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Mitochondria: Structure and Role in Respiration

Stefan Krauss, Beth Israel Deaconess Medical Center and Harvard Medical School, USA
Mitochondria fulfill various important roles in cellular metabolism. Of note they play a central role in cellular energy metabolism.

Introductory article
Article Contents
. Introduction . Mitochondrial Architecture . Physical Organization of Mitochondrial Enzymes in Metabolism . H 1 Gradients . The ATP Synthase . Major Transport Systems . Integration of Mitochondrial Functions with Cytoplasmic Metabolic Pathways . Summary

Mitochondria have captured the interest of biochemists for more than 50 years. They have been studied intensively in the past decades, not least because they are abundant and can be isolated easily from dierent tissues. Mitochondria have lately moved into the spotlight of other exciting areas, namely the study of apoptosis, evolutionary biology and molecular medicine. Originally, it was the realization that mitochondria play a central role in cellular energy metabolism that attracted the attention of cell physiologists and physiological chemists, and led to Nobel Prizewinning work such as Peter Mitchells chemiosmotic theory. Since the days of classical physiological chemistry, bioenergetics research has gone a long way. Contributions from structural biology, biophysics and mathematical biology increase our still incomplete understanding of mitochondrial metabolism and its regulation in ever more detail.

Mitochondrial Architecture
Mitochondria are about 0.51 mm in diameter and up to 7 mm long. Their shape and number per cell depends on the particular tissue. They may appear as spheres, rods or lamentous bodies, but the general architecture is the same (Figure 1). The number of mitochondria per cells varies depending on the energy requirements: tissues with a high

Outer membrane

Inner membrane

capacity to perform aerobic metabolic functions such as skeletal muscle or kidney will have a larger number of mitochondria. Mitochondria have two membranes, each composed of a phospholipid bilayer. The two membranes are quite distinct in appearance and in physico-chemical properties, thus determining the biochemical function of each membrane. The inner membrane encloses and convolutes into the mitochondrial matrix, forming cristae. This serves to increase the surface of the inner membrane, which carries the main enzymatic machinery of oxidative phosphorylation. The inner and outer membranes are characterized by dierent phospholipid compositions and protein-to-lipid ratios. For the outer membrane, this ratio is about 50:50, and it is thought that the protein has very little enzymatic or transport function. In the inner membrane, the proteinto-lipid ratio is 80:20. The outer membrane is widely permeable to ions and larger molecules. The inner mitochondrial membrane is much less permeable to ions and small molecules than the outer membrane, therefore providing compartmentalization through separation of the matrix from the cytosolic environment. This compartmentalization is a central feature of the conversion of free energy derived from oxidizable substrates. The inner mitochondrial membrane is, in fact, an electrical insulator and chemical barrier. Sophisticated ion transporters exist to allow specic molecules to cross this barrier. There are several antiport systems embedded in the inner membrane, allowing exchange of anions between the cytosol and the mitochondrial matrix. Examples of these are a phosphate-OH 2 exchanger, the adenine nucleotide translocase (which specically exchanges adenosine diphosphate (ADP) for adenosine triphosphate (ATP), mono-, di- and tricarboxylate carriers and the aspartateglutamate shuttle.

Figure 1 Mitochondrial architecture.


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Mitochondria: Structure and Role in Respiration

Physical Organization of Mitochondrial Enzymes in Metabolism

The mitochondrial matrix contains a range of enzymes, which form parts of major metabolic pathways such as carbohydrate, lipid and amino acid oxidation and urea and haem biosynthesis. The enzymatic machinery of oxidative phosphorylation, leading to ATP production, is mainly located in the inner mitochondrial membrane.

Thermodynamic calculations show that oxidation of NADH2 by O2 is sucient to drive the synthesis of several moles of ATP. The mitochondrial electron transport chain, which features components of successively increasing reduction potentials (i.e. reducing power), is designed to split this large change in free energy into smaller components. So how is the free energy derived from oxidation of NADH and FADH2 used by the mitochondrion to produce ATP?

Citric acid cycle

The citric acid cycle (also termed the tricarboxylic acid cycle or Krebs cycle) achieves the complete oxidation of acetylCoA, which is a central intermediate produced by various catabolic pathways such as glycolysis and fatty acid oxidation. The enzymes of this cycle are primarily located in the mitochondrial matrix, with the exception of succinate dehydrogenase, which is bound to the inner membrane, forming part of complex II of the electron transport chain. The physical closeness of the citric acid cycle and the reactions of oxidative phosphorylation in the mitochondrial matrix makes sense: The main function of the citric acid cycle is to oxidize the acetyl component of acetylCoA to two molecules of carbon dioxide and concomitantly to conserve the liberated free energy in the form of NADH (reduced form of nicotinamideadenine dinucleotide) and FADH2 (reduced form of avin adenine dinucleotide). Most of the cells needs for the cellular energy carrier ATP (adenosine 5-triphosphate) are then met by mitochondrial oxidative phosphorylation, for which NADH and FADH2 are the substrates.

The chemiosmotic theory the general mechanistic principle of oxidative phosphorylation

During the past 50 years, a number of concepts have emerged to explain how free energy derived from the oxidation of substrates may be conserved by the cell to drive cellular processes, in particular how electron transport and oxidative phosphorylation are coupled. Of these hypotheses, Edward Slaters chemical coupling hypothesis and Paul Boyers conformational coupling hypothesis have received considerable attention, although it is Peter Mitchells chemiosmotic theory that has become widely accepted. The chemiosmotic theory seems to be consistent with experimental evidence. It postulates the coupling of respiration and ATP synthesis in mitochondria. The free energy of electron transport is conserved by the proton gradient, which is built up by the enzymatic complexes of the electron transport chain. These complexes pump protons from the matrix to the intermembrane space and create an electrochemical gradient across the inner membrane. This gradient then enables the ATPase to synthesize ATP. Complex I (or NADHcoenzyme Q reductase) passes electrons from NADH to CoQ. Several ironsulfur clusters are involved in the electron transport process. The two coenzymes of complex I, avin mononucleotide (FMN) and CoQ are able to accommodate up to two electrons each in stable conformations and donate one or two electrons to the cytochromes of complex III. It is thought that four protons are pumped per pair of electrons. Complex II (succinatecoenzyme Q reductase) contains succinate dehydrogenase and three small hydrophobic subunits. It is anchored in the membrane, facing the mitochondrial matrix. It directly passes electrons from succinate, an intermediate of the citric acid cycle, using FAD as coenzyme, three ironsulfur clusters and cytochrome b560. It has no proton-pumping activity. Complex III (coenzyme Qcytochrome c reductase) transfers electrons from reduced CoQ to cytochrome c. It contains two b-cytochromes, one cytochrome c1, and an ironsulfur cluster. Two protons are pumped per pair of electrons.

Oxidative phosphorylation
Oxidative phosphorylation relies on a series of respiratory complexes (called the electron transport chain) which are embedded in the inner mitochondrial membrane. Figure 2 gives a schematic representation of the electron transport chain. Note that this representation is conceptual, the complexes are thought to be laterally mobile within the membrane. Recent work suggests that some or all of the complexes may form large aggregates or supercomplexes.

H 1 Gradients
Electrons arrive at the electron transport chain in the form of NADH and FADH2 (Figure 2; note that complex II is not shown for simplicity). The electron-transporting complexes pass electrons derived from these reducing equivalents, NADH and FADH2, via protein-bound redox centres onto a nal recipient, oxygen, thus forming water.

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Mitochondria: Structure and Role in Respiration

CYTOSOL H + H + cyt c H + H + H +

Inner mitochondrial membrane

QH2 F0 Proton leak


1/2 O2 H2O +2H+

F1 ATP H + H +

ADP + Pi H + H (complex II not shown) + H +

Complex I

Complex III

Complex IV

ATP synthase

Figure 2 Oxidative phosphorylation physical organization of the components of the electron transport chain and the chemiosmotic proton circuit. As electrons (derived from NADH or FADH2) are transported down the chain (green line), protons are being pumped from the matrix to the cytosolic side of the inner mitochondrial membrane, thus establishing a proton gradient. This gradient may be used by the ATP synthase to form ATP, or it may be dissipated via the proton leak pathway, thus generating heat.

Complex IV (cytochrome c oxidase) catalyses the last step of electron transfer: the reduction of oxygen to water. Complex IV translocates four protons per pair of electrons. Proton translocation is an endergonic process (i.e. it requires energy) because it occurs against an electrochemical gradient. The precise protein translocation mechanism is still subject to research. In what has been known as the proton pump mechanism, the transfer of electrons results in conformational changes to the involved complexes. In complex III, the Q cycle facilitates electron transport and proton translocation. Here, CoQ is reduced in two steps. Ubisemiquinone, carrying one electron, is reduced by complex I and accepts a proton from the matrix. QH2 then diuses to the intermembrane space, where two protons are subsequently released to the intermembrane space. One electron is recycled to facilitate proton uptake, thus forming ubisemiquinone at the matrix side of the membrane, whereas another electron is passed onto cytochrome c1. The electrochemical gradient (Dp, also termed proton motive force) resulting from the protontranslocating activities of complexes I, III and IV has two components: the electrical potential (DCm) and a pH component: Dp 5 DCm 2 DpH Dp is usually given in millivolts. Mitochondria isolated from hepatocytes usually have membrane potentials of around 170 mV.

Proton leak
Mitochondrial proton leak, also known as the proton conductance pathway, is an established phenomenon, which is not fully understood. Some protons which are pumped by the electron transport chain leak back into the matrix, bypassing the ATP synthase (see Figure 2). This means that some of the free energy conserved in the proton gradient is dissipated and lost to the cell. Mitochondria in brown adipose tissue feature an uncoupling protein (UCP1) which catalyses proton leak and exploits the energy dissipation for (regulated) heat generation. However, proteins with high sequence homology to UCP1 have been identied in other tissues such as skeletal muscle, and although their role as uncouplers is not generally established, this has led some researchers to believe that at least part of the proton conductance pathway may be enzymatically catalysed and regulated. The physiological importance of proton leak is not entirely clear, but it is thought that it may act as a valve in situations where Dp becomes unnaturally high, and reduce the number of reactive oxygen species (free radicals) generated by certain processes in the electron transport chain. A very active area of research is the exploration of the potential of proton leak in the regulation of body weight.

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Mitochondria: Structure and Role in Respiration

The ATP Synthase

The ATP synthase, or F1F0-ATPase, utilizes the proton motive force, Dp, to convert ADP and phosphate to ATP, thereby coupling electron transport and proton pumping to ATP synthesis (Figure 2). This multisubunit transmembrane protein, which is the most complex structure in the inner mitochondrial membrane, has attracted considerable experimental attention in recent years. In 1997 Paul Boyer and John Walker were awarded the Nobel Prize for their elucidation of the enzymatic mechanism underlying the synthesis of ATP. The chemiosmotic theory requires that the ATPase is located in the inner membrane in such a way that the protons pumped by complexes I, III and IV of the electron transport chain are allowed to return to the matrix (chemiosmotic proton circuit). It has been shown that puried ATPase molecules, when inserted into articial membrane vesicles, will synthesize ATP when an electrochemical gradient is established across the membrane. The bovine enzyme contains 16 dierent proteins and is over 500 kDa in size. The membrane sector (F0) contains the proton channel and is connected to the catalytic F1 component via a stalk consisting of two parallel structures, the rotor and the stator. The F1 component points into the matrix side of the membrane. It has ve subunits (a e) in a stoichiometry 3:3:1:1:1. The head of the F1 subunit, which is visible on electron micrographs as a bulb-like structure pointing into the matrix, is a hexamer of alternating a and b subunits. It is now generally thought that the ATPase operates by rotational catalysis. Subunits a and b are homologous, they both bind nucleotides but only b has catalytic activity. Within the catalytic component F1, there are therefore three active sites. Paul Boyer suggested a binding exchange mechanism, according to which each site would pass through a cycle of three dierent states: open (the empty state), loose (where ADP and phosphate are bound), and tight (tightly bound ATP), each site being in a dierent state at any given moment. During ATP synthesis, protons are being translocated into the matrix. It is not yet known how the molecular mechanism works. The synthesized ATP molecules are released into the matrix and can be transported to the cytosol by the adenine nucleotide translocase.

metabolites and ions between the matrix and the cytosol is possible. This is facilitated by a number of transport systems. These systems can be electroneutral or electrogenic (typically used to translocate polyanionic species, e.g. the adenine nucleotide translocase), or they may be driven by DCm (e.g. during electrical uniport of cations), or by DpH (e.g. the Pi2 /OH 2 exchanger). Metabolites carried across the inner mitochondrial membrane are predominantly in anionic form. The range of anion transporters in the inner mitochondrial membrane depends on the tissue and its particular function. Common to all mitochondria are the adenine nucleotide carrier, which exchanges cytoplasmic ADP for ATP generated during oxidative phosphorylation, the phosphate transporter and the pyruvate carrier. Other carriers transport or exchange intermediates of the citric acid cycle, the urea cycle (di- and tricarboxylate carrier, 2-oxoglutarate carrier, glutamateaspartate carrier), and fatty acyl esters of carnitine. The transport of monovalent cations is tightly controlled. The mitochondrial membrane potential would drive accumulation of ions such as K 1 if these could accidentally leak into the matrix, with the result that the mitochondria would swell (due to concomitant uptake of water). To prevent this, mitochondria use a transporter that exchanges matrix K 1 or Na 1 for H 1 , maintaining a concentration of these ions that is much lower than in the cytosol.

The mono-, di- and tricarboxylate carriers

Citric acid cycle intermediates are used by cells for fatty acid synthesis and gluconeogenesis, which are largely cytosolic pathways. The di- and tricarboxylate carriers mediate the electroneutral (net) export of citric acid cycle intermediates for this purpose. The dicarboxylate carrier 2 exchanges malate or succinate for HPO2 4 , the tricarboxylate carrier exchanges citrate and isocitrate (plus one proton) for malate. The monocarboxylate transporter exchanges cytosolic pyruvate for OH 2 .

Ca2 1 and Pi transport

2 Pi (H2PO4 ) is produced when ATP is used to drive cytosolic processes, and must be returned to the mitochondrial matrix to allow ATP generation. The phosphate transporter is ubiquitous in mitochondria of all cell types, 2 either in exchange for OH 2 ions or and exchanges H2PO4 by symport with a proton, which in either case is electroneutral. This carrier is highly active, and the concomitant proton translocation explains the inuence of DpH on the distribution of Pi across the membrane. Ca2 1 acts as intracellular second messenger in cell signalling, aecting a wide range of metabolic reactions including intramitochondrial ones. Also, mitochondria

Major Transport Systems

Mitochondrial transport
Because of its composition, the inner mitochondrial membrane is impermeable to most solutes and ions. While the design of the inner membrane is geared to maintain a high Dp, it is also essential that exchange of certain

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Mitochondria: Structure and Role in Respiration

have an important function to buer cytosolic Ca2 1 concentrations (when Ca2 1 concentrations are in excess of 1 mmol L 2 1, mitochondria readily accumulate the cation). Consequently, intracellular Ca2 1 concentrations must be tightly controlled. Inux and eux of Ca2 1 are mediated independently by dierent transport systems. Ca2 1 enters mitochondria via a uniporter. The eux is mediated by electroneutral antiport with H 1 in liver, and with Na 1 in mitochondria from heart, brain and brown adipose tissue (the latter is itself coupled to a Na 1 /H 1 exchanger). Under steady-state conditions, the uniporter and the antiporter work together at relatively low activity to give symmetrical cycling of calcium which is driven by the chemiosmotic proton circuit. When the cytosolic Ca2 1 concentration rises, the uptake exceeds expulsion from the matrix and net uptake occurs. The uptake of Ca2 1 lowers DCm, which results in a transient stimulation of the proton pumping respiratory chain (uncoupling eect), thus increasing DpH. Ca2 1 acts as a regulator of mitochondrial function. It causes isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to lower the Km for their substrates (and, therefore, increase their anity for them). Also aected by Ca2 1 is pyruvate dehydrogenase phosphatase, the enzyme that dephosphorylates the pyruvate dehydrogenase complex, thus increasing its Vmax. Recent work has shown that mitochondria are not only responding to simple increases in Ca2 1 , but are able to decode rather complex (frequency modulated) cytosolic Ca2 1 signals which regulate the rate of oxidative phosphorylation.

oxidation is formed in the matrix, but NADH produced in glycolysis is cytosolic. Examples of such substrate transport shuttles are the a-glycerol phosphate shuttle of insect ight muscle and the mammalian malateaspartate shuttle. These shuttles have in common that they involve reciprocal transfer of oxidized and reduced species of various redox couples, thus accomplishing the net transfer of reducing equivalents across the membrane.

Integration of Mitochondrial Functions with Cytoplasmic Metabolic Pathways

The citric acid cycle is a pathway central not only to mitochondrial metabolism, but in many ways also to cellular metabolism as a whole. In the light of mitochondrial oxidative phosphorylation, its major function is catabolic as it completely oxidizes the carbon atoms in acetylCoA and conserves free energy. Cycle intermediates are, however, precursors and substrates of anabolic pathways such as gluconeogenesis as well as cholesterol, fatty acid, porphyrin and protein biosynthesis. Figure 3 summarizes some of the interrelationships between major mitochondrial and cytosolic metabolic
Glucose Pyruvate Acetyl-CoA Fatty acid oxidation Ketone bodies HMGCoA

Amino acids

Amino acids

The carnitine palmitoyltransferase system

Fatty acids are oxidized in the mitochondrion. Long-chain fatty acylCoA cannot readily cross the inner mitochondrial membrane, but mitochondria feature a shuttle system called the carnitine palmitoyl transferase system. Overt carnitine palmitoyltransferase (or CPT I) transfers the acyl portion to carnitine. A special carnitine carrier protein then translocates acylcarnitine to the matrix side of the membrane in exchange for carnitine. Finally, CPT II then transfers the acyl group to CoA from the mitochondrial pool.


Carbon source Oxaloacetate for biosynthetic pathways Malate Citrate (fatty acids, sterols) Fumarate 2-Oxoglutarate


Asparate tyrosine phenylalanine

NADH/ FADH2 Succinyl-CoA Amino acids Oxidative phosphorylation

Substrate transport shuttles: transfer of electrons from cytoplasmic NADH to the respiratory chain
The mitochondrial inner membrane contains a number of substrate transport shuttles. These shuttles function to transport reducing equivalents across the inner mitochondrial membrane, as none of the nucleotides involved in cellular redox reactions (NAD(P) 1 , NAD(P)H, FAD, FADH2, CoA) are permeable to the inner mitochondrial membrane. Most of the NADH derived from glucose

Porphyrin Odd chain fatty acid biosynthesis and branched chain (-aminolevulinate) 2-oxoacid metabolism Isoleucine methionine valine Catabolic pathways Anabolic (biosynthetic) pathways


Figure 3 Integration of cytosolic and mitochondrial pathways. The citric acid cycle has integrative functions in a complex network of cellular biosynthetic and degradative processes. A cell may derive energy (in the form of ATP) from different carbon sources including carbohydrates, amino acids (proteins) and lipids. Conversely, breakdown products and intermediates of oxidative metabolism may be used for biosynthetic pathways.

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pathways. This representation is far from complete and can only give an indication of the complexity of such interrelationships. The cellular concentrations of citric acid cycle intermediates exceed the catalytic amounts that would be needed to drive the cycle. This is because the citric acid cycle has important functions in the integration of dierent pathways, providing intermediates for a range of biosynthetic pathways and a sink for degradation products. SuccinylCoA is a breakdown product of odd chain fatty acid oxidation and branched chain 2-oxoacid metabolism, and also of a few amino acids (Figure 3). It serves as a precursor for porphyrin (i.e. haem) biosynthesis. This pathway is partially located in the mitochondrial matrix. Citrate is used as precursor for fatty acid and cholesterol synthesis. It also has regulatory properties: it is known to allosterically inhibit phosphofructokinase, and to stimulate acetylCoA carboxylase. It is a source of cytosolic reducing equivalents for reductive biosyntheses. The particular relationships between dierent pathways are dictated by the requirements of the cell, and are subject to hormonal regulation. The fate of carbohydrates in the well-fed stage is usually complete oxidation via glycolysis (cytosolic) to the stage of pyruvate, conversion into acetyl CoA and subsequent oxidation in the citric acid cycle, thus providing reducing equivalents which are the substrates for oxidative phosphorylation. Amino acids are used for protein biosynthesis or are oxidized in peripheral tissues. Excess amino acids can be oxidized entirely to carbon dioxide and water; alternatively the intermediates can be used as substrates for fatty acid biosynthesis whereby the nitrogen is converted to urea. During fasting, glycogen stores in muscle and liver are depleted, which also yields glucose that can be oxidized. Prolonged starvation causes increased breakdown of fats and proteins, and the free amino acids are then used as carbon sources for the citric acid cycle which they enter either as acetylCoA or cycle intermediates. Glucose, which is a major fuel for the brain, can be synthesized in gluconeogenesis from malate or oxaloacetate (using the malateaspartate shuttle) to provide energy for peripheral tissues.

Mitochondria play a central role in energy metabolism of cells. They usually provide most of the ATP by oxidative phosphorylation. A major consequence of the architecture of mitochondria is the impermeability of the inner membrane that facilitates the generation of a proton gradient, called the proton motive force. The oxidative processes cells use to degrade fuel molecules yield NADH and FADH2 which are used as electron donors for the electron transport chain. The components of the chain are located in the inner mitochondrial membrane and include four complexes and some electron carriers. While electrons are transported along the chain, three of the four complexes act as proton pumps, expel protons from the matrix and build up the proton motive force. Another enzyme, the ATPase, utilizes the proton gradient to form ATP from ADP and Pi, thus allowing the protons to return to the matrix. The coupling of electron transport (i.e. oxidative processes) and ATP synthesis via the proton gradient is the main postulate of the chemiosmotic theory. Due to the impermeability of the inner mitochondrial membrane to most solutes, a range of transporters exists which allow exchange of ions and metabolites (mostly in anionic form) between matrix and cytosol. These transporters also help to integrate mitochondrial and cytosolic metabolic pathways. The citric acid cycle can be seen as the centre of a range of metabolic processes. It is amphibolic, i.e. it serves both catabolic and anabolic purposes depending on the particular requirements of a cell as dened by function and physiological state.

Further Reading
Boyer PD (1997) The ATP synthase a splendid molecular machine. Annual Review of Biochemistry 66: 717749. Nicholls DG and Ferguson SJ (1992) Bioenergetics 2. London: Academic Press. Saraste M (1999) Oxidative phosphorylation at the n de sie`cle. Science 283: 14881493. Abrahams JP, Leslie AG, Lutter R and Walker JE (1994) Structure at 2.8 resolution of F1-ATPase from bovine heart mitochondria. Nature A 370: 621628. For further information on the structure and function of the ATP synthase, see also and follow the links to the 1997 Nobel Prize for Chemistry.

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