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Mitochondria (singular: mitochondrion) are double-membrane-bound organelles found in nearly all eukaryotic cells. They are the main site of aerobic respiration in most eukaryotes, where they couple the oxidation of nutrients to the synthesis of adenosine triphosphate (ATP) through oxidative phosphorylation. They also take part in the metabolism of amino acids, lipids, and nucleotides, in the synthesis of iron-sulfur clusters and heme, in calcium signaling, in innate immune signaling, and in programmed cell death. They are not the only source of ATP in cells, because glycolysis in the cytosol also makes ATP, and a few eukaryotes have lost mitochondria or have reduced forms of them (hydrogenosomes and mitosomes), and Monocercomonoides has none. Mitochondria descend from an alphaproteobacterial endosymbiont, and they keep a small genome of their own.

Mitochondria are often drawn as isolated bean-shaped organelles, but in living cells they form dynamic networks that fuse, divide, and move along the cytoskeleton. Their number per cell varies from one to thousands, depending on the cell type and its energy demand. A mitochondrion has four compartments:

• Outer membrane. A lipid bilayer that is permeable to ions and to molecules of up to about 5 kDa because of abundant channels, in particular the voltage-dependent anion channel (VDAC, also called mitochondrial porin). Protein complexes in the outer membrane import proteins (the TOM complex) and assemble _β_-barrel proteins (the SAM complex). The outer membrane is also a site of contact with the endoplasmic reticulum, where lipids and calcium are exchanged.
• Inner membrane. A bilayer that is impermeable to most ions and small molecules, which allows the maintenance of a proton gradient. It has a high content of protein (about 75 percent by mass) and of the phospholipid cardiolipin. Specific carriers transport metabolites, such as the ADP/ATP carrier and the phosphate carrier. The inner membrane contains the complexes of the electron transport chain and ATP synthase. Cardiolipin is not a seal against protons by itself, but it stabilizes the respiratory complexes and supercomplexes and affects the curvature of the membrane.
• Cristae. The inner membrane is folded into invaginations called cristae, which greatly increase its area. Their shape is maintained by the MICOS complex, by dimers of ATP synthase that bend the membrane at the tips of the folds, and by the protein OPA1. The cristae are connected to the part of the inner membrane that is next to the outer membrane (the inner boundary membrane) by narrow cristae junctions, and the shape of the cristae changes with the metabolic state.
• Matrix. The compartment within the inner membrane is a dense solution of proteins with hundreds of enzymes, such as those of the citric acid cycle and of the beta oxidation of fatty acids, the mitochondrial genome, and mitochondrial ribosomes.
• Intermembrane space. The narrow space between the two membranes. It collects the protons that are pumped by the respiratory chain, and it contains cytochrome c, which transfers electrons in the respiratory chain and is also released to start apoptosis.

Typical mitochondria are about 0.5 to 1 μm wide and a few micrometers long, though the length of the tubules in a network can be much greater. The mitochondrial volume can be a large fraction of the cell, for example about 20 to 30 percent in some heart muscle cells.

The electron transport chain

Substrates are oxidized by the citric acid cycle, the oxidation of fatty acids, and other pathways, and electrons are collected by NADH and FADH₂. These carry the electrons to the respiratory chain in the inner membrane. NADH donates electrons to Complex I (NADH:ubiquinone oxidoreductase), and FADH₂ donates them through Complex II (succinate dehydrogenase) and other dehydrogenases, so that both reduce ubiquinone. Ubiquinol passes electrons to Complex III (cytochrome bc₁), then to cytochrome c, and then to Complex IV (cytochrome c oxidase), which reduces molecular oxygen (O₂) to water. As electrons pass through Complexes I, III, and IV, the energy released is used to pump protons from the matrix to the intermembrane space. Complex II does not pump protons. In many species, the complexes associate in supercomplexes (respirasomes), although how much this matters for function is debated.

Quantitative Bioenergetics of the Mammalian Mitochondrial Respiratory Complexes

The proton-motive force and ATP synthesis

The proton pumping creates an electrochemical gradient across the inner membrane, the proton-motive force:

where Δψ is the electrical potential difference across the membrane (the matrix side is negative, about −150 to −180 mV), ΔpH is the difference in pH (the matrix is more alkaline by about 0.5 to 0.8 units), R is the gas constant, T is the absolute temperature, and F is the Faraday constant. At 37 °C, 2.303 _RT_/_F_ is about 61.5 mV per pH unit, so that ΔpH contributes about 30 to 50 mV and the total is about 180 to 220 mV, mostly from the electrical term. The process is the chemiosmotic coupling that was proposed by Peter Mitchell. The protons flow back to the matrix through the F_O part of ATP synthase, which turns a ring of c subunits (8 in mammals) and drives the rotation of the central stalk in the F₁ head. The rotation causes conformational changes in three catalytic sites, which produce ATP from ADP and phosphate, as described in Boyer's binding-change mechanism. The synthase rotates at roughly 100 to 150 revolutions per second under physiological conditions, and not "thousands of revolutions per minute" in the sense of a macroscopic motor (it is about 6,000 to 9,000 revolutions per minute, which is of that order, but it is a small molecular machine in a viscous environment, and the speed depends on conditions).

The yield of ATP is about 2.5 ATP per NADH and about 1.5 per FADH₂, taking into account about 8/3 ≈ 2.7 protons per ATP made by the synthase and about one more proton for the export of the ATP and import of phosphate. Complete oxidation of one glucose molecule yields about 30 to 32 ATP in total. For example, with the usual values of 10 protons pumped per NADH, one NADH gives 10 / 3.7 ≈ 2.7 ATP in a simple calculation, and the accepted consensus value is 2.5 because of some proton leak and slippage.

Proton leak and thermogenesis

Not all of the proton flow passes through ATP synthase. Some protons leak across the inner membrane, a process that accounts for a substantial part of the resting metabolic rate in some tissues, and that depends partly on the ADP/ATP carrier. In brown adipose tissue, uncoupling protein 1 (UCP1, thermogenin) is activated by fatty acids and provides a regulated proton channel. The energy of the gradient is then released as heat, which is the basis of non-shivering thermogenesis in infants, small mammals, and hibernators. It is a regulated process and not a sabotage of the organelle. The heat that is made in this way is released as thermal energy in the tissue, not as "thermal radiation". Some plants, fungi, and protists have an alternative oxidase that transfers electrons from ubiquinol to oxygen without proton pumping at Complexes III and IV.

Reactive oxygen species

A small fraction of the electrons (of the order of 0.1 to 2 percent of the oxygen consumed, although the value is debated and depends on the conditions) leaks to oxygen and forms superoxide, mainly at Complexes I and III. Superoxide is converted by superoxide dismutase to hydrogen peroxide, which is removed by peroxidases and catalase. Reactive oxygen species (ROS) damage lipids, proteins, and DNA at high levels, but they also act as signals at lower levels.

• Metabolism. Mitochondria contain the enzymes of the citric acid cycle, the oxidation of pyruvate and of fatty acids, parts of the urea cycle, the synthesis of heme, and the synthesis of iron-sulfur clusters, which are needed by many proteins in the cell. They take part in the metabolism of amino acids and in the synthesis of some lipids, such as cardiolipin.
• Calcium signaling. Mitochondria take up calcium through the mitochondrial calcium uniporter (MCU), which is driven by the membrane potential. This buffers cytosolic calcium and activates dehydrogenases of the matrix, and an excess of calcium can trigger the opening of the permeability transition pore and cell death.
• Innate immunity and signaling. MAVS, a protein of the outer membrane, is a hub for antiviral signaling after the detection of RNA by RIG-I and MDA5. Released mitochondrial DNA can activate the cGAS-STING pathway and the inflammasome, and it acts as a damage signal.
• Heat and other roles. Specialized mitochondria are found in steroid-producing cells, where the first steps of steroid hormone synthesis occur in the organelle.

Organization

Mitochondria have their own genome (mtDNA). In humans, it is a circular molecule of 16,569 base pairs with 37 genes, which encode 13 proteins (all subunits of the respiratory complexes and of ATP synthase), 22 transfer RNAs, and 2 ribosomal RNAs. The majority of the roughly 1,000 to 1,500 proteins of the organelle are encoded in the nucleus, made in the cytosol, and imported. The mtDNA is organized in nucleoprotein complexes called nucleoids. The main protein is TFAM, and it does not have histones. The size and form of mtDNA differ widely among eukaryotes, from about 6 kb in Plasmodium to hundreds of kb in plants, with linear and multiple molecules in some groups. The mitochondrial ribosomes of mammals are 55S and not 70S, and they have a high proportion of protein. In bacteria, ribosomes are 70S. In other groups, such as yeasts, the mitochondrial ribosomes are closer to 70S, so the statement that they are the same as the ribosomes of prokaryotes is not true. The mitochondrial genetic code deviates from the standard code (for example UGA codes for tryptophan in vertebrate mitochondria).

Inheritance, mutation, and heteroplasmy

In most animals, mtDNA is inherited from the mother, because the paternal mitochondria are eliminated or diluted after fertilization. Biparental inheritance has been reported in rare cases. mtDNA accumulates mutations faster than nuclear DNA in animals, with a rate that is about 10 times higher, in part because of the replication errors of the mitochondrial DNA polymerase gamma, and in part because of oxidative damage, although the contribution of ROS to mutations is less than was once believed. The lack of histones is not the reason for the high rate. mtDNA has limited recombination and repair compared with the nucleus, which results in the accumulation of deleterious mutations. In plants, the rate is low.

A cell has hundreds to thousands of copies of mtDNA, and a cell can contain a mixture of normal and mutant copies, which is called heteroplasmy. A mutation usually causes disease only when its fraction exceeds a threshold, often about 60 to 90 percent, which depends on the mutation and the tissue (the threshold effect). Tissues with a high demand for energy, such as the brain, heart, skeletal muscle, and retina, are most often affected. Examples of diseases that are caused by mtDNA mutations are Leber hereditary optic neuropathy, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes), and MERRF. Mutations in the nuclear genes for mitochondrial proteins also cause mitochondrial disease, and together mitochondrial diseases are among the most common inherited metabolic disorders. A fraction of mutations is removed in the germline by selection, and techniques of mitochondrial replacement have been developed to prevent the transmission of mutant mtDNA, and they are regulated. The heteroplasmy level changes over time, and it can be described by a random drift in which the variance of the change in fraction h per generation of a segregating unit is approximately _h_(1 − h) / n_e, where n_e is the effective number of segregating units. The "bottleneck" in the germline, with a small n_e, explains the large differences in heteroplasmy among siblings.

• Fusion. Fusion of the outer membranes requires the mitofusins Mfn1 and Mfn2, and fusion of the inner membranes requires OPA1. Fusion allows mixing of contents, which complements damaged components and helps to maintain mtDNA.
• Fission. The dynamin-related protein Drp1 is recruited to the outer membrane by receptors and forms a ring that constricts the organelle, often at sites of contact with the endoplasmic reticulum. Fission allows distribution of mitochondria in cell division and the isolation of damaged parts.
• Mitophagy. Damaged mitochondria that have lost their membrane potential are marked for degradation by the kinase PINK1 and the ubiquitin ligase Parkin, and are removed by autophagy and lysosomes. Mutations in the genes PINK1 and PRKN (Parkin) cause early-onset forms of Parkinson's disease. Other receptors also work in pathways that are independent of Parkin.
• Biogenesis. The number of mitochondria is regulated by the transcriptional coactivator PGC-1_α_ and other factors, in response to exercise, cold, and energy demand.
• Transport. Mitochondria are moved along microtubules by kinesin and dynein motors and are anchored where energy is needed, for example in axons and at synapses.

Apoptosis

In the intrinsic pathway of apoptosis, signals of stress cause the proteins BAX and BAK to form pores in the outer membrane (mitochondrial outer membrane permeabilization, MOMP). Cytochrome c and other proteins are released into the cytosol, where cytochrome c binds to APAF-1 and forms the apoptosome, which activates caspase-9 and then the executioner caspases, and the cell dies in an ordered way. The BCL-2 family of proteins controls MOMP. Pro-survival members such as BCL-2 and BCL-XL inhibit BAX and BAK, and other members (BH3-only proteins) promote activation. Apoptosis helps to remove damaged and potentially cancerous cells, but its role in cancer is one of several, and it is not the "ultimate" executioner of cells, because there are other forms of cell death, including necroptosis and pyroptosis, and apoptosis also can occur by extrinsic receptor pathways without the mitochondrion.

Endosymbiotic Theory and Gene Transfer

Mitochondria evolved from an endosymbiotic alphaproteobacterium, as proposed by Lynn Margulis in 1967 (with earlier ideas by Konstantin Mereschkowski and Ivan Wallin) in the endosymbiotic theory. Evidence includes the presence of a genome that is related to bacterial genomes, an inner membrane with cardiolipin, bacterial-type features such as the initiation of translation with _N_-formylmethionine, _β_-barrel proteins of the outer membrane, division by fission that involves relatives of bacterial proteins, and phylogenetic trees that place mitochondrial genes within the alphaproteobacteria. During evolution, most of the genes of the endosymbiont were lost or transferred to the nucleus, which is called endosymbiotic gene transfer.

The nature of the host and the timing are debated. Many recent phylogenomic analyses place the eukaryotic nuclear lineage within the Asgard archaea (such as the Heimdallarchaeia), and the cultivation of Prometheoarchaeum syntrophicum in 2020 supported models of syntrophic interactions. The hypothesis that the host "engulfed" the bacterium by phagocytosis is one of several models, and others propose that the bacterium was taken up in a symbiosis with a host that did not yet have a phagocytic lifestyle. The mitochondrion is thought to have been present in the last eukaryotic common ancestor, which lived in the range of 1.2 to 2 billion years ago, although the date is uncertain, and the question of whether the mitochondrion came before or after other eukaryotic features, such as the nucleus and the cytoskeleton ("mito-early" or "mito-late"), is disputed.

Energy and the size of cells

A bacterium makes ATP at its plasma membrane, and the surface-to-volume ratio of a sphere of radius r is:

so that, as a cell grows, the available membrane area per unit volume falls. This limits the rate of energy production per unit volume. Nick Lane and William Martin proposed in 2010 that the internalization of the energy-producing membranes in mitochondria allowed eukaryotes to have much larger genomes, and a much larger energy budget per gene, than prokaryotes. In their analysis, the power per gene of eukaryotes is about 10³ to 10⁵ times that of prokaryotes. This view is influential and is also criticized. Others argue that energy is not the main limit to genome size or that the large eukaryotic genomes arose through other mechanisms and that the bacteria with large cells, such as Thiomargarita and Epulopiscium, use other strategies. The idea that energy was limiting is therefore a hypothesis and not a settled conclusion. Eukaryotic cells also have a larger volume than bacteria, but the cell volume (about 10³ to 10⁵ times more) is only one component. The surface-to-volume argument is also not complete, because the energy produced is proportional to the membrane area, and the number of mitochondrial membranes can be increased by the folding of the cristae, which supports the proposal.

Mitochondria are studied by electron microscopy and cryo-electron tomography, by fluorescence microscopy with dyes that depend on membrane potential (such as TMRM), by respirometry (the oxygen consumption rate in intact cells, such as in the Seahorse assay, or in isolated mitochondria with high-resolution respirometers), by proteomics of isolated organelles (MitoCarta), and by sequencing of mtDNA. They are separated from tissue by differential centrifugation.

Why have mitochondria retained a small 16.6-kilobase genome instead of transferring all genes to the nucleus?

According to the Co-location for Redox Regulation (CoRR) and extreme hydrophobicity hypotheses, the 13 protein-coding genes retained in human mtDNA encode highly hydrophobic core membrane subunits of Complexes I, III, IV, and V (such as MTCO1 and MTCYB). If synthesized in the cytosol, these polytopic transmembrane helices would aggregate or misroute to the endoplasmic reticulum, and local synthesis allows rapid, organelle-specific redox control of electron transport.

How does the PINK1-Parkin pathway selectively identify and clear damaged mitochondria?

In healthy mitochondria with an intact inner membrane potential (Δψ ≈ −180 mV), the serine/threonine kinase PINK1 is continuously imported via TOM/TIM complexes and cleaved by the intramembrane protease PARL. When a mitochondrion depolarizes, import stalls and full-length PINK1 accumulates on the outer membrane, where it phosphorylates ubiquitin at Ser65 to recruit and activate the E3 ligase Parkin, coating the damaged organelle in polyubiquitin chains for autophagosomal engulfment.

Why is the theoretical P/O ratio of NADH approximately 2.5 ATP rather than the historical integer value of 3?

Oxidation of one NADH molecule translocates 10 H⁺ across the inner membrane (4 at Complex I, 4 at Complex III, and 2 at Complex IV). Mammalian F₁F_O-ATP synthase possesses an 8-subunit _c_-ring rotor, requiring 8/3 ≈ 2.67 H⁺ per synthesized ATP, plus 1 H⁺ for electrogenic ATP^4-/ADP^3- exchange and H₂PO₄^- symport (3.67 H⁺ total per ATP), yielding 10 / 3.67 ≈ 2.72 ATP (or ~2.5 after accounting for basal proton leak).

• Mitochondrial DNA
• Oxidative phosphorylation
• Endosymbiotic theory
• Mitochondrial disease
• Electron transport chain
• ATP synthase
• Apoptosis