Mitochondrion organelle
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The
mitochondria are capsule-shaped cellular organelles that generate energy from aerobic (oxygen-utilizing) metabolism utilizing respiratory chain and ATP syntheses enzymes. Most animal cells contain between a few hundred and a
few thousand mitochondria. The most mitochondria are found in the cells that are most metabolically active: neurons and muscle cells, where mitochondria make up about 40% of cell volume. About 10% of the body weight of a human adult is
mitochondria.
A mitochondrion has two membranes. The outer membrane contains small pores (porins, also known as Voltage-Dependent Anion Channels,
VDACs) that are freely permeable to ions and other molecules smaller than 10 kiloDaltons in size. The inner membrane is highly impermeable, even to protons (H+ ions). The proton gradient across the inner
membrane is used by ATP synthetase enzyme to generate ATP molecules. The region between the outer membrane and the inner membrane is more positively charged (
P−phase) because of the higher proton concentration, whereas the inside of the inner membrane is more negatively charged (
N−phase, the matrix). It is in the matrix that the Krebs citric acid cycle occurs. There can be tens of thousands of respiratory chain and associated ATP synthase molecules embedded in the inner membrane of a mitochondrion,
especially in metabolically active cells that have their inner membranes most highly folded into cristae that increase surface area.
The inner membrane contains a number of active molecule carriers, including a phosphate (
Pi = H2PO4-) carrier and the
Adenine Nucleotide Transporter (ANT). The ANT imports ADP molecules into the matrix for ATP synthesis in exchange for ATP molecules which are exported for energy use throughout the cell (like portable batteries). The
respiratory chain ("electron transport chain") attached to the inner wall of the inner membrane is composed of 4 protein complexes. These protein complexes are identified as
Complex I, II, III and IV. Complex II consists of only four peptides, two of which comprise the Krebs citric acid cycle protein succinate dehydrogenase, and two of which anchor the complex to the inner mitochondrial membrane.
Complex I and Complex II independently supply electrons to Complex III, which supplies electrons to Complex IV. Soluble carriers are used to transport electrons to and from Complex III. The soluble carrier
transporting electrons from Complex I & II to Complex III is
Coenzyme Q (CoQ). The soluble carrier that transports electrons from Complex III to Complex IV is
cytochrome−c. For this reason Complex III is also known as
cytochrome−c reductase and Complex IV is also known as
cytochrome−c oxidase. Complex IV combines its electrons (which are actually hydrogen atoms) with oxygen to form water. The energy released by the oxidations in the respiratory chain are used to pump protons outside the
inner mitochondrial membrane.
Protons pumped out of mitochondrial matrix
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Re-entering protons drive "ATP turbine"
The inner mitochondrial membrane is fairly impermeable to H+ ions ("protons") and thus is able to function much like a hydroelectric dam. Respiratory enzymes (Complex I, III & IV) pump protons out of
the inner mitochondrial matrix, building proton pressure outside the "dam" (the membrane). The proton pressure ("proton-motive force") across the inner membrane is composed of two components: a pH difference and an
electrical potential (membrane potential), which is the most important component. The pH difference is small, amounting to only about 0.5 pH units. The membrane potential of the mitochondrial membrane is about twice as great as that
of a large nerve fiber, amounting to over 200 milliVolts.
Complex V (F0F1−ATP synthase) is the "hydroelectric turbine" that utilizes the energy of the proton flow into the matrix through the "turbine" to synthesize ATP. The ATP synthase
(Complex V) "rotary motor" is the smallest known natural nanomachine. It uses proton-motive force to drive the endothermic reaction:
ADP + Pi => ATP
The combined result of respiratory (oxidative) steps and the ATP-creation (phosphorylation of ADP) step is called
oxidative phosphorylation. Normally respiration (oxygen consumption) and phosphorylation (ATP production) are tightly
coupled, ie, the amount of ATP produced corresponds to the amount of oxygen consumed -- referred to as
state 3 respiration. In the absence of ADP (eg, in a resting state), however, any respiration that occurs will be due to "proton leak" through the inner mitochondrial membrane rather than due to ATP production --
referred to as
state 4 respiration. (State 1, state 2 and state 5 are experimental conditions of more historical interest than metabolic interest.)
In state 4 respiration protons flowing directly through the inner membrane rather than through the "ATP turbine" (Complex V) produce heat energy rather than ATP energy.
Uncoupling proteins are weak acids that dissolve inner membrane lipids thereby increasing the uncoupling of oxidation from phosphorylation. Uncoupling respiration from phosphorylation to produce heat is useful for small rodents,
naked newborn babies, and hibernating & cold-acclimated animals, all of which contain "brown fat". Uncoupling is also useful for fever production.
UCP1 is the UnCoupling Protein found in "brown fat", fat which has been made brown by high concentrations of mitochondria.
UCP2 has broad tissue distribution and seems to function in stress response, but its expression is less than 1% of UCP1.
UCP3 is found in muscle and is regulated by thyroid hormone (T3).
The function of UCP1 is to generate heat ("thermogenesis"). Claims have been made that UCP3 generates little heat, but functions to reduce free radical damage by lowering protein gratient during periods of high metabolic
activity. Mice with higher UCP3 have shown higher metabolic intensity (17% greater resting oxygen consumption) and 36% longer lifespan [AGING CELL; Speakman,JR; 3(3):87-95 (2004)]. Proton leak has not been shown to be a factor in
CRAN (The fact that dieting-resistant obese subjects have been shown to have smaller amounts of UCP3 would seem to indicate that thermogenesis from UCP3 is not negligible.
Increasing insulin levels associated with aging and type−2 diabetes stimulates nitric oxide synthetase resulting in peroxynitrite [THE INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY 34:1340-1354 (2002)]. Lipid
peroxidation of the inner mitochondrial membrane by peroxynitrite can increase proton leak independent of uncoupling protein. Peroxynitrite can also degrade function of respiratory enzymes [JOURNAL OF NEUROCHEMISTRY 70:2195-2202
(1998)] and inactivate mitochondrial superoxide dismutase (Mn−SOD) enzyme [PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (USA) 93(21):<?XML:NAMESPACE PREFIX = SKYPE />
11853-11858
(1996)].
Mitochondria are the only cellular organelles with their own DNA. (There is no other cellular DNA outside the nucleus apart from the DNA of mitochondria.) Mitochondrial DNA (
mtDNA) in humans are circular strands of 16,569 nucleic acids that code for 37 genes -- 22 transfer RNAs, 2 ribosomal RNAs and 13 transmembrane proteins. There are nearly 1,500 other gene products in mitochondria, which are
coded-for by nuclear DNA (
nDNA). In contrast to nDNA, the mtDNA is derived almost entirely from the mother. Each cell contains many mitochondria, but the total mtDNA in a cell represents less than 1% of the amount of DNA found in the nucleus.
Inner Membrane mtDNA-coded Proteins in Complex I, III, IV & V
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Re-entering protons drive "ATP turbine"
Each mitochondrion contains 2-to-12 identical copies of mitochondrial DNA (2-to-12 circular strands). Each mtDNA strand codes for 13 proteins, all of which are transmembrane subunits of Complex I, III, IV or V.
Of the 13 mtDNA proteins, 7 are in Complex I, 1 is in Complex III, 3 are in Complex IV and 2 are in Complex V. A distinctive feature of the 13 proteins coded-for by the mtDNA is that they are hydrophobic (not
easily dissolved in water), suggesting that it might be difficult to synthesize & transport them in the watery cytoplasm. For this reason it has seemed improbable that the mtDNA for these proteins could be moved to the nucleus where
they would be better protected & repaired. But one of the Complex V (ATPase) mtDNA-coded proteins has been successfully synthesized in the nucleus and utilized in the mitochondria for a mammalian cell [REJUVENATION RESEARCH;
Zullo,SJ; 8(1):18-28 (2005)] giving hope to the idea that all 13 mtDNA proteins might eventually be moved to the nucleus. An alternate hypothesis, however, claims that the mtDNA genes are of value in providing rapid local synthesis
of proteins required for oxidative phosphorylation. Oxidative stress due to insufficient oxidative phosphorylation capability could signal mitochondrial transcription factors to induce production of mtDNA-coded proteins that are then
implanted into the inner membrane where they attract the nDNA-coded proteins required for complete assembly of the complexes [PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY; Allen,JF; 358(1429):19-38 (2003)].
Complex I, which has 7 mtDNA-coded proteins (more than a quarter of all the proteins in the Complex), ages most rapidly. Substantia nigra neurons have increased susceptibility to Complex I defects -- which may be responsible
for Parkinson's Disease [NEUROBIOLOGY OF AGING; Smigrodzki,R; 25:1273-1281 (2004)]. By contrast, Complex II (which has no mtDNA-coded proteins) and Complex III (which has only one) are relatively unaffected by aging.
Cytochrome−c oxidase (between Complex III and Complex IV) activity declines with age, resulting in increased production of superoxide and hydrogen peroxide. Diseases due to mutated mtDNA have the greatest effect on cells producing
the most energy -- cells of brain and muscle -- hence mitochondrial diseases are often
encephalomyopathies . A very common syndrome of mitochondrial disease is
Mitochondria
Encephalomyopathy,
Lactic
Acidosis &
Stroke (
MELAS).
Homoplasmy describes the original condition of all of a person's mtDNA being the same, but as mtDNA mutations occur and the mutated mtDNA replicates, cells, tissues and even mitochondria can have a mixture of mtDNA types, a
condition known as
heteroplasmy.
An estimated 1−2% of oxygen used by mitochondria will normally "leak" from the respiratory chain to form superoxide [JOURNAL OF NEUROCHEMISTRY 59:1609-1623 (1992) & JOURNAL OF INTERNAL MEDICINE 238:405-421 (1995)].
The pro-inflammatory cytokine
Tumor Necrosis Factor−alpha (
TNF−α, associated with the metabolic syndrome) induces increased free radical production from the respiratory chain . Aging is associated with decreased oxidative phosphorylation coupling efficiency and increased
superoxide production. Free radicals can damage the mitochondrial inner membrane, creating a positive feedback-loop for increased free-radical creation. The "viscious cycle" theory that free radical damage to mitochondrial DNA
leads to mitochondria that produce more superoxide has been questioned. The most damaged mitochondria are consumed by lysosomes whereas the more defective mitochondria (which produce less ATP as well as less superoxide) remain to
reproduce themselves [REJUVENATION RESEARCH; de Grey,A; 8(1):13-17 (2005)]. But the efficiency of lysosomes to consume malfunctioning mitochondria declines with age, resulting in more mitochondria producing higher levels of
superoxide. Mitochondria of older organisms are fewer in number, larger in size and less efficient (produce less energy & more superoxide).
Coenzyme Q (CoQ, in humans CoQ10) is also known as
ubiquinone, so-called because it is "ubiquitous" (universally-found) in almost all cellular organisms, with the exception of gram-positive bacteria and some fungi. CoQ is an essential component of the mitochondrial
respiratory chain. From Complex I or Complex II dehydrogenase CoQ is reduced to CoQH2 and subsequently oxidized in two steps -- first to
.CoQ
− and then to CoQ. But
.CoQ
− is unstable and can easily errantly transfer an electron to an O2 molecule resulting in superoxide ion (
.O2−).
.O2− from Complex III escapes Mitochondria
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Complex I has been believed to generate
.O2− in one of the iron-sulfur clusters, which would go to the mitochondrial matrix where it could be neutralized by Mn−SOD. Experiments on isolated mitochondria identified the site of
superoxide generation to be at the flavine mononucleotide moiety of Complex but claims have been made that experiments on isolated mitochondria are misleading An experiment on isolated synaptosomes indicated that Complex I
inhibition increases H2O2 production . Most of the
.O2− generated from Complex III comes from
.CoQ
−, with about half going to the matrix to be neutralized and half floating toward the cytoplasm Thus,
.O2− from Complex I & III can cause lipid peroxidation of the inner mitochondrial membrane and mtDNA damage, whereas
.O2− from Complex III can damage the whole cell, including nDNA. Membrane potentials below 140 mV (potential resulting from the proton gradients across the inner mitochondrial
membrane) are not associated with
.O2
−, but above 140 mV
.O2
− generation increases exponentially with potential. Uncoupling proteins can be a device for reducing proton pressure (membrane potential), thereby reducing superoxide production.
Voltage drops between Complexes
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Higher voltage drops between energy states in the Complexes also result in greater capacity for superoxide generation. This may account for the high superoxide production associated with Complex I, which has a high voltage drop in
transferring its electrons to Complex III.
Oxidative damage to particular mitochondrial proteins in the flight muscles of houseflies has been identified as a biomarker of aging for those insects. Specifically, adenine nucleotide transferase enzyme in mitochondrial membranes and
the citric acid cycle enzyme aconitaseare particularly vulnerable to oxidative damage and are used to identify the "physiological age" of houseflies. Aconitase is readily oxidized by superoxide, a process that generates hydroxyl
radical
CoQ forms an important part of the antioxidant defense against these superoxide radicals [BIOCHEMISTRY AND CELL BIOLOGY 70:390-403 (1992)]. The Mn−SOD (SuperOxide Dismutase) of mitochondria can be induced to higher concentrations
by oxidative stress (in contrast to the cytoplasmic Cu/Zn−SOD which is constitutive rather than induced). Heart mitochondria also contains catalase (which is confined to peroxisomes in most other tissues) [BIOSCIENCE REPORTS
17(1):3-8 (1997)].
Associated with aging is a decline in the amount of CoQ in organs. A person 80 years old will typically have about half as much CoQ10 in the heart, lungs and spleen as a 20-year-old [LIPIDS 24(7):579-584 (1989)]. Declines in
functional mitochondria & CoQ10 with age is most damaging to those organs that have the highest energy demands per gram of tissue, namely: the heart, kidney, brain, liver and skeletal muscle, in that order [JOURNAL OF
INTERNAL MEDICINE 238:405-421 (1995)]. Neurons are the largest cells in the body and have the highest metabolic demands, with 70% of ATP produced required to maintain the sodium-potassium pump. Clinically, damage to brain and muscle
tissue are the first symptoms of mitochondrial disease. Mitochondria in the brain tissue of Alzheimer's Disease patients is particularly damaged. Therapy has included the B−vitamins that act as coenzymes in the respiratory chain
(thiamine, riboflavin, niacinamide) and CoQ10 [ACTA NEUROLOGICA SCANDINAVIA 92:273-280 (1995)].
mtDNA deletion mutations accumulate in post-mitotic cells with age [BIOCHIMICA ET BIOPHYSICA ACTA 410:183-193 (1999)]. The "
mitochondrial theory of aging" postulates that damage to mtDNA and organelles by free radicals leads to loss of mitochondrial function and loss of cellular energy (with loss of cellular function). Mutations in mtDNA occur at
10-20 times the rate seen in nuclear DNA. A significant portion of "photoaging" of the skin may be due to mtDNA deletions from singlet oxygen induced by ultraviolet light . Unlike nuclear DNA, mtDNA has no protective
histone proteins. And DNA repair is less efficient in mitochondria than in the nucleus. These factors account for the more rapid aging seen with Complex I & III as compared to Complex II & IV. Aging
mitochondria become enlarged and, if they can be engulfed by lysosomes, are resistant to degredation and contribute to lipofuscin formation
A comparison of 7 non-primate mammals (mouse, hamster, rat, guinea-pig, rabbit, pig and cow) showed that the rate of mitochondrial superoxide and hydrogen peroxide production in heart & kidney were inversely correlated with
maximum life span [FREE RADICAL BIOLOGY & MEDICINE 15:621-627 (1993)]. A similar study of 8 non-primate mammals showed a direct correlation between maximum lifespan and oxidative damage to mtDNA in heart & brain. There was a
4-fold difference in levels of oxidative damage and a 13-fold difference in longevity, supportive of the idea that mtDNA oxidative damage is but one of several causes of aging.
A comparison of the heart mitochondria in rats (4-year lifespan) and pigeons (35-year lifespan) showed that pigeon mitochondria leak fewer free-radicals than rat mitochondria, despite the fact that both animals have similar metabolic
rate and cardiac output. Pigeon heart mitochondria (Complexes I & III) showed a 4.6% free radical leak compared to a 16% free radical leak in rat heart mitochondria [MECHANISMS OF AGING AND DEVELOPMENT 98(2):95-111
(1997)]. Hummingbirds use thousands of calories in a day (more than most humans) and have relatively long lifespans (the broad-tailed hummingbird
Selasphorus platycerus has a maximum lifespan in excess of 8 years). Birds have less unsaturation (oxidizability) in their mitochondrial membranes and have higher levels of small-molecule antioxidants, such as ascorbate & uric
acid. Even for mammals there is a direct relationship between mitochondrial membrane saturation and maximum lifespan [JOURNAL OF LIPID RESEARCH; Pamplona,R; 39(10):1989-1994 (1998)].
Free-radicals from mitochondria result in damage to cellular protein, lipids and DNA throughout the cell. This damage has been implicated as a cause of aging. If the fatty acids entering the mitochondria for energy-yielding oxidation
have been peroxidized in the blood, this places an additional burden on antioxidant defenses. The greatest damage occurs in the mitochondria themselves, including damage to the respiratory chain protein complexes (leading to higher levels
of superoxide production), damage to the mitochondrial membrane (leading to membrane leakage of calcium ions and other substances) and damage to mitochondrial DNA (leading to further damage to mitochondrial protein complexes). An
experiment in yeast that improved the accuracy of mitochondrial protein synthesis demonstrated a 27% longer mean life span .
Mitochronrial Permaeability
Transition Port (MPTP)
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Mitochondria play a key role in
apoptosis ("cell suicide"). Release of cytochrome−c from mitochondria into the cytoplasm is the event which initiates apoptotic cell destruction by caspase enzymes. Release of cytochrome−c into the cytoplasm
can occur either by a Ca2+−dependent mechanism or a Ca2+−independent mechanism. In the
Ca2+−dependent case Ca2+ overload in the mitochondrion triggers opening of the Mitochondrial Permeability Transition Pore (
MPTP), which penetrates both the outer and inner membranes making a channel between the mitochondrial matrix and the cytosol outside the mitochondrion. The MPTP is a complex consisting of three proteins,
VDAC (porin) of the outer membrane,
ANT (Adenine Nucleotide Translocator) of the inner membrane and cyclophilin−D.
Cyclophilin−D protein binds to ANT to promote MPTP formation possibly by increasing the sensitivity of the MPTP components to the effects of Ca2+ . The entry of large solutes and accompanying water into
the matrix causes the mitochondrion to swell and burst, releasing cytochrome−c into the cytoplasm.
The
Ca2+−independent case requires two separate events for cytochrome−c release: (1) formation of large pores in the outer mitochondrial membrane by
Bax/Bak proteins and (2) release of cytochrome−c from the inner mitochondrial membrane . The Ca2+−independent case can lead to apoptosis, whereas the Ca2+−dependent case is
invariably associated with necrosis. In apoptosis the MPTP opens only briefly (if it opens at all), whereas in necrosis the MPTP remains open. Apoptosis requires ATP energy, but ATP energy is depleted if the MPTP remains open [NATURE;
Halestrap,A; 434:578-579 (2005)]. The threshold amount of Ca2+ which causes MPTP opening in lymphocytes, brain and liver of old mice is significantly lower than that of young mice [BIOCHEMICAL AND BIOPHYSICAL RESEARCH
COMMUNICATIONS; Mather,M; 273(2):603-608 (2000)].
Cardiolipin
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Cytochrome−c is normally held to the inner mitochondrial membrane by the lipid
cardiolipin (diphosphatidylglycerol). Cardiolipin composes 10% of the inner mitochondrial membrane and is present at lower concentrations in the outer mitochondrial membrane (especially near contact sites between the two
membranes). This distinctive lipid is found only in mitochondrial membranes. Mitochondrial membrane cardiolipin content declines with age, resulting in a decline in cytochrome−c activity. 40% lower cardiolipin content and 35% lower
cytochrome−c activity has been demonstrated in old rats compared to young rats. Restoration of membrane cardiolipin content restored cytochrome−c activity [FEBS LETTERS; Paradies,G; 406(1-2):136-138 (1997)].
Oxidation of cardiolipin releases cytochrome−c from the inner mitochondrial membrane, but cytochrome−c will not be released into the cytoplasm to induce apoptosis without the formation of large pores in the outer
mitochondrial membrane by
Bax/Bak protein.
Bax/Bak membrane permeabilization occurs preferentially at cardiolipin-rich contact sites between the outer and inner mitochondrial membrane But
Bax/Bak permeabilization of the outer membrane alone may be sufficient to induce apoptosis.
If only one or a few mitochondria release cytochrome−c apoptosis may not occur, but the damaged mitochondria would themselves be degraded. By this means a few aberrant mitochondria which are producing excessive free radicals can
be eliminated.
(For more about mechanisms of
apoptosis see
Cellular Senescence and Apoptosis in Aging)
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