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Abstract: Mitochondria are unique organelles found in nearly all eukaryotic cells. They are the sites of cellular respiration, the catabolic process that generates ATP by extracting energy from sugars, fats and other fuels with the help of oxygen. Most biochemists agree that 36 molecules of ATP can be produced for each glucose molecule during cellular respiration as result of Krebs cycle reactions, the electron transport system, and chemiosmosis. Also, two ATP molecules are produced through glycolysis, so the grand total is 38 molecules of ATP. Mitochondria also replicate by a splitting process that seems to have been evolved from that in bacteria and they have a genome structure consisting of a one circular DNA molecule as is common in the majority of prokaryotes. Finally, these organelles contain transfer RNAs, ribosomes that are more similar to prokaryotic than to eukaryotic ribosomes, and other equipment needed to transcribe and translate their DNA and proteins

Introduction: Mitochondria are tiny organelles ranging from 0.5m to 10 m in length and 1 m in diameter. They are found in nearly all eukaryotic cells and are responsible for energy production. They act like the energy factories of the cell located in the cytoplasm. Mitochondria tend to be concentrated near cellular structures that required large inputs of energy, such as the flagellum.1 The structure of mitochondria is similar to that of bacteria and is generally inclusive of mitochondria in all plant, animal and lower eukaryotic cells. Mitochondria are oval in shape, are about 1-10m long, and have both and an outer and an inner membrane, folded structures called cristae, a space located between the outer and inner mitochondrial membrane is called the

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intermembrane space. , and the space enclosed by the inner membrane is called the mitochondrial matrix.1 The mitochondria uses enzymes to help convert food material into adenosine triphosphate (ATP), which can be directly used by the cell as an energy source. The food we eat is oxidized to produce high-energy electrons that are converted to stored energy. This energy is stored in high energy phosphate bonds in a molecule called adenosine triphosphate, or ATP, ATP is converted from adenosine diphosphate by adding the phosphate group with the high-energy bond. Various reactions in the cell can either use energy (whereby the ATP is converted back to ADP, releasing the high energy bond) or produce it (whereby the ATP is produced from ADP). 1 The major functional role of the mitochondria is very important in cellular respiration, the catabolic process that generates ATP by extracting energy from sugars, fats and other fuels with the help of oxygen, and it is here in the cell where it takes place. Overall, the summary of the process of cellular respiration involves the transfer of electrons through a series of membrane-bound protein carries (the electron transport chain) also known as the oxidative phosphorylation system. Electrons are then removed from the membrane carriers by reducing some terminal electron acceptor such as oxygen in the case of aerobic respiration or nitrogen, sulfate or carbon dioxide such as in the case of anaerobic respiration. Pyruvate and fatty acyl-coenzyme A (CoA) are transported into mitochondria by specific transporter proteins and are metabolized by the citric acid cycle. NADH and FADH2 are produced by the citric acid cycle and electrons are transferred from NADH and FADH2 to O2 by a series of electron carriers in the inner mitochondrial membrane. A proton motive force is created by this electron transfer, and protons, moving back down their electrochemical gradient into the matrix, power the ATP synthase to produce ATP. 1

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One of the most unique attributes of mitochondria is the fact that they possess their own genome and ribosomes, independent from that of the cell. Mitochondrial DNA, coupled with the similarities in structure of mitochondria and bacteria have led to the theory of serial endosymbiosis which holds that mitochondria (and chloroplasts in plant cells) were originally small prokaryotes living within ancient cells.1 Results and Discussion: Taken a deeper look into the structure of the mitochondria; the outer membrane is smooth and limits the organelle by being simple in protein composition and serves to maintain the shape of the mitochondria. It is freely permeable to small molecules and ions, but it blocks off passages of proteins and other macromolecules because of the many channels formed by the large amounts of the trans membrane protein porin. Porins can form channels within the outer membrane through which molecules that are less than 5000 daltons can pass freely.2 The inner membrane of the mitochondria is folded into shelf like structures called cristae. The cristae are formed by large folds that provide a much larger inner membrane surface area for maximizing the amount inner membrane-bound metabolic enzymes that can exist in one mitochondrion. The cristae can also serve to increase the inner membrane area to volume ratio, so matrix metabolism do not have to go far to run into proteins localized to the inner membrane.2 It does not allow the passage of small ions and so it maintains a closed space within the cell. The inner membrane and outer membrane effectively divide the mitochondria into two internal compartments. The space located between the outer and inner mitochondrial membrane is called the intermembrane space. Several enzymes that utilize ATP, such as creatine kinase and adenylate kinase, reside here. Because of the extreme permeability of the mitochondrial outer membrane to small molecules such as metabolites, for the purpose of discussing metabolism the intermembrane space is equivalent to

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the cytosol of the cell.3 The space enclosed by the inner membrane is called the mitochondrial matrix. Within the mitochondrial matrix there are RNA, circular mitochondrial DNA, proteins, and ribosomes. This semi-autonomous organelle has the machinery for the production of some of its own proteins; it has its own DNA and can reproduce by splitting itself. The metabolic steps of cellular respiration occur in the inner membrane. In general, the outer membrane of the mitochondria consists of membrane proteins that are responsible for moving molecules and ions in and out of the organelle. The inner membrane however is considerable more complex and is composed of five key membrane proteins: NADH dehydrogenase, Succinate dehydrogenase, Cytochrome c reductase, Cytochrome c oxidase, and ATP synthase.1 The following the figure shown below illustrates the major structural components of the mitochondria explained above:

Mitochondria serves as sites for the production of cellular energy for the Krebss cycle, and the electron transport chain and oxidative phosphorylation processes takes place on the inner mitochondrial membrane, which borders the mitochondrial matrix and the intermembrane space.

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Animals and other organisms obtain the energy available in carbohydrates through the process of cellular respiration. Cells take carbohydrates into their cytoplasm, and through a complex series of metabolic processes, they break down the carbohydrates and release the energy. The overall mechanism of cellular respiration involves four processes: glycolysis, in which glucose molecules are broken down to form pyruvic acid molecules; the Krebs cycle (citric acid cycle), in which pyruvic acid is further broken down and the energy in its molecule is used to form highenergy compounds, such as nicotinamide adenine dinucleotide (NADH); the electron transport system, in which electrons are transported along a series of coenzymes and cytochromes and the energy in the electrons is released; and chemiosmosis, in which the energy given off by electrons pumps protons across a membrane and provides the energy for ATP synthesis.3 The general chemical equation for cellular respiration is: C6H12O6 + 6O2 6H2O + 6CO2 + energy (1)

The diagram below shows an overview of cellular respiration

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A few things must happen in the cytoplasm of a cell before the Krebs cycle can begin to work. This little process is the anaerobic respiration of glucose (glycolysis) into two molecules of pyruvic acid. Glucose + 2NAD + 2ADP +2Phosphate groups --> 2 pyruvic acid + 2NADH + 2ATP (2) The glycolysis pathway begins when 2 ATP molecules that are phosphorylated and the energy derived is used to activate the breakdown of the glucose molecule. The phosphate groups are added to the new intermediate molecules present in the pathway. At this point two molecules of NAD are reduced by two pairs of hydrogen atoms. Each pair of hydrogen atoms donates two electrons to each NAD molecule, thus reducing it. Each reduced NAD binds one hydrogen proton and leaves one hydrogen ion. So, 2 NADH molecules and 2 hydrogen ions are created. Finally, at the end of the reaction as energy is released, the oxidation of an aldehyde group to a carboxylic acid group in the intermediate molecules produces enough energy to convert 4 molecules of ADP to 4 ATP; what is left are 2 molecules of pyruvic acid. Since 2 ATP molecules were used to activate the reaction, the net gain of the reaction is 2 molecules of ATP, 2 molecules of NADH and 2 molecules of pyruvic acid. The molecules of pyruvic acid then enter the mitochondrion from the cytoplasm and the Krebs cycle is initiated. After the glycolysis takes place in the cell's cytoplasm, the pyruvic acid molecules travel into the interior of the mitochondrion. Once the pyruvic acid is inside, carbon dioxide is enzymatically removed from each three-carbon pyruvic acid molecule to form acetic acid. The enzyme then combines the acetic acid with an enzyme, coenzyme A, to produce acetyl coenzyme A, also known as acetyl CoA. Once acetyl CoA is formed, the Krebs cycle begins.3 The cycle is split into eight steps, each of which will be explained below.

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Step 1: The acetic acid subunit of acetyl CoA is combined with oxaloacetate to form a molecule of citrate. The acetyl coenzyme A acts only as a transporter of acetic acid from one enzyme to another. After Step 1, the coenzyme is released by hydrolysis so that it may combine with another acetic acid molecule to begin the Krebs cycle again. Step 2: The citric acid molecule undergoes an isomerization. A hydroxyl group and a hydrogen molecule are removed from the citrate structure in the form of water. The two carbons form a double bond until the water molecule is added back. Only now, the hydroxyl group and hydrogen molecule are reversed with respect to the original structure of the citrate molecule. Thus, isocitrate is formed. Step 3: In this step, the isocitrate molecule is oxidized by a NAD molecule. The NAD molecule is reduced by the hydrogen atom and the hydroxyl group. The NAD binds with a hydrogen atom and carries off the other hydrogen atom leaving a carbonyl group. This structure is very unstable, so a molecule of CO2 is released creating alpha-ketoglutarate. Step 4: In this step, our friend, coenzyme A, returns to oxidize the alpha-ketoglutarate molecule. A molecule of NAD is reduced again to form NADH and leaves with another hydrogen. This instability causes a carbonyl group to be released as carbon dioxide and a thioester bond is formed in its place between the former alpha-ketoglutarate and coenzyme A to create a molecule of succinyl-coenzyme A complex. Step 5: A water molecule sheds its hydrogen atoms to coenzyme A. Then, a free-floating phosphate group displaces coenzyme A and forms a bond with the succinyl complex. The phosphate is then transferred to a molecule of GDP to produce an energy molecule of GTP. It leaves behind a molecule of succinate.

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Step 6: In this step, succinate is oxidized by a molecule of FAD (Flavin adenine dinucleotide). The FAD removes two hydrogen atoms from the succinate and forces a double bond to form between the two carbon atoms, thus creating fumarate. Step 7: An enzyme adds water to the fumarate molecule to form malate. The malate is created by adding one hydrogen atom to a carbon atom and then adding a hydroxyl group to a carbon next to a terminal carbonyl group. Step 8: In this final step, the malate molecule is oxidized by a NAD molecule. The carbon that carried the hydroxyl group is now converted into a carbonyl group. The end product is oxaloacetate which then combines with acetyl-coenzyme A and begins the Krebs cycle all over again.3 The eight steps shown above are illustrated in diagram below:

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Embedded in the inner membrane are proteins and complexes of molecules that are involved in the process called electron transport. The electron transport system (ETS), as it is called, accepts energy from carriers in the matrix and stores it to a form that can be used to phosphorylate ADP. Two energy carriers are known to donate energy to the ETS, namely nicotine adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD). Reduced NAD carries energy to complex I (NADH-Coenzyme Q Reductase) of the electron transport chain. FAD is a bound part of the succinate dehydrogenase complex (complex II). What happens when NADH binds to complex I? It binds to a prosthetic group called flavin mononucleotide (FMN), and is immediately re-oxidized to NAD. NAD is "recycled," acting as an energy shuttle. What happens to the hydrogen atom that comes off the NADH? FMN receives the hydrogen from the NADH and two electrons. It also picks up a proton from the matrix. In this reduced form, it passes the electrons to iron-sulfur clusters that are part of the complex, and forces two protons into the intermembrane space. The obligatory forcing of protons into the intermembrane space is a key concept. Electrons cannot pass through complex I without accomplishing proton translocation. If you prevent the proton translocation, you prevent electron transport. If you prevent electron transport, you prevent proton translocation. The events must happen together or not at all.5 Electron transport carriers are specific, in that each carrier accepts electrons (and associated free energy) from a specific type of proceeding carrier. Electrons pass from complex I to a carrier (Coenzyme Q) embedded by itself in the membrane. From Coenzyme Q electrons are passed to a complex III which is associated with another proton translocation event. Note that the path of electrons is from Complex I to Coenzyme Q to Complex III. Complex II, the succinate dehydrogenase complex, is a separate starting point, and is not a part of the NADH pathway.

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From Complex III the pathway is to cytochrome c then to a Complex IV (cytochrome oxidase complex). More protons are translocated by Complex IV, and it is at this site that oxygen binds, along with protons, and using the electron pair and remaining free energy, oxygen is reduced to water. Since molecular oxygen is diatomic, it actually takes two electron pairs and two cytochrome oxidase complexes to complete the reaction sequence for the reduction of oxygen. This last step in electron transport serves the critical function of removing electrons from the system so that electron transport can operate continuously. The reduction of oxygen is not an end in itself. Oxygen serves as an electron acceptor, clearing the way for carriers in the sequence to be reoxidized so that electron transport can continue. In your mitochondria, in the absence of oxygen, or in the presence of a poison such as cyanide, there is no outlet for electrons. All carriers remain reduced and Krebs products become out of balance because some Krebs reactions require NAD or FAD and some do not. The purpose of electron transport is to conserve energy in the form of a chemiosmotic gradient. The gradient, in turn, can be exploited for the phosphorylation of ADP as well as for other purposes. With the cessation of aerobic metabolism cell damage is immediate and irreversible. From succinate, the sequence is Complex II to Coenzyme Q to Complex III to cytochrome c to Complex IV. Thus there is a common electron transport pathway beyond the entry point, either Complex I or Complex II. Protons are not translocated at Complex II. There isn't sufficient free energy available from the succinate dehydrogenase reaction to reduce NAD or to pump protons at more than two sites.4

The energy needed to push protons out of the matrix and into the intermembrane space comes from the oxidation of either reduced NAD (NADH) or reduced FAD (FADH2). In the case of NADH, passage of an electron pair to Coenzyme Q provides a "pulling" force. That is, NADH is

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much less electronegative than Coenzyme Q, while the iron-sulfur protein carriers in between are intermediate in electronegativity. The amount of available free energy is 69.5 kJ/mole of NADH (KiloJoules per mole). The efficiency of electron transport can be represented by the standard reduction potential difference, namely the voltage generated by a redox reaction under standard biochemical conditions.

(3) The standard reduction potential of NADH is -0.315V, while that of coenzyme Q is 0.045V (difference of 0.345 V). Therefore there is a strong 'pull' by Coenzyme Q on electrons through the components of Complex I.5 Just for the sake of understanding the principles, let Complex I (NADH dehydrogenase complex), embedded in an intact inner membrane, be the only component of an experimental electron transport system. We'll simply take the electrons from Coenzyme Q when they reach it, so the system can keep going. The removal of protons from the matrix and deposition of protons in the intermembrane space creates a concentration difference of protons across the inner membrane. This is called the chemiosmotic gradient. As the gradient builds up, more and more energy is required to push protons across. When the amount of energy required to push protons reaches 69.5 kJ/mole, electron transport has to stop. In fact, the second law of thermodynamics requires that electron transport stop before the gradient builds up to that point. If there was no way of draining energy from the system, electron transport could not continue despite the presence of adequate substrate. However, a mitochondrion is always in a steady state of respiration, in which the energy lost by processes that dissipate the gradient is constantly replaced by electron transport.4 ATP synthase consists of two functional units, one that conducts the passage of protons (F0) and one that catalyzes the phosphorylation of ADP (F1). Both units must be functional for ATP

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synthesis to take place. The F0 subunit (actually "F sub-zero," the zero is a subscript) can be pictured as a rotor while the F1 subunit remains stationary. F0 includes a "gamma" subunit that rotates as protons are driven through the channel created by the c ring component of F0. The following is a simplistic description of the Boyer model for proton-driven ATP synthesis. The F1 subunit includes three "beta" subunits that are identical in structure but not in form. The conformation of each beta subunit changes as the gamma subunit of F0 rotates. At any given time one beta subunit is in the "loose" (L) conformation, which binds ADP and inorganic phosphate. In that conformation the subunit is constrained so that it does not release either molecule. A beta subunit in "tight" (T) conformation binds ATP with such tenacity that it readily converts ADP and inorganic phosphate to ATP. A subunit in the T conformation cannot release ATP, however. In the "open" (O) conformation a beta subunit releases bound nucleotides. Even if there is no proton gradient one beta subunit will be in the T form with bound ATP, which forms spontaneously even in the absence of a proton gradient. The role of the gradient is to cause the release of bound ATP, not to cause its synthesis. Once ATP is released, binding of ADP and inorganic phosphate is spontaneous. Of course, both reactants must be present for the system to operate. A complete cycle takes place as follows. As protons are driven through the c ring their passage causes rotation of the gamma subunit. As the subunit rotates it causes conversion of the T form (with bound ATP) to the O form. ATP is then released. Meantime, the subunit in L form (which holds bound ADP and inorganic phosphate) is converted to the T form which results in conversion of ADP and phosphate to ATP. The subunit that had been in the O conformation is converted into the L form, binding and "locking" ADP and inorganic phosphate in place. For ATP synthesis to continue ADP and inorganic phosphate must both be available and the ETS must be capable of conducting electron transport and storing energy as a chemiosmotic gradient.

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Each proton pump translocates a specific number of protons (from 2 to 4) with each passage of an electron pair. A specific number of protons must be driven through the F0 subunit of ATP synthase to accomplish one complete cycle. Because some energy stored in the gradient is always lost as heat or exploited for processes other than ATP synthesis, there is not a one to one correspondence between number of protons translocated by the ETS and number of protons entering the matrix through ATP synthase.4 The following picture shown below illustrates everything explained above:

The energy production of cellular respiration is substantial. Most biochemists agree that 36 molecules of ATP can be produced for each glucose molecule during cellular respiration as result of Krebs cycle reactions, the electron transport system, and chemiosmosis. Also, two ATP molecules are produced through glycolysis, so the grand total is 38 molecules of ATP.4

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One of the most unique attributes of mitochondria is the fact that they possess their own genome and ribosomes, independent from that of the cell. Mitochondrial DNA (mtDNA) is a closed circular duplex molecule with super helical twists. It is important to note that the circular form of mtDNA is characteristic of bacteria, not of eukaryistic DNA. On a whole, plants have the largest mitochondrial genomes and animal cells have the smallest. Due to its small size of about 16,500 nucleotides long and its limited information content, the mitochondrial genome is fairly simple compared to other eukaryotic DNAs.1 Mitochondrial DNA, coupled with the similarities in structure of mitochondria and bacteria have led to the theory of serial endosymbiosis which holds that mitochondria (and chloroplasts in plant cells) were originally small prokaryotes living within ancient cells. The ancestors of mitochondria are believed to have been aerobic heterotrophic prokaryotes that entered the cell as undigested prey or internal parasites. Symbiosis eventually became mutually beneficial as the prokaryote was provided with a place to live and the eukaryote, now being an anaerobe, could better function in a steadily increasing aerobic world due to the mitochondrias ability to use oxygen as an advantage to the cell. There is a host of evidence supporting the theory of serial endosymbiosis. Endosymbiotic relationships exist in the modern world, suggesting that they would have been possible in the ancient world as well. Mitochondria are similar in size to bacteria and the inner membrane of mitochondria possesses several enzymes and transport systems found on the plasma membranes of modern prokaryotes. Mitochondria also replicate by a splitting process that seems to have been evolved from that in bacteria and they have a genome structure consisting of a one circular DNA molecule as is common in the majority of prokaryotes. Finally, these organelles contain

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transfer RNAs, ribosomes that are more similar to prokaryotic than to eukaryotic ribosomes, and other equipment needed to transcribe and translate their DNA and proteins.1 Conclusion: The structure of mitochondria is similar to that of bacteria and is generally inclusive of mitochondria in all plant, animal and lower eukaryotic cells. Mitochondria are oval in shape, are about 1-10m long, and have both and an outer and an inner membrane, folded structures called cristae, a space located between the outer and inner mitochondrial membrane is called the intermembrane space. , and the space enclosed by the inner membrane is called the mitochondrial matrix. The energy production of cellular respiration is substantial. Most biochemists agree that 36 molecules of ATP can be produced for each glucose molecule during cellular respiration as result of Krebs cycle reactions, the electron transport system, and chemiosmosis. Also, two ATP molecules are produced through glycolysis, so the grand total is 38 molecules of ATP. There is a host of evidence supporting the theory of serial endosymbiosis. Endosymbiotic relationships exist in the modern world, suggesting that they would have been possible in the ancient world as well. References: 1. Goodman, S. R; Organelles Structure and Function by Mitochondria. In Medical Cell Biology 3rd Ed; Elsevier Inc., London, 2008. 2. Starr, C.; Taggart, R.; How Cells Release Stored Energy. In Biology, The Unity and Diversity of Life 9th Ed; Brooks/Cole Thomson Learning, Inc., California, 2001. 3. The Krebs Cycle, http://incolor.inetnebr.com/mcanaday/main.htm (accessed Nov. 2011) 4. The Electron Transport System of Mitochondria, http://www.ruf.rice.edu/~bioslabs/studies/mitochondria/mitets.html (accessed Nov. 2011)

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