Citric Acid Cycle and Oxidative Phosphorylation
The citric acid cycle, also called the Krebs cycle or the tricarboxylic acid (TCA)
cycle, is in the mitochondria. Although oxygen is not directly required in the
cycle, the pathway will not occur anaerobically because NADH and FADH2 will
accumulate if oxygen is not available for the electron transport chain.
The primary function of the citric acid cycle is oxidation of acetyl-CoA to carbon
dioxide. The energy released from this oxidation is saved as NADH, FADH2, and
guanosine triphosphate (GTP) . The overall result of the cycle is represented by
the following reaction:
The cycle is central to the oxidation of any fuel that yields acetyl-CoA, including
glucose, fatty acids, ketone bodies, ketogenic amino acids, and alcohol. There
is no hormonal control of the cycle, as activity is necessary irrespective of the
fed or fasting state. Control is exerted by the energy status of the cell.
Key points
:
1 . Isocitrate dehydrogenase, the major control enzyme, is inhibited by NADH and
ATP and activated by ADP.
2. a-Ketoglutarate dehydrogenase is similar to the pyruvate dehydrogenase
complex. It requires thiamine, lipoic acid, CoA, FAD, and NAD. Lack of thiamine
slows oxidation of acetyl-CoA in the citric acid cycle.
3. Succinyl-CoA synthetase (succinate thiokinase) catalyzes a substrate-level
phosphorylation of GDP to GTP.
4. Succinate dehydrogenase is on the inner mitochondrial membrane, where
it also functions as complex II of the electron transport chain.
5. Citrate synthase condenses the incoming acetyl group with oxaloacetate to
form citrate.
The mitochondrial electron transport chain (ETC) carries out the following two
Reactions
Sources of NADH, FADH2, and 02
Many enzymes in the mitochondria, including those of the citric acid cycle and
pyruvate dehydrogenase, produce NADH, all of which can be oxidized in the
electron transport chain and in the process, capture energy for ATP synthesis by
oxidative phosphorylation. 02 is delivered to tissues by hemoglobin. The majority
of oxygen required in a tissue is consumed in the electron transport chain ETC.
Its function is to accept electrons at the end of the chain, and the water formed
is added to the cellular water. This scheme is shown below
Electron Transport Chain
NADH is oxidized by NADH dehydrogenase (complex I), delivering its electrons
into the chain and returning as NAD to enzymes that require it. The electrons are
passed along a series of protein and lipid carriers that serve as the wire. These
include, in order:
• NADH dehydrogenase ( complex I) accepts electrons from NADH
• Coenzyme Q (a lipid)
• Cytochrome b/c1 (an Fe/heme protein; complex III)
• Cytochrome c (an Fe/heme protein)
• Cytochrome a/a3 (a Cu/heme protein; cytochrome oxidase, complex IV)
transfers electrons to oxygen
Proton Gradient
The electricity generated by the ETC is used to run proton pumps (translocators),
which drive protons from the matrix space across the inner membrane into the
inter membrane space, creating a small proton (or pH) gradient. This is similar
to pumping any ion, such as Na+, across a membrane to create a gradient. The
three major complexes I, III, and IV (NADH dehydrogenase, cytochrome b/c1
and cytochrome a/ a3) each translocate protons in this way as the electricity
passes through them. The end result is that a proton gradient is normally
maintained across the mitochondrial inner membrane. If proton channels open,
the protons run back into the matrix. Such proton channels are part of the
oxidative phosphorylation complex.
Oxidative Phosphorylation
ATP synthesis by oxidative phosphorylation uses the energy of the proton
gradient and is carried out by the F0F1 ATP synthase complex, which spans the
inner membrane as shown previously. As protons flow into the mitochondria
through the F0 component, their energy is used by the F1 component (ATP
synthase) to phosphorylate ADP using Pi. On average, when an NADH is oxidized
in the ETC, sufficient energy is contributed to the proton gradient for the
phosphorylation of 3 ATP by F0F1 ATP synthase. FADH2 oxidation provides
enough energy for approximately 2 ATP.
Inhibitors
The ETC is coupled to oxidative phosphorylation so that their activities rise and
fall together. Inhibitors of any step effectively inhibit the whole coupled process,
resulting in:
• Decreased oxygen consumption
• Increased intracellular NADH/NAD and FADH2/FAD ratios
• Decreased ATP
Important inhibitors include cyanide and carbon monoxide.
Cyanide
Cyanide is a deadly poison because it binds irreversibly to cytochrome a/a3,
preventing electron transfer to oxygen, and producing many of the same
changes seen in tissue hypoxia. Sources of cyanide include:
• Burning polyurethane (foam stuffing in furniture and mattresses)
• Byproduct of nitroprusside (released slowly; thiosulfate can be used to
destroy the cyanide)
Nitrites may be used as an antidote for cyanide poisoning if given rapidly. They
convert hemoglobin to methemoglobin, which binds cyanide in the blood before
reaching the tissues. Oxygen is also given, if possible.
Carbon monoxide
Carbon monoxide binds to cytochrome a/a3but less tightly than cyanide. It also
binds to hemoglobin, displacing oxygen. Symptoms include headache, nausea,
tachycardia, and tachypnea. Lips and cheeks turn a cherry-red color. Respiratory
depression and coma result in death if not treated by giving oxygen. Sources of
carbon monoxide include:
• Propane heaters and gas grills
• Vehicle exhaust
• Tobacco smoke
• House fires
• Methylene chloride-based paint strippers
Other inhibitors include antimycin (cytochrome b/c1 ), doxorubicin (CoQ), and
oligomycin (F0).
Uncouplers
Uncouplers decrease the proton gradient, causing:
• Decreased ATP synthesis
• Increased oxygen consumption
• Increased oxidation of NADH
Because the rate of the ETC increases, with no ATP synthesis, energy is released
as heat. Important uncouplers include 2,4-dinitrophenol (2,4-DNP) and aspirin
(and other salicylates). Brown adipose tissue contains a natural uncoupling
protein (UCP, formerly called thermogenin), which allows energy loss as heat to
maintain a basal temperature around the kidneys, neck, breastplate, and
scapulae in newborns.
Reactive Oxygen Species
When molecular oxygen (02) is partially reduced, unstable products called
reactive oxygen species (ROS) are formed. These react rapidly with lipids to
cause peroxidation, with proteins, and with other substrates, resulting in
denaturation and precipitation in tissues. Reactive oxygen species include:
• Superoxide (O:t)
• Hydrogen peroxide (H202)
• Hydroxyl radical (OH")
The polymorphonuclear neutrophil produces these substances to kill bacteria in
the protective space of the phagolysosome during the oxidative burst
accompanying phagocytosis. Production of these same ROS can occur at a
slower rate wherever there is oxygen in high concentration. Small quantities of
ROS are inevitable by-products of the electron transport chain in mitochondria.
These small quantities are normally destroyed by protective enzymes such as
catalase. The rate of ROS production can increase dramatically under certain
conditions, such as reperfusion injury in a tissue that has been temporarily
deprived of oxygen. ATP levels will be low and NADH levels high in a tissue
deprived of oxygen (as in an MI) . When oxygen is suddenly introduced, there is
a burst of activity in the ETC, generating incompletely reduced ROS.
Defenses against ROS accumulation are particularly important in highly aerobic
tissues and include superoxide dismutase and catalase. In the special case of
erythrocytes, large amounts of superoxide are generated by the spontaneous
dissociation of the oxygen from hemoglobin (occurrence is 0.5-3% of the total
hemoglobin per day). The products are methemoglobin and superoxide. The
processes that adequately detoxify the superoxide require a variety of enzymes
and compounds, including superoxide dismutase, catalase, as well as
glutathione peroxidase,
vitamin E in membranes, and vitamin C in the cytoplasm. Low levels of any of
these detoxifying substances result in hemolysis. For example, inadequate
production of NADPH in glucose 6-phosphate dehydrogenase deficiency results
in accumulation of the destructive hydrogen peroxide (Chapter 14).
Mutations in Mitochondrial DNA
The circular mitochondrial chromosome encodes 1 3 of the more than 80
proteins that comprise the major complexes of oxidative phosphorylation as well
as 2 2 tRNAs and 2 rRNAs. Mutations in these genes affect highly aerobic tissues
(nerves, muscle), and the diseases exhibit characteristic mitochondrial pedigrees
(maternal inheritance) . Key characteristics of most mitochondrial DNA (mtDNA)
diseases are lactic acidosis and massive proliferation of mitochondria in muscle,
resulting in ragged red fibers. Examples of mtDNA diseases are:
• Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
(MELAS)
• Leber hereditary optic neuropathy
• Ragged red muscle fiber disease
Coordinate Regulation of the Citric Acid Cycle and Oxidative
Phosphorylation
The rates of oxidative phosphorylation and the citric acid cycle are closely
coordinated, and are dependent mainly on the availability of 02 and ADP. If 02 is
limited, the rate of oxidative phosphorylation decreases, and the concentrations
of NADH and FADH2 increase. The accumulation of NADH, in turn, inhibits the
citric acid cycle. The coordinated regulation of these pathways is known as
"respiratory control:'
In the presence of adequate 02, the rate of oxidative phosphorylation is
dependent on the availability of ADP. The concentrations of ADP and ATP are
reciprocally related; an accumulation of ADP is accompanied by a decrease in
ATP and the amount of energy available to the cell. Therefore, ADP
accumulation signals the need for ATP synthesis. ADP allosterically activates
isocitrate dehydrogenase, thereby increasing the rate of the citric acid cycle and
the production of NADH and FADH2. The elevated levels of these reduced
coenzymes, in turn, increase the rate of electron transport and ATP synthesis.
Electron Transport Chain
NADH is oxidized by NADH dehydrogenase (complex I), delivering its electrons
into the chain and returning as NAD to enzymes that require it. The electrons are
passed along a series of protein and lipid carriers that serve as the wire. These
include, in order:
• NADH dehydrogenase ( complex I) accepts electrons from NADH
• Coenzyme Q (a lipid)
• Cytochrome b/c1 (an Fe/heme protein; complex III)
• Cytochrome c (an Fe/heme protein)
• Cytochrome a/a3(a Cu/heme protein; cytochrome oxidase, complex IV)
transfers electrons to oxygen