Mitochondrion is the “department of energy (DOE)” of cell and the place for production, storage and supplement of energy. About 80% of energy required for cell life activities is provided by mitochondrion.
Mitochondria are linear or granular under the microscope. In general, the diameter of mitochondria is around 0.5~1.0 μm, and the length of mitochondria are around 1.5~3.0 μm. The “megamitochondria” can be seen in the skeletal muscle with the length of 8~10 μm. Additionally, the size of the mitochondria is closely related to the physiological states of cell (e.g. osmotic pressure, pH and temperature), such as the shapes of which are bubble in low osmotic pressure environment and linear in high osmotic pressure environment.
Typically, the number of mitochondria varies greatly depending on the types of cell. There are a lot of mitochondria in the cells that are high in metabolism, whereas there are fewer of them, such as every myocardial cell and liver cell contain an average of 2, 000 mitochondria, spermatozoa contains only a small amount of mitochondria with an average of 25, but the number of mitochondria in the oocyte is up to 300, 000. The number of mitochondria is relatively stable in the same type of cell.
Under the electron microscope, we can see that the mitochondrion is composed by outer membrane, inner membrane, intermembrane space and matrix space. The mitochondrion is a closed vesicle structure with composed by two layers of unit membrane. The inner membrane and outer membrane show intussusception state and form a structure of vesicle-in-vesicle, but they cannot connect with each other.
The average thickness of outer membrane is 5~7 nm. The outer membrane is of high permeability and has the characteristics of smooth and leveling. There are many channels on the membrane, which composed by the transport protein of porin. The diameter of the channels is about 2~3 nm, and substance with molecular weight less than 10,000 could pass through.
The ratio of lipid and protein in outer membrane is about 1: 1. The marker enzyme of outer membrane is monoamine oxidase.
The average thickness of inner membrane is 4.5 nm with low permeability. Inner membrane has the permselectivity. Only small and uncharged molecule whose molecular weight under 150 could pass through the inner membrane. Some of the larger molecules pass through the mitochondrial matrix via specific membrane translocation proteins.
The inner membrane of mitochondrion in-fold inwards to form the cristae. At the same time, the cristae expands the superficial area of inner membrane, which supply a lot of attachment surfaces for the enzyme to increase the metabolic efficiency.
There is electron transport respiratory chain inlaid in the inner membrane. Abundant elementary particles which are attach to the inner membrane and cristae. The elementary particle is the key device for the energy released during the electron transfer of the respiratory chain applying to the phosphorylation of ADP to generate ATP.
The ratio of lipid and protein in inner membrane is about 0.25: 1. The protein content of inner membrane is higher than that of other membrane components. Cytochrome C oxidase is the marker enzyme in inner membrane.
I. Elementary particle
It is a petiolate spherical granule attached on the mitochondrial inner membrane and cristae membrane matrix surface, and the chemical essence is F1F0 ATP synthase complex. The elementary particle is coupled with the electron transport chain on their function, and they are the apparatus for synthesizing ATP.
1. Head sector
Head sector, also called coupled factor F1, is composed of α3β3γδε, nine subunits of five types. The F1F0 ATP synthetase system of the head sector is equal to energy transition apparatus. When F1F0 ATP synthetase system is activated and the ADP phosphorylation to become ATP (ADP+Pi→ATP), the released energy will be stored in ATP.
2. Stalk
It is an Oligomycin Sensitivity Conferring Protein (OSCP) to connect F1 and F0 with the function of regulating the proton channel. The synthesis of ATP can be stopped by the interference of oligomycin. Oligomycin itself cannot directly have an effect on F1 factors, but by acting on the stalk protein, and then pass through the stalk to inhibit F1 factors.
3. Base Piece
It is a hydrophobic protein complex embedded in the inner membrane, and also called coupled factor F0. It not only plays the role of connecting the F1 and the inner membrane, but also acts as the transmembrane channel for the proton H+ flowing through F1.
II. Electron transport respiratory chain
It is a group of enzyme complex in the inner membrane which can transport H+ and transmit e-, including nicotinamide adenine dinucleotide I (NAD), flavin adenine dinucleotide (FAD), coenzyme Q, and cytochrome b, c1, c, a and a3. They are sequenced as a chain connected with each other at the inner membrane with the function of dissociating the hydrogen (equaling to a proton and an electron) released from the oxidative decomposition into H+ and e-. The electron passes through the electron transport respiratory chain enzyme system step by step, finally transmits into oxygen, after that, the 1/2 O2 combines with e- to become O2-, and then O2- combines with two H+ from the matrix to form H2O. In this process, the energy is released, too.
The electron transport respiratory chain could also act as the proton pump at the same time. When e- is passed through step by step, H+ is transferred from matrix space into intermembrane space, causing the concentration gradient difference of H+ in both sides of inner membrane. With the help of this potential energy from the H+ concentration difference, the H+ in the intermembrane space can regurgitate by following the concentration gradient difference and drive the F1F0ATP synthetase system at the head of elementary particle to catalyze the formation of ATP.
Intermembrane space, which is the lacuna between inner membrane and outer membrane, communicates with inter cristae space. At the contact site of the inner membrane and outer membrane with the membrane gap narrowing, called translocation contact site. The translocation contact site has many channel proteins and specific receptors, which are the channels for proteins and other substances in and out of the mitochondrion.
Matrix space is surrounded by the inner membrane and filled with the matrix, which is the place for oxido-reduction, containing various enzymes involving in tricarboxylic acid cycle, fatty acid oxidation, amino acid decomposition and so on. In addition, it also contains mtDNA, RNA, ribosomes, etc., which constitute a relatively independent genetic information replication, transcription and translation system
Mitochondria origin from the old anaerobic eukaryotic cell commensalised with the early bacteria. They established mutually beneficial relationships with host cells, and then evolved into mitochondria during the long-term evolution.
The precursor of eukaryotic cell is a highly evolved aerobic bacterium. A double-layer membrane can be formed by invagination, folding, and confluence of the increasing membrane and being surrounded by other membrane structure. Then the genome is surrounded in and finally evolved into mitochondrion with the function of respiration.
I. Intermediate wall division
The inner membrane of mitochondrion folds towards the centre, or one crista extends to the other edge of mitochondrial inner membrane to form the michitsura crista. So that, one mitochondrion is divided into two parts and completely divided.
II. Contraction division
Part of the mitochondrion central contracts and extends toward two edges membrane, making the center became a very narrow neck. Then the entire mitochondrion is at the state of dumbbell shape, and finally splits into two new mitochondria.
The mitochondrion gives birth to several small buds, and then buds split away from mater and evolve into mitochondrion.
In higher animals, mitochondrion is the only organelle that contains extra nuclear genetic materials. Mitochondrion not only has DNA, but also has a set of independent genetic information expression system. There are mtDNA, RNA, ribosomes and enzymes involved in protein synthesis existing in the mitochondrial matrix, so that it can be capable of independent expression and synthesis of protein.
Mtiochondrial genome is a double-stranded closed circular DNA molecule, called mtDNA. mtDNA does not bind to protein, and so it is a nudity and multi-copied DNA. mtDNA has tight structure and rare the non-coding sequences, which can replicate, transcribe and translate itself. But mtRNA has small molecule weight and little information. Additionally, genetic code of mtDNA is not exactly the same as the nuclear code.
The whole length of human mtDNA is 16,569 bp, and it totally codes 37 genes, including 22 tRNAs, 2 rRNA (12S rRNA and 16S rRNA) and 13 proteins encoding genes. There are two cicular strands in the mtDNA, one strand is heavy chain (H), another is light chain (L). Both of the two strands have coding function, and codes of the two strands are different from each other. Light chain (L) only codes 8 kinds of tRNA and one peptide, and the rest 14 kinds of tRNA, 2 kinds of rRNA, and 12 kinds of peptide are all coded by heavy chain(H).
The proteins coded by mtDNA are all the key enzyme or subunit for the completeness of electron transport and the process of oxidative phosphorylation. But the mitochondrial genome has only a small amount of genetic information and can only code 1% of proteins needed by itself.
More than 99% of proteins and enzymes in mitochondria are encoded by nuclear DNA. The ribosomal big/small subunits of mitochondrion and the enzymes needed for replication, transcription and translation of mtDNA are all coded by nuclear DNA. They are synthesized at the cytoplasmic free ribosome and then transported to mitochondrion installation or implement their function.
Therefore, the expression of mitochondrial genetic information is co-controlled by two sets of genetic systems: the mitochondrial genome and the nuclear genome. That is to say the mitochondrion is a semi-autonomous organelle.
MTS is a stretch of special amino acid sequence (about 20-80 amino acids) located at the N-terminus of the precursor protein amino acid, which can import precursor into mitochondrion.
The precursor protein is transmembrane under the guidance of MTS. MTS firstly combines with the mitochondrial receptor in the outer membrane, and then goes into the matrix through the translocation contact site between the inner and outer mitochondrial membrane. After that, MTS is removed immediately by the hydrolytic enzyme in the matrix. Because of the MTS can only act as the guide of precursor protein, so it is also called leader peptide.
Molecular chaperone is a type of newly recognizable peptide chain in the cell, which is the protein molecule and can help the new peptide chain to unfold, transmembrane and refold. It does not participate in the formation of the final product, so called “molecular chaperone”, such as heat shock protein 70 (hsp70).
During the transmembrane, precursor undergoes a process of unfolding and refolding been mature assembling, which is a process of energy dissipation with the participation of molecular chaperone. In this way, it is possible to effectively prevent the protein from being decomposed or condensed during the transport process.
Three functions of molecular chaperone:
I. Help the precursor protein to unfold
After being compounded in the cytoplasmic free ribosome, precursor protein combines with the molecular chaperone of Hsp70. Molecular chaperone has the function of unfoldase that can prevent protein molecule from folding and aggregating. As the densely-folded molecule can't pass through mitochondrial membrane, so Hsp70 is vital for transportation of precursor protein.
II. Assist precursor protein to transmembrane
When the unfold precursor protein comes to the surface of mitochondrion, precursor protein separates from Hsp70 and then combines with the specificity receptor covered with the mitochondrial membrane. Then the receptor couples with the channel protein and forms transmembrane channel at the translocation contact site.
Guide peptides waveringly do the Brownian movement unsteadily in the transmembrane channel. In this process, a type of mitochondrial matrix heat shock protein 70 (mthsp70), also called molecular chaperone, can crosslink with guide peptides that enter into the mitochondrial cavity. Once guide peptide enters into mitochondrial cavity, one molecule mthsp70 will combine with it immediately to prevent guide peptide from moving backward. Mthsp70 must haul the guide peptides and meanwhile attach to both guide peptides and mitochondrial membrane. This arrangement makes mthsp70 produce towing power by change the structure. With guiding peptides combine more mthsp70, mthsp70 haul the guide peptides and precursor protein enters transfer orbit.
III. Help the precursor protein to refold
After precursor protein transferring into mitochondrial matrix, guide peptides are hydrolyzed immediately by guide peptide hydrolyses. Meanwhile, mthsp70 helps to refold precursor protein as fold-factor to regain it native conformation, so that it can restore natural conformation to perform functions.
Cell respiration is also called cellular oxidation or biological oxidation. It is the process that, with the participation of O2, a various of energy-supplied macromoleculars (e.g. glucose, lipid and protein) in the cells can be completely oxidized to produce CO2 and H2O, and at the same time, the energy released from the catabolism is saved in the ATP. The mitochondrion is the key place for the cellular respiration and the energy transition. The main function of mitochondrion is to carry out the three carboxylic acid cycle and oxidative phosphorylation to synthesize ATP
Features
1. The essence of biological oxidation is a series of redox reactions catalyzed by enzyme system in mitochondrion.
2. The energy produced by biological oxidation is saved in the high energy phosphate bond of ATP.
3. The reaction process is carried out step by step, and energy is gradually released.
4. The whole process is carried out under the condition of constant temperature (37℃) and constant voltage, and requires water to participate.
Energy transition molecule-ATP
ATP is a kind of high energy phosphate compound. When biological oxidation occurs, the released energy is saved in the high energy phosphate bond of ATP through the ADP phosphorylation, and so that achieves the energy conversion. When the cell requires energy to perform its function, the high energy phosphate bond hydrolysis and release energy to meet the need of the cellular life activities.
Short formula for energy release and energy storage reaction of ATP:
Dephosphorylation (energy release)
A-P～P～P ————-→ A-P～P + Pi + energy
（ATP） phosphorylation (energy storage) （ADP）
Energy substances such as sugar, fat, protein, etc, taken from food are firstly digested into amino acids, fatty acids, monosaccharides and other small molecules, and then can be absorbed by cells.
Taking glucose as an example, cell oxidation is divided into four major steps: glycolysis, generation of acetyl coenzyme A (CoA), tricarboxylic acid cycle (TAC cycle) and electron transport and coupling oxidative phosphorylation.
Place: cytosol
At the function of glycolysis enzyme system, one molecule of glucose produces two molecules of pyruvate, and at the same time takes off 2 couples of H+, which are completely given to hydrogen carrier NAD+ (nicotinamide adenine dinucleotide) to carry, and then forms two molecules of NADH+H+ and purely produces two molecules of ATP.
Glucose (C6H12O6) + 2NAD+ + 2ADP + 2Pi → 2 pyruvates (CH3COCOOH) + 2NADH + 2H+ + 2ATP
Place: mitochondrial matrix
When the cell is in aerobic condition, the pyruvate passes through mitochondrial membrane and goes into mitochondrial matrix. Under the effect of the pyruvate dehydrogenase system, pyruvate oxidates (dehydrogenize and decarboxylate), combines with CoA (coenzyme A), and then produces the acetyl CoA (acetyl coenzyme A). The dropped two pairs of H+ are delivered to hydrogen carrier.
2 pyruvates (CH3COCOOH) + 2CoA (HSCoA) +2NAD+ → 2 (CoA) CH3CO-SCoA + 2NADH + 2H+ + 2CO2
When the cells are in anoxic state, pyruvate can continue its anaerobic glycolysis, called lactic acid fermentation, producing two molecules of lactic acids and two molecules of ATP. This is the energy supply form for anaerobic organisms or aerobic organisms lacking of oxygen supplement. For example, the over fatigued muscles cells produce too much lactic acid by anaerobic glycolysis and make feel ache.
Place: mitochondrial matrix
TCA is an ubiquitous metabolic pathway in aerobic organisms. And TCA is also a common pathway for the complete oxidation and decomposition of glucose, amino acids and fatty acids. The name of TAC cycle is given because of the major intermediate metabolite of citric acid in this cycle has three carboxyls.
Acetyl CoA firstly combines with the oxaloacetate (OxAc) to form citric acid and enters into a loop system formed by a series of reactions, and then the citric acid enters TAC cycle.
Under the effects of a series of enzyme system, Citric acid undergoes dehydrogenation and decarboxylation, reproduces the OxAc at the end edge of cycle, OxAc combines with another acetyl CoA to form citric acid, and then starts the next cycle. The taken-off H+ are delivered to hydrogen carrier, such as NAD+ and FAD.
2 acetyl CoA (CH3CO-SCoA) + 6NAD++ 2FAD + 2ADP + 2Pi + 6H2O → 2 OxAc + 6NADH + 6H++ 2FADH2 + 2ATP+ 4CO2
During the glycolysis to TAC cycle, one molecule of glucose can totally produce four molecules of ATP and six molecules of CO2. Meanwhile, it takes off 12 couples of H+, among which 10 couples are hydrogen carriers in the form of NAD+ and 2 couples are hydrogen carriers in the form of FAD+ (Flavin Adenine Dinucleotide). And the energy is transmitted to NAD+ and FAD+, and then makes reduced to NADH+H+ and FADH2, respectively.
Place: mitochondrial inner membrane
I. Electron transport
It refers to the electronic movement of redox reactions in living organisms. The finally complete oxidation product of the macromolecule is CO2 and H2O. However, not the formation of H2O but H is seen in the above reaction. The entire oxidative process do not finish until these H combines with O to produce H2O.
H cannot directly combine with O2. H firstly dissociates into H+ and e-, the election passes through the electron transport respiratory chain in the mitochondrial inner membrane step by step, and finally transmit to oxygen. e combines with 1/2O2 to form O2-, Then O2- combines with 2H+ in the matrix to form H2O.
II. Oxidative phosphorylation
All the hydrogen atoms taken-off in every phases of cellular oxidation are dissociated, transmit step by step through the electron transport respiratory chain of inner membrane, and finally combine with oxygen to form water. The energy released in the electron transport process is used to catalyze ADP phosphorylation to produce ATP by the ATP synthetase complex and make the energy transition, this is called oxidative phosphorylation.
At the electron transport respiratory chain, there are three coupling sites for energy release and ATP formation. Each site releases energy to make one molecule of ADP phosphorylated to produce one molecule of ATP.
e- is transmitted by the electron transport respiratory chain step by step, and meanwhile acts as the proton pump. H+ is continuously pumped from the matrix space into intermembrane space, which leads to the gradient differences of H+ concentration in both sides of mitochondrial inner membrane. With the help of potential energy held in the H+ concentration differences, the pumped proton counter flows along the concentration gradient by proton channel, drives the F1F0 ATP synthetase system of granule's head and catalytics phosphorylation of ADP to produce ATP.
One molecule of glucose is completely phosphorylated in the cell to form CO2 and O2, and produces 32 pure ATPs. In the cytoplasm, the glycolysis can produce purely 2 molecules of ATP, tricarboxylic acid cycle could produce 2 molecules of ATP, and the inner membrane respiratory chain produce 28 molecules of ATP.
Chemiosmotic coupling hypothesis is firstly proposed by English biochemist Peter Mitchell in 1961, and he won the Nobel Prize in Chemistry in 1978.
Chemiosmotic coupling hypothesis explains the production of the electrochemical proton gradient potential energy (or called proton motive force, PMF), and explains that the electrochemical proton gradient is the mechanism of electron transport couples with the oxidative phosphorylation.
The synthesis of ATP from ADP and Pi is under the catalysis of F0F1ATP synthase. However, how exactly does F1 factor utilize electrochemical gradient potential energy of H+ to make ADP and Pi establish covalent bonds to form ATP? In 1998, the biochemist Paul D. Boyer proposed the binding-change mechanism and won the Nobel Prize in Chemistry in 1997.
Coupling factor F1 is composed by α3β3γδε five subunits. 3α subunit and 3β subunit form 3α3β hexamer, which sequences in interior phases around the same center axis like the “orange slice”. γ subunit, δ subunit and ε subunit combine together with stronger affinity to form “axis” and “trochanter (rotor)” inserted in the 3α3β hexamer center. It can not only make F1 couples with F0 coupling factor, but also regulate the three β subunits' conformational change by rotating.
In the synthetic process of ATP, under the driving rotation of “axis” and “trochanter”, three β subunits of F1 are at different conformational states at the same time. That is to say, each β subunit must experience three different states (the tight combined state, loosen combined state, and empty state) orderly when catalyzing synthesizes one molecule of ATP. Driven by proton stream, when 3α3β hexamer rotates 120°, every β subunit will occur conformational change accordingly. On the one hand, when substrates (ADP and Pi) combine with the active sites of F1, the state changes from loosen to tight, and it is benefit for ADP to react with Pi to synthesize ATP; On the other hand, when ATP combines with active site of F1, its state changes from tight to loose, and it is benefit for the release of production (ATP).
Binding-change mechanism explains the function of coupling factor F1 on ATP synthesis. From the aspect of structure, ATP synthase complex is like an exactly accurate molecule “water turbine” machine. When H+ counterflows from the F0 proton channel through the concentration gradient, the turbine (basal lamina F0 coupling factor) and wheels (head sector F1 coupling factor) are driven to roll, causing the conformational change of leaf (F1 coupling factor of head sector), ADP tightly combines with Pi to produce ATP. When the conformational change occurs again on the leaf (F1coupling factor of head sector) from the tight state to loosen state, ATP is released into matrix, and the proton breaks away from the enzyme into matrix. The ADP and Pi needed for the oxidative phosphorylation are transported from cytoplasm to mitochondrial matrix, while the synthesized ATP is needed to transport to outer of mitochondrion. There are specific transport proteins to transport these materials in-and- out of the mitochondrion.
The diseases caused by the defects of mitochondrial structure and function are called mitochondrial disorders, which mainly refer to the diseases caused by changes in mitochondrial genes. Mitochondrial disorders mainly affect the brain and muscle system, so they are also called mitochondrial encephalomyopathy.
The mitochondrion is sensitive to the change of environmental factors. Some environmental factors, such as ischemia, hypoxia, toxicants, drugs, radiation, microwave and so on, can directly cause mitochondrial structure and functional abnormalities. The mitochondrion can be used as an important basis for cytopathological examination or an indicator of environmental factors.
When the body is ischemic, the intracellular oxygen pressure will also accordingly decrease. In the ischemic and hypoxic environment, oxidative phosphorylation of mitochondrial function is weakened or even stops immediately. The level of intracellular ATP decreases rapidly and could decrease to zero in one minute if the ischemia lasts 30 to 60 minutes, there will be a point of no return, the mitochondrial membrane's permeability change, the cells will die inevitably. Metabolic active cells, such as liver cells and cardiomyocytes are more pronounced, are causing not only swelling but also the disintegration of crista leading to disappears, and so many obvious empty vacuoles are seen under the light microscope.
The cyanide and the cytochrome oxidase are of high affinity, after the combination of the two, the activity of the cytochrome oxidase will lose and the respiratory chain will be stopped. The carbon monoxide (CO) causes the anoxia and the cellular respiratory will be stopped.
Mitochondrial inherited diseases are a variety of diseases caused by mtDNA mutation, and have the characteristics of maternal inheritance. Because of the whole mtDNA come from oocyte. So that, the mutative mtDNA is just transmitted by mother, but not father.
I. High rate of mutation: mtDNA is bare and the lack of DNA damage repair system, so its mutation rate is high, resulting in the difference of mtDNA sequences between individuals and populations.
II. Polyplasmy: There are thousands of mtDNA copies in cell mitochondrion, which are randomly and unequally allocated into daughter cells during cell division, resulting in the number of copies varies greatly, ranging from several to hundreds of thousands.
III. Heteroplasmy: It includes sequence-based heteroplasmy and length-based heteroplasmy. DNA mutations will result in the presence of both wild-type and mutant sequences in the cell.
IV. Threshold effect: Only when the mutated mtDNA reaches its threshold that will show the disease symptom.
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