Chapter 15.

Mitochondria
Mitochondria (Greek word mitos = thread) were first observed (in the XIXth century) in light microscopy as granules inside the cytoplasm, but their ultrastructure and functions were discovered around the mid XX century, when electron microscopy and procedures for isolating intact mitochondria were developed. They are power plants of eukaryotic cells, providing energy requested by cellular metabolic processes and other energy-demanding activities. The main function of mitochondria is to produce ATP (adenosine triphosphate) in a process called oxidative phosphorylation.

15.1 Morphology and Ultrastructure

Number. Mitochondria are present in various number from one to thousands/cell depending on the metabolic activity of cells (1 in a few algae and protozoa, 10-24 in sperm cells, 300 in renal cells, 1500 in hepatocytes). Mitochondria are even more numerous in muscle cells, which require a large amount of ATP to fuel their contraction (in heart muscle cells, for example, mitochondria may occupy as much as 30 percent of the total cell volume). Localization. Localisation of mitochondria in cells corresponds to areas with high activity: - in muscles, between myofilaments (supplying ATP requested by muscular contraction), - in neurons, in synaptic regions (supplying ATP requested by membrane repolarisation), - in flagella, around axoneme (supplying ATP requested by movement), - in pancreas, near RER (supplying ATP requested by protein synthesis), - or near sources of substrates for oxidations (droplets of lipids) Morphology. In light microscopy mitochondria appear in oval shape, of 0.5-1 m (short axis) and 1-3 or even 10 m (long axis). In many cell types mitochondria are very mobile they can be in permanent movement, streaming with the cytoplasm (they can form long moving chains in association with the microtubules), and changing shape. They may grow in length, undergo fusions and fissions (they divide). Ultrastructure of mitochondria was established by electron microscopy. G.E. Palade had the main contribution he discovered the foldings of the inner membranes (called by him cristae). Sjstrand discovered the outer membrane and thus the Palade-Sjstrand model of mitochondrial ultrastructure was established.

A mitochondrion has double membrane: an outer (smooth) membrane, and an inner membrane with foldings (mitochondrial cristae). Between the two membranes lies intermembrane space, and the inner membrane encloses mitochondrial matrix. As ultrastructural details, on the matrix face of the inner membrane globular proteins may be seen by negative staining, which correspond to a part of ATP-synthase complex; in the matrix may be observed circular DNA, ribosomes and electrondense granules Ca2+ precipitates. The number and shape of the mitochondrial cristae are different depending on cell type, and their surface is larger as the metabolism of the cell is more intense: - mitochondria with a high number of large, perpendicular cristae in myocardium; - mitochondria with longitudinal cristae (like small tubules) in adrenal cortex; - mitochondria with overlapped cristae in neurons - mitochondria with a few small cristae in hepatocytes After mitochondria isolation by differential centrifugation, they become spherical and may display 2 different conformational states: - orthodox conformation (similar to the aspect of mitochondria in the intact cells) - condensed conformation (the intermembrane space is larger and the matrix is more condensed)

15.2 Chemical Composition, Metabolic Processes and Enzymes Localised in Mitochondria

Chemical composition. The outer membrane consists of lipids (this mitochondrial membrane is relatively rich in cholesterol 5%) and proteins in the same ratio and is freely permeable to most organic and inorganic molecules with molecular weight up to 6,000 Da. This high permeability is conferred by porins transmembrane proteins that form aqueous channels. Thus, for most solutes, the intermembrane space is continuous with the cytosol. The inner mitochondrial membrane is composed of about 25% lipids and 75% proteins, and is almost completely impermeable to most hydrophilic substances, excepting a group of molecules transported by specific carrier systems. The inner membrane has no cholesterol and is rich in cardiolipin (diphosphatidylglycerol), a mitochondrial-specific phospholipid. Both features are characteristics of bacterial plasma membranes, from which the inner mitochondrial membrane is presumably derived by evolution. Cardiolipin, concentrated at this level, reduces permeability of the phospholipid bilayer to protons, establishing a concentration gradient across the membrane, important in order to generate ATP. In freeze-fracture electron micrographs of the inner mitochondrial membrane some transmembrane protein complexes were identified and using other analythical methods over 60

different polypeptides have been described within the complex structure of this membrane. All these proteins are involved in transmembrane transport of different substances, in electron transport and oxidation reaction (along the respiratory chain), and in ATP synthesis. Metabolic processes localised in mitochondria are biosynthetic processes and processes of the energetic metabolism. General biosynthetic processes are biosynthesis of DNA, biosynthesis of mRNA, and biosynthesis of proteins. Among specific biosynthetic processes could be mentioned some steps in the synthesis of steroid hormones in the adrenal cortex. Energetic metabolism processes. In a first stage, the food is digested and the resulted products (amino acids, glucose, free fatty acids) are absorbed inside the cytosol where the glucose is converted into pyruvate. Amino acids and the free fatty acids are next transported into the mitochondrial matrix, and are transformed into Acetyl-Coenzyme A. Acetyl-Coenzyme A enters the citric acid cycle, and as result of its transformation 2CO2, 3NADH (nicotinamide adenine dinucleotide) and 1FADH2 (flavine adenine dinucleotide) will be produced. In the inner membrane NADH and FADH2 are oxidized by enzymes of the respiratory chain in order to obtain ATP in the ATP-synthase. Localisation of enzymes in mitochondria. The marker enzyme for the outer membrane is monoamine oxidase (MAO), involved in the oxidation of serotonin, triptamin, etc. The intermembrane space, which is structureless under the electron microscope contains adenylate kinase (catalyses the reaction: AMP + ATP ! 2ADP resulting in ADP which will enter back in the matrix and participate in the ATP synthesis). This way, AMP could be also used in the ATP production. In the inner membrane the complexes of the respiratory chain, ATP-synthase and the transporters of ions are located. The marker for the inner membrane is cytocrome-C oxydase (an enzyme of the respiratory chain). The most important enzymes in the mitochondrial matrix are pyruvate-dehydrogenase, enzymes involved in the -oxidations of fatty acids, and in the citric cycle, and of course, enzymes involved in synthesis of DNA, RNA, and proteins.

15.3 Molecular Structure of the Respiratory Chain

In mitochondria the chemical energy is obtained from reaction: H2 + 1/2O2 ! H2O

which occurs in small steps

First of all, protons are separated from electrons: NADH and FADH2 are oxidised in the inner mitochondrial membrane, and electrons are captured into the electron-transport chain, moved along it

and donated to molecular oxygen. The electron-transport chain, located in the inner mitochondrial membrane is made up of a series of electron carriers (redox enzymes); there are around 15 distinct electron transporters, but only two of them perform independently their function: coenzyme Q (or ubiquinone a hydrophobic unit enclosed in the membrane lipid bilayer) and cytochrome c (a hydrophilic protein located on the outer face of the inner mitochondrial membrane); the others are organised in 4 complexes: Complex I. Complex I (NADH-dehydrogenase) is the largest mitochondrial complex (m.w. over 600 kDa) which contains about 30 different polypeptides. It has a transmembrane orientation and is made up of many Fe/S proteins and a FMN (flavin mono nucleotide). This complex takes the electrons from NADH and delivers them to ubiqiunone. Complex II. Complex II (succinate-dehydrogenase) is located on the outer face of the inner membrane (oriented towards the intramembrane space). It contains a cytochrome b, succinate dehydrogenase and many other polypeptides. This complex takes the electrons from FADH2 and delivers them to ubiquinone. Complex III. Complex III (complex of cytochromes bc1) is also a transmembrane complex. It consists of 8 subunits, three of them having a higher importance: cytochrome b, cytochrome c1 and a Fe/S protein. This complex passes the electrons from ubiqiunone to cytochrome c. Complex IV. Complex IV (cytochrome c oxidase), is a transmembrane enzyme (m.w. = 400 kDa) composed of some protein subunits and two cytocromes: a1 and a3. It also contains 2 Cu atoms involved in the redox reactions. This complex takes the electrons from cytochrome c and delivers them to an oxygen molecule: O2 + 4H+ + 4e ! 2H2O The major complexes move freely in membrane with a relatively high speed. Coenzyme Q and cytochrome c move 10 times faster transporting the electrons between the complexes, and the delivery of electrons is performed by their random colliding. However the ratio of the different complexes in the membrane is different: 1 (Complex I) : 3 (Complex III) : 7 (Complex IV) : 9 cyt. c : 50 Co.Q The electrons are transported along the respiratory chain according to redox potential of the complexes. Because oxygen is used as a final electron acceptor, the oxidative activities of mitochondria are frequently termed respiration, and the electron-transport chain respiratory chain. Cytochrome oxidase uses around 90% of the entire O2 used by cells.

15.4 Mechanism of Oxidative Phosphorylation. The Chemiosmotic Theory

While in prokaryotes the respiratory chain is enclosed in the plasma membrane and the ATP synthesis occurs at the membrane-cytosol interface, in eukaryotes the ATP synthesis occurs in the inner mitochondrial membrane. Mitchell (1961) suggested that in complexes I, III and IV, the transport of electrons is coupled to translations of protons from the matrix to the intermembrane space (and consequently to the cytosol because of the porins). This transfer of protons, on one hand, and the impermeability of the inner mitochondrial membrane for protons, on the other hand, result in an electrochemical gradient between the two sides of the membrane. This gradient has two components: a pH gradient, and an electric membrane potential (the inner side becomes negative), and is used as a source of energy in the FoF1complex of ATP-synthase to synthesise the ATP. The ATP-synthase, or FoF1-ATP-ase is present in high amount in mitochondria. It is organised in two structurally and functionally distinct subunits, which can be experimentally separated using high salt concentrations. One of these subunits is Fo (o = oligomycin, a drug that inhibits the electron flow in ATP-ase). It is embedded in the inner membrane (a hydrophobic protein complex) and has a channel for protons. The Fo subunit of the ATP-synthase consists of three different polypeptides (termed: a 1 molecule, b 2 molecules and c, 10 molecules are present). Of these polypeptides, polypeptide b2 project out of the membrane to form the stalk. The other unit of the ATP-synthase is the F1 unit, which appears as a globular head on the matrix face of the inner membrane. It represents the catalytic region of the enzyme. The F1 headpiece is made up of 5 different polypeptides (3 molecules of -polypeptide, 3 molecules of , and 1 molecule of , and polypeptides). - and -polypeptides alternate in the structure of F1 unit giving a triaxial symmetry. The energy of the electrochemical gradient is used by the FoF1 complex in order to synthesise ATP starting from ADP and Pi after the following reaction:

ADP + Pi

ATP + H2O

The synthesis of each ATP molecule requires 3 protons.

The molecular mechanism of ATP synthesis Polypeptides b1, and c from the Fo subunit and the F1 subunit represent a functional unit called rotor; The other polypeptides from the Fo subunit, together with the thin arm (b2) represent a stator. Protons pass between the rotor and the stator into the matrix, and their energy is used by rotor to rotate. Because the stator the thin arm of the b2 molecule is in contact with F1 subunit at the level of " molecule, the rotation movement will generate mechanic energy. This energy is then converted in the F1 headpiece where it triggers conformational changes followed by Pi binding to ADP. After the protons induced conformational changes in the catalytic site, they are discharged in the matrix where will interact with oxygen molecules that previously accepted the electrons transported along the respiratory chain. The result consists in the formation of water molecules. Thus, the synthesis of ATP according to this mechanism requires the integrity of the inner mitochondrial membrane. Certain substances such as 2,4-dinytrophenol (2,4-DNP) uncouple the process of phosphorylation from respiration (the electron transport). The transport of electrons along the respiratory chain does not occur normally, but at a higher rate, because the cell needs ATP that the inefficient mitochondria cannot generate. The impossibility of mitochondria to produce ATP is given by the decrease or even the absence of the proton gradient induced by 2,4-DNP.

Inhibitors of the Mitochondrial Respiration The electron-transport along the respiratory chain can be blocked by certain compounds that have affinity for one complex or another. Many substances like rothenone, piericidine or amytal, inhibit the activity of the complex I stopping the influx of electrons from NADH in the respiratory chain. In this particular case, the mitochondrial respiration continues at a lower rate using only the electrons obtained from FADH2 delivered by complex II. Antimycine and mixothiazol, which operate at the level of complex III and carbon monoxide and cyanides that arrest the transfer of electrons to oxygen, totally interrupt the respiration and consecutively cause the cell death and in very short time the death of the entire organism (if it is present in higher doses), since the cells dont stow ATP.

15.5 Transport of Ions Across the Inner Mitochondrial Membrane

a) Translocation of ATP and ADP. ATP and ADP are transported in opposite directions by ATP-ADP translocase (antiport system). ATP synthesised in the matrix is delivered to the intermembrane space and is exchanged with ADP, which is imported in the matrix.

b) Transport of anions: pyruvate (from glycolysis) is transported into the matrix to be converted to acetyl-CoA, and the inorganic phosphate (Pi) is also translocated inside the matrix to be used in the ADP phosphorylation. c) Transport of aspartate and glutamate has a very important physiological significance. Because cytosolic NADH cannot pass into the matrix, the used NADH must be continuously refreshed through reducing reactions. The NADH generated by glycolysis and a series of intermediates and their mitochondrial transporters are used in this purpose. The cytosolic NADH is oxidised to NAD+ with the reduction of oxaloacetate to malate. Malate enters the matrix it is exchanged with -ketoglutarate (antiport system). Inside the matrix, malate reduces NAD+ to NADH and returns to oxaloacetate, which will be converted to aspartate the form able to return in the cytosol. In the cytosol, aspartate is reconverted to oxaloacetate and the cycle can start again. For this entire cycle to function, ketoglutarate arrived into the cytosol, is transformed to glutamate - able to return in the matrix (through the aspartate-glutamate antiport) where, of course will turn again into -ketoglutarate. Both the malate--ketoglutarate and aspartate-glutamate antiport systems are located in the inner mitochondrial membrane. d) Transport of fatty acids from cytosol into the matrix where are metabolised by #-oxidation. Their transport is performed by carnitine a specific mobile molecule in the inner mitochondrial membrane. e) Transport of Ca2+ ions by the Ca-ATPase into the matrix is very important for the control of the Ca2+ concentration in the cytosol.