the process, produces reduced cofactors (three molecules of nadh and one molecule of fadh2) that are a source of electrons for the electron transport chain, and a molecule of gtp (which is readily converted to an atp). o2 and nadh: energy-releasing reactions main articles: oxidative phosphorylation and electron transport chain electron transport chain in the mitochondrial intermembrane space the electrons from nadh and fadh2 are transferred to oxygen (o2) and hydrogen (protons) in several steps via an electron transport chain. nadh and fadh2 molecules are produced within the matrix via the citric acid cycle and in the cytoplasm by glycolysis. reducing equivalents from the cytoplasm can be imported via the malate-aspartate shuttle system of antiporter proteins or fed into the electron transport chain using a glycerol phosphate shuttle. the major energy-releasing reactions that make the mitochondrion the "powerhouse of the cell" occur at protein complexes i, iii and iv in the inner mitochondrial membrane (nadh dehydrogenase (ubiquinone), cytochrome c reductase, and cytochrome c oxidase). at complex iv, o2 reacts with the reduced form of iron in cytochrome c: o 2 + 4 h + ( aq ) + 4 fe 2 + ( cyt c ) ⟶ 2 h 2 o + 4 fe 3 + ( cyt c ) +4h+(aq)+4fe^(cyt\,c)->2h2o+4fe^(cyt\,c)}}} δ r g o ′ = − 218 kj/mol g^=-218 releasing a lot of free energy from the reactants without breaking bonds of an organic fuel. the free energy put in to remove an electron from fe2+ is released at complex iii when fe3+ of cytochrome c reacts to oxidize ubiquinol (qh2): 2 fe 3 + ( cyt c ) + qh 2 ⟶ 2 fe 2 + ( cyt c ) + q + 2 h + ( aq ) (cyt\,c)+qh2->2fe^(cyt\,c)+q+2h+(aq)}}} δ r g o ′ = − 30 kj/mol g^=-30 the ubiquinone (q) generated reacts, in complex i, with nadh: q + h + ( aq ) + nadh ⟶ qh 2 + nad + + nadh -> qh2 + nad+ δ r g o ′ = − 81 kj/mol g^=-81 while the reactions are controlled by an electron transport chain, free electrons are not amongst the reactants or products in the three reactions shown and therefore do not affect the free energy released, which is used to pump protons (h+) into the intermembrane space. this process is efficient, but a small percentage of electrons may prematurely reduce oxygen, forming reactive oxygen species such as superoxide. this can cause oxidative stress in the mitochondria and may contribute to the decline in mitochondrial function associated with aging. as the proton concentration increases in the intermembrane space, a strong electrochemical gradient is established across the inner membrane. the protons can return to the matrix through the atp synthase complex, and their potential energy is used to synthesize atp from adp and inorganic phosphate (pi). this process is called chemiosmosis, and was first described by peter mitchell, who was awarded the 1978 nobel prize in chemistry for his work. later, part of the 1997 nobel prize in chemistry was awarded to paul d. boyer and john e. walker for their clarification of the working mechanism of atp synthase. heat production under certain conditions, protons can re-enter the mitochondrial matrix without contributing to atp synthesis. this process is known as proton leak or mitochondrial uncoupling and is due to the facilitated diffusion of protons into the matrix. the process results in the unharnessed potential energy of the proton electrochemical gradient being released as heat. the process is mediated by a proton channel called thermogenin, or ucp1. thermogenin is primarily found in brown adipose tissue, or brown fat, and is responsible for non-shivering thermogenesis. brown adipose tissue is found in mammals, and is at its highest levels in early life and in hibernating animals. in humans, brown adipose tissue is present at birth and decreases with age. storage of calcium ions transmission electron micrograph of a chondrocyte, stained for calcium, showing its nucleus (n) and mitochondria (m) the concentrations of free calcium in the cell can regulate an array of reactions and is important for signal transduction in the cell. mitochondria can transiently store calcium, a contributing process for the cell's homeostasis of calcium. their ability to rapidly take in calcium for later release makes them good "cytosolic buffers" for calcium. the endoplasmic reticulum (er) is the most significant storage site of calcium, and there is a significant interplay between the mitochondrion and er with regard to calcium. the calcium is taken up into the matrix by the mitochondrial calcium uniporter on the inner mitochondrial membrane. it is primarily driven by the mitochondrial membrane potential. release of this calcium back into the cell's interior can occur via a sodium-calcium exchange protein or via "calcium-induced-calcium-release" pathways. this can initiate calcium spikes or calcium waves with large changes in the membrane potential. these can activate a series of second messenger system proteins that can coordinate processes such as neurotransmitter release in nerve cells and release of hormones in endocrine cells. ca2+ influx to the mitochondrial matrix has recently been implicated as a mechanism to regulate respiratory bioenergetics by allowing the electrochemical potential across the membrane to transiently "pulse" from δψ-dominated to ph-dominated, facilitating a reduction of oxidative stress. in neurons, concomitant increases in cytosolic and mitochondrial calcium act to synchronize neuronal activity with mitochondrial energy metabolism. mitochondrial matrix calcium levels can reach the tens of micromolar levels, which is necessary for the activation of isocitrate dehydrogenase, one of the key regulatory enzymes of the krebs cycle. cellular proliferation regulation the relationship between cellular proliferation and mitochondria has been investigated. tumor cells require ample atp to synthesize bioactive compounds such as lipids, proteins, and nucleotides for rapid proliferation. the majority of atp in tumor cells is generated via the oxidative phosphorylation pathway (oxphos). interference with oxphos cause cell cycle arrest suggesting that mitochondria play a role in cell proliferation. mitochondrial atp production is also vital for cell division and differentiation in infection in addition to basic functions in the cell including the regulation of cell volume, solute concentration, and cellular architecture. atp levels differ at various stages of the cell cycle suggesting that there is a relationship between the abundance of atp and the cell's ability to enter a new cell cycle. atp's role in the basic functions of the cell make the cell cycle sensitive to changes in the availability of mitochondrial derived atp. the variation in atp levels at different stages of the cell cycle support the hypothesis that mitochondria play an important role in cell cycle regulation. although the specific mechanisms between mitochondria and the cell cycle regulation is not well understood, studies have shown that low energy cell cycle checkpoints monitor the energy capability before committing to another round of cell division. additional functions mitochondria play a central role in many other metabolic tasks, such as: signaling through mitochondrial reactive oxygen species regulation of the membrane potential apoptosis-programmed cell death calcium signaling (including calcium-evoked apoptosis) regulation of cellular metabolism certain heme synthesis reactions (see also: porphyrin) steroid synthesis hormonal signaling – mitochondria are sensitive and responsive to hormones, in part by the action of mitochondrial estrogen receptors (mters). these receptors have been found in various tissues and cell types, including brain and heart immune signaling neuronal mitochondria also contribute to cellular quality control by reporting neuronal status towards microglia through specialised somatic-junctions. mitochondria of developing neurons contribute to intercellular signaling towards microglia, which communication is indispensable for proper regulation of brain development. some mitochondrial functions are performed only in specific types of cells. for example, mitochondria in liver cells contain enzymes that allow them to detoxify ammonia, a waste product of protein metabolism. a mutation in the genes regulating any of these functions can result in mitochondrial diseases. mitochondrial proteins (proteins transcribed from mitochondrial dna) vary depending on the tissue and the species. in humans, 615 distinct types of proteins have been identified from cardiac mitochondria, whereas in rats, 940 proteins have been reported. the mitochondrial proteome is thought to be dynamically regulated. organization and distribution typical mitochondrial network (green) in two human cells (hela cells) mitochondria (or related structures) are found in all eukaryotes (except the oxymonad monocercomonoides). although commonly depicted as bean-like structures they form a highly dynamic network in the majority of cells where they constantly undergo fission and fusion. the population of all the mitochondria of a given cell constitutes the chondriome. mitochondria vary in number and location according to cell type. a single mitochondrion is often found in unicellular organisms, while human liver cells have about 1000–2000 mitochondria per cell, making up 1/5 of the cell volume. the mitochondrial content of otherwise similar cells can vary substantially in size and membrane potential, with differences arising from sources including uneven partitioning at cell division, leading to extrinsic differences in atp levels and downstream cellular processes. the mitochondria can be found nestled between myofibrils of muscle or wrapped around the sperm flagellum. often, they form a complex 3d branching network inside the cell with the cytoskeleton. the association with the cytoskeleton determines mitochondrial shape, which can affect the function as well: different structures of the mitochondrial network may afford the population a variety of physical, chemical, and signalling advantages or disadvantages. mitochondria in cells are always distributed along microtubules and the distribution of these organelles is also correlated with the endoplasmic reticulum. recent evidence suggests that vimentin, one of the components of the cytoskeleton, is also critical to the association with the cytoskeleton. mitochondria-associated er membrane (mam) main article: mitochondria associated membranes (mam) the mitochondria-associated er membrane (mam) is another structural element that is increasingly recognized for its critical role in cellular physiology and homeostasis. once considered a technical snag in cell fractionation techniques, the alleged er vesicle contaminants that invariably appeared in the mitochondrial fraction have been re-identified as membranous structures derived from the mam—the interface between mitochondria and the er. physical coupling between these two organelles had previously been observed in electron micrographs and has more recently been probed with fluorescence microscopy. such studies estimate that at the mam, which may comprise up to 20% of the mitochondrial outer membrane, the er and mitochondria are separated by a mere 10–25 nm and held together by protein tethering complexes. purified mam from subcellular fractionation is enriched in enzymes involved in phospholipid exchange, in addition to channels associated with ca2+ signaling. these hints of a prominent role for the mam in the regulation of cellular lipid stores and signal transduction have been borne out, with significant implications for mitochondrial-associated cellular phenomena, as discussed below. not only has the mam provided insight into the mechanistic basis underlying such physiological processes as intrinsic apoptosis and the propagation of calcium signaling, but it also favors a more refined view of the mitochondria. though often seen as static, isolated 'powerhouses' hijacked for cellular metabolism through an ancient endosymbiotic event, the evolution of the mam underscores the extent to which mitochondria have been integrated into overall cellular physiology, with intimate physical and functional coupling to the endomembrane system. phospholipid transfer the mam is enriched in enzymes involved in lipid biosynthesis, such as phosphatidylserine synthase on the er face and phosphatidylserine decarboxylase on the mitochondrial face. because mitochondria are dynamic organelles constantly undergoing fission and fusion events, they require a constant and well-regulated supply of phospholipids for membrane integrity. but mitochondria are not only a destination for the phospholipids they finish synthesis of; rather, this organelle also plays a role in inter-organelle trafficking of the intermediates and products of phospholipid biosynthetic pathways, ceramide and cholesterol metabolism, and glycosphingolipid anabolism. such trafficking capacity depends on the mam, which has been shown to facilitate transfer of lipid intermediates between organelles. in contrast to the standard vesicular mechanism of lipid transfer, evidence indicates that the physical proximity of the er and mitochondrial membranes at the mam allows for lipid flipping between opposed bilayers. despite this unusual and seemingly energetically unfavorable mechanism, such transport does not require atp. instead, in yeast, it has been shown to be dependent on a multiprotein tethering structure termed the er-mitochondria encounter structure, or ermes, although it remains unclear whether this structure directly mediates lipid transfer or is required to keep the membranes in sufficiently close proximity to lower the energy