the enzyme and transfers the electrons to the electron transport
chain directly.
This process is made possible by the localization of the enzyme catalyzing
this step inside the inner membrane of the mitochondrion.
Step 7.
Water is added to fumarate during step seven, and malate is produced.
The last step in the citric acid cycle regenerates oxaloacetate by oxidizing
malate. Another molecule of NADH is produced in the process.
Products of the Citric Acid Cycle
Two carbon atoms come into the citric acid cycle from each acetyl group,
representing four out of the six carbons of one glucose molecule.
Two carbon dioxide molecules are released on each turn of the cycle;
however, these do not necessarily contain the most recently added carbon
atoms.
The two acetyl carbon atoms will eventually be released on later turns of the
cycle; thus, all six carbon atoms from the original glucose molecule are
eventually incorporated into carbon dioxide.
Each turn of the cycle forms three NADH molecules and one
FADH2 molecule. These carriers will connect with the last portion of
aerobic respiration to produce ATP molecules.
One GTP or ATP is also made in each cycle.
Several of the intermediate compounds in the citric acid cycle can be used in
synthesizing non-essential amino acids; therefore, the cycle is amphibolic
(both catabolic and anabolic).
2. Oxidative phosphorylation
The electron transport chain (ETC) is the stage of aerobic respiration that
uses free oxygen as the final electron acceptor of the electrons removed
during glucose metabolism in glycolysis and the citric acid cycle.
The ETC is located in membrane of the mitochondrial cristae, an area with
many folds that increase the surface area available for chemical reactions.
Electrons carried by NADH and FADH2 are delivered to electron acceptor
proteins embedded in the membrane as they move toward the final electron
acceptor, O2, forming water.
The electrons pass through a series of redox reactions, using free energy at
three points to transport hydrogen ions across the membrane.
This process contributes to the formation of the H+ gradient used in
chemiosmosis. As the protons are driven down their concentration gradient
through ATP synthase, ATP is generated from ADP and inorganic
phosphate. Under aerobic conditions, the stages of cellular
respiration can generate 36-38 ATP.
Most of the ATP generated during the aerobic catabolism of glucose,
however, is not generated directly from these pathways.
Rather, it is derived from a process that begins with moving electrons
through a series of electron transporters that undergo redox reactions.
This causes hydrogen ions to accumulate within the intermembranous space.
Therefore, a concentration gradient forms in which hydrogen ions diffuse
out of the intermembranous space into the mitochondrial matrix by passing
through ATP synthase.
The current of hydrogen ions powers the catalytic action of ATP synthase,
which phosphorylates ADP, producing ATP.
Electron Transport Chain
The electron transport chain is the last component of aerobic respiration and
is the only part of glucose metabolism that uses atmospheric oxygen.
Oxygen continuously diffuses into plants; in animals, it enters the body
through the respiratory system.
Electron transport is a series of redox reactions that resemble a relay race or
bucket brigade in that electrons are passed rapidly from one component to
the next, to the endpoint of the chain where the electrons reduce molecular
oxygen, producing water.
There are four complexes composed of proteins, and the aggregation of
these four complexes, together with associated mobile, accessory electron
carriers, is called the electron transport chain.
The electron transport chain is present in multiple copies in the inner
mitochondrial membrane of eukaryotes and the plasma membrane of
prokaryotes.
Complex I
To start, two electrons are carried to the first complex aboard NADH. This
complex, labelled I, is composed of flavin mononucleotide (FMN) and an
iron-sulfur (Fe-S)-containing protein.
FMN, which is derived from vitamin B2, also called riboflavin, is one of
several prosthetic groups or co-factors in the electron transport chain.
A prosthetic group is a non-protein molecule required for the activity of a
protein. Prosthetic groups are organic or inorganic, non-peptide molecules
bound to a protein that facilitate its function; prosthetic groups include co-
enzymes, which are the prosthetic groups of enzymes.
The enzyme in complex I is NADH dehydrogenase and is composed of 44
separate polypeptide chains. Complex I can pump four hydrogen ions across
the membrane from the matrix into the intermembrane space, and it is in this
way that the hydrogen ion gradient is established and maintained between
the two compartments separated by the inner mitochondrial membrane.
Q and Complex II
Complex II directly receives FADH2, which does not pass through Complex
I. The compound connecting the first and second complexes to the third
is ubiquinone (Q).
The Q molecule is lipid soluble and freely moves through the hydrophobic
core of the membrane. Once it is reduced, (QH2), ubiquinone delivers its
electrons to the next complex in the electron transport chain.
Q receives the electrons derived from NADH from complex I, and the
electrons derived from FADH2 from complex II.
This enzyme and FADH2 form a small complex that delivers electrons
directly to the electron transport chain, bypassing the first complex.
Since these electrons bypass and thus do not energize the proton pump in the
first complex, fewer ATP molecules are made from the FADH2 electrons.
The number of ATP molecules ultimately obtained is directly proportional to
the number of protons pumped across the inner mitochondrial membrane.
Complex III
The third complex is composed of cytochrome b, another Fe-S protein,
Rieske center (2Fe-2S center), and cytochrome c proteins; this complex is
also called cytochrome oxidoreductase.
Cytochrome proteins have a prosthetic group of heme. The heme molecule is
similar to the heme in hemoglobin, but it carries electrons, not oxygen.
As a result, the iron ion at its core is reduced and oxidized as it passes the
electrons, fluctuating between different oxidation states: Fe++ (reduced) and
Fe+++ (oxidized).
The heme molecules in the cytochromes have slightly different
characteristics due to the effects of the different proteins binding them,
giving slightly different characteristics to each complex.
Complex III pumps protons through the membrane and passes its electrons
to cytochrome c for transport to the fourth complex of proteins and enzymes
(cytochrome c is the acceptor of electrons from Q; however, whereas Q
carries pairs of electrons, cytochrome c can accept only one at a time).
Complex IV
The fourth complex is composed of cytochrome proteins c, a, and a3. This
complex contains two heme groups (one in each of the two cytochromes, a,
and a3) and three copper ions (a pair of CuA and one CuB in cytochrome a3).
The cytochromes hold an oxygen molecule very tightly between the iron and
copper ions until the oxygen is completely reduced. The reduced oxygen
then picks up two hydrogen ions from the surrounding medium to make
water (H2O).
The removal of the hydrogen ions from the system contributes to the ion
gradient used in the process of chemiosmosis.
Chemiosmosis
In chemiosmosis, the free energy from the series of redox reactions just
described is used to pump hydrogen ions (protons) across the membrane.
The uneven distribution of H+ ions across the membrane establishes both
concentration and electrical gradients (thus, an electrochemical gradient),
owing to the hydrogen ions’ positive charge and their aggregation on one
side of the membrane.
If the membrane were open to diffusion by the hydrogen ions, the ions
would tend to diffuse back across into the matrix, driven by their
electrochemical gradient.
Recall that many ions cannot diffuse through the nonpolar regions of
phospholipid membranes without the aid of ion channels.
Similarly, hydrogen ions in the matrix space can only pass through the inner
mitochondrial membrane through an integral membrane protein called ATP
synthase.
This complex protein acts as a tiny generator, turned by the force of the
hydrogen ions diffusing through it, down their electrochemical gradient. The
turning of parts of this molecular machine facilitates the addition of
phosphate to ADP, forming ATP, using the potential energy of the hydrogen
ion gradient.
Chemiosmosis is used to generate 90 per cent of the ATP made during
aerobic glucose catabolism; it is also the method used in the light reactions
of photosynthesis to harness the energy of sunlight in the process of
photophosphorylation.
Recall that the production of ATP using the process of chemiosmosis in
mitochondria is called oxidative phosphorylation. The overall result of these
reactions is the production of ATP from the energy of the electrons removed
from hydrogen atoms.
These atoms were originally part of a glucose molecule. At the end of the
pathway, the electrons are used to reduce an oxygen molecule to oxygen
ions. The extra electrons on the oxygen attract hydrogen ions (protons) from
the surrounding medium, and water is formed.
ATP Yield
The number of ATP molecules generated from the catabolism of glucose
varies. For example, the number of hydrogen ions that the electron transport
chain complexes can pump through the membrane varies between species.
Another source of variance stems from the shuttle of electrons across the
membranes of the mitochondria. (The NADH generated from glycolysis
cannot easily enter mitochondria.)
Thus, electrons are picked up on the inside of mitochondria by either
NAD+ or FAD+. As you have learned earlier, these FAD+ molecules can
transport fewer ions; consequently, fewer ATP molecules are generated
when FAD+ acts as a carrier. NAD+ is used as the electron transporter in the
liver and FAD+ acts in the brain.
Another factor that affects the yield of ATP molecules generated from
glucose is the fact that intermediate compounds in these pathways are used
for other purposes.
Glucose catabolism connects with the pathways that build or break down all
other biochemical compounds in cells, and the result is somewhat messier
than the ideal situations described thus far. For example, sugars other than
glucose are fed into the glycolytic pathway for energy extraction.
Moreover, the five-carbon sugars that form nucleic acids are made from
intermediates in glycolysis. Certain nonessential amino acids can be made
from intermediates of both glycolysis and the citric acid cycle.
Lipids, such as cholesterol and triglycerides, are also made from
intermediates in these pathways, and both amino acids and triglycerides are
broken down for energy through these pathways. Overall, in living systems,
these pathways of glucose catabolism extract about 34 per cent of the energy
contained in glucose.
3. Metabolism without oxygen
If oxygen is not present, ATP is only produced by substrate-level
phosphorylation.
Without oxygen, organisms must use another electron acceptor. Most
organisms will use some form of fermentation to accomplish the
regeneration of NAD+ to ensure the continuation of glycolysis.
In alcohol fermentation, pyruvate from glycolysis is converted to ethyl
alcohol; during lactic acid fermentation, pyruvate is reduced to form
lactate as an end-product.
Without fermentation and anaerobic respiration, we wouldn’t have yogurt
or soy sauce. Nor would our muscle cells cramp from the buildup of
lactate when we exercise vigorously, and oxygen is scarce.
In aerobic respiration, the final electron acceptor is an oxygen molecule,
O2. If aerobic respiration occurs, then ATP will be produced using the
energy of high-energy electrons carried by NADH or FADH2 to the
electron transport chain. If aerobic respiration does not occur, NADH
must be reoxidized to NAD+ for reuse as an electron carrier for the
glycolytic pathway to continue. Some living systems use an organic
molecule as the final electron acceptor.
Processes that use an organic molecule to regenerate NAD+ from NADH
are collectively referred to as fermentation.
In contrast, in some living systems, the electron transport chain (ETC)
uses an inorganic molecule as a final electron acceptor, which is
called anaerobic cellular respiration. Both processes allow organisms
to convert energy for their use in the absence of oxygen.
Anaerobic Cellular Respiration
Certain prokaryotes, including some species of bacteria and Archaea, use
anaerobic respiration. For example, the group of Archaea called
methanogens reduces carbon dioxide to methane to oxidize NADH.
These microorganisms are found in the soil and in the digestive tracts of
ruminants, such as cows and sheep. Similarly, sulfate-reducing bacteria and
Archaea, most of which are anaerobic, reduce sulfate to hydrogen sulfide to
regenerate NAD+ from NADH.
Lactic Acid Fermentation
The fermentation method used by animals and certain bacteria, like those in
yoghurt, is lactic acid fermentation.
This type of fermentation is used routinely in mammalian red blood cells
and in skeletal muscle that has an insufficient oxygen supply to allow
aerobic respiration to continue (that is, in muscles used to the point of
fatigue).
In muscles, lactic acid accumulation must be removed by blood circulation
and the lactate brought to the liver for further metabolism.
The chemical reactions of lactic acid fermentation are the following:
Pyruvic acid+NADH↔lactic acid+NAD+
The enzyme used in this reaction is lactate dehydrogenase (LDH).
The reaction can proceed in either direction, but the reaction from left to
right is inhibited by acidic conditions.
Such lactic acid accumulation was once believed to cause muscle stiffness,
fatigue, and soreness, although more recent research disputes this
hypothesis.
Once the lactic acid has been removed from the muscle and circulated to the
liver, it can be reconverted into pyruvic acid and further catabolized for
energy.
Alcohol Fermentation
Another familiar fermentation process is alcohol fermentation which
produces ethanol the alcohol.
The first chemical reaction of alcohol fermentation is the following
(CO2 does not participate in the second reaction):
pyruvic acid+H+→CO2+acetaldehyde+NADH+H+→ethanol+NAD+
The first reaction is catalyzed by pyruvate decarboxylase, a cytoplasmic
enzyme, with a coenzyme of thiamine pyrophosphate (TPP, derived from
vitamin B1 and also called thiamine).
A carboxyl group is removed from pyruvic acid, releasing carbon dioxide as
a gas. The loss of carbon dioxide reduces the size of the molecule by one
carbon, making acetaldehyde.
The second reaction is catalyzed by alcohol dehydrogenase to oxidize
NADH to NAD+ and reduce acetaldehyde to ethanol.
The fermentation of pyruvic acid by yeast produces ethanol.
Ethanol tolerance of yeast is variable, ranging from about 5 percent to 21
percent, depending on the yeast strain and environmental conditions.
Other Types of Fermentation
Other fermentation methods occur in bacteria. Many prokaryotes are
facultatively anaerobic. This means that they can switch between aerobic
respiration and fermentation, depending on the availability of oxygen.
Certain prokaryotes, like Clostridia, are obligate anaerobes. Obligate
anaerobes live and grow in the absence of molecular oxygen. Oxygen is a
poison to these microorganisms and kills them on exposure.
It should be noted that all forms of fermentation, except lactic acid
fermentation, produce gas. The production of particular types of gas is used
as an indicator of the fermentation of specific carbohydrates, which plays a
role in the laboratory identification of the bacteria.
Various methods of fermentation are used by assorted organisms to ensure
an adequate supply of NAD+ for the sixth step in glycolysis. Without these
pathways, that step would not occur and no ATP would be harvested from
the breakdown of glucose.