**3. Mitochondria – the relationship between the structure and the function**

Mitochondria are multifunction organelles, which play a key role in both the proper functioning of the cell and normal cell death scenario (Kuznetsov & Margreiter, 2009, as cited in McBride et al., 2006). Their main role is the production of adenosine triphosphate (ATP) through metabolic processes involving tricarboxylic acid cycle (TCA) and the electron transport chain (ETC). Most cellular ATP is generated in the process of oxidative phosphorylation, which is possible thanks to the 'sophisticated machinery' located in the inner mitochondrial membrane. Mitochondria participate in the regulation of redox state and calcium homeostasis in cell. Cations of calcium regulate some mitochondrial processes, such as enzyme activity, i.e. pyruvate dehydrogenase, or metabolic rate. These organelles participate in biosynthesis of amino acids, vitamin cofactors, fatty acids and neurotransmitters (Waldbaum & Patel, 2009). Many other biochemical reactions are associated with the functioning of these structures, including synthesis of heme group and some steps of steroid synthesis. Also, a part of the processes occurring in the urea cycle take place there (Pinti et al. 2010). Mitochondria have critical function in the control of apoptotic and necrotic cell death and in most types of cells they are also a major site of reactive oxygen species (ROS) generation (Duchen, 2004). ROS are involved in many signaling pathways. Most of them are second messengers that trigger different cellular events, such as cytokine secretion or activation of transcription factors, but in excess they can contribute to the formation of defects in mitochondria, as well as in a whole cell (Edeas et al., 2010a).

### **3.1 Mitochondrial structure and biogenesis**

Mitochondria are encapsulated by two membranes, each with different structure and function, separated by intermembrane space, in which some important proteins involved in the mitochondrial bioenergetics and/or cell death are located (Fig. 3) (Duchen, 2004, Borutaite, 2010). The outer membrane contains porins, which make it permeable to molecules smaller than 5-6 kDa (Waldbaum & Patel, 2009). Compounds such as water, O2, CO2, and NH3 easily pass through the membrane, but hydrophilic metabolites and all inorganic ions in order to get over this membrane require the participation of specific channels and carrier proteins. Such a transport is generally based on the exchange of molecules, i.e. ADP is exchanged for ATP and Pi (inorganic phosphate) for OH- (Szewczyk & Wojtczak, 2002). The mitochondrial inner membrane contains enzymes facilitating an oxidative phosphorylation (OXPHOS). This complex of enzymes consists of four oxidoreductases involved in respiratory electron transport (Complexes I - IV) and the ATP synthase complex (Complex V).

Fig. 3. Mitochondria structure and components.

Until recently, the inner membrane has been described as a multiple infolded structure forming cristae and containing numerous mitochondrial proteins (Duchen, 2004). However, electron tomographic analyses of a variety of mitochondria (both isolated and observed *in situ* in various cell types) have provided overwhelming evidence showing the need of some changes in the perception of the structure of these organelles. These infoldings or rather invaginations are not randomly spaced in the membrane, as often considered, but resemble microcompartments, which face each other in the peripheral region of the membrane. The narrow junctions are wide enough to pass metabolites and many soluble proteins (Mannella, 2008). However, the number of cristae junctions and the morphology of the intercristal space depends on the metabolic state of mitochondria (Logan, 2006). Isolated mitochondria usually occur in one of two morphologic states, condensed or orthodox. The first one is characterized by contracted, very dense matrix and wide cristae. In the second state matrix is expanded and cristae compartments are more compact. Osmotic and metabolic changes in mitochondria are responsible for these alterations. It is believed that mitochondrial inner membrane topology is regulated by the cell to improve mitochondria capacity in their response to stimuli (Mannella, 2008).

Tissue cells contain from a few dozen to several thousands of mitochondria and their number is associated with cell energy demands. Organs such as heart, muscles or brain contain the largest number of mitochondria. Mitochondria are very dynamic structures, which can divide, undergo fusion and can take the form of the network of elongated and interconnected laments. The phenomena of fission and fusion have an impact on mitochondrial shape, size and number (Logan, 2006). The division and replication of mitochondria is under control of the nucleus and is somehow associated with division and replication of nuclear DNA. Replication of mitochondria requires coordination between the process of mtDNA replication and synthesis of proteins encoded in both genomes (nucleus and mitochondrial). The production of both types of proteins must be synchronized to preserve their functionality.

### **3.2 Electron transport chain and ATP synthesis**

132 Biomedical Science, Engineering and Technology

secretion or activation of transcription factors, but in excess they can contribute to the

Mitochondria are encapsulated by two membranes, each with different structure and function, separated by intermembrane space, in which some important proteins involved in the mitochondrial bioenergetics and/or cell death are located (Fig. 3) (Duchen, 2004, Borutaite, 2010). The outer membrane contains porins, which make it permeable to molecules smaller than 5-6 kDa (Waldbaum & Patel, 2009). Compounds such as water, O2, CO2, and NH3 easily pass through the membrane, but hydrophilic metabolites and all inorganic ions in order to get over this membrane require the participation of specific channels and carrier proteins. Such a transport is generally based on the exchange of molecules, i.e. ADP is exchanged for ATP and Pi (inorganic phosphate) for OH- (Szewczyk & Wojtczak, 2002). The mitochondrial inner membrane contains enzymes facilitating an oxidative phosphorylation (OXPHOS). This complex of enzymes consists of four oxidoreductases involved in respiratory electron transport (Complexes I - IV) and the ATP

Until recently, the inner membrane has been described as a multiple infolded structure forming cristae and containing numerous mitochondrial proteins (Duchen, 2004). However, electron tomographic analyses of a variety of mitochondria (both isolated and observed *in situ* in various cell types) have provided overwhelming evidence showing the need of some changes in the perception of the structure of these organelles. These infoldings or rather invaginations are not randomly spaced in the membrane, as often considered, but resemble microcompartments, which face each other in the peripheral region of the membrane. The narrow junctions are wide enough to pass metabolites and many soluble proteins (Mannella, 2008). However, the number of cristae junctions and the morphology of the intercristal space depends on the metabolic state of mitochondria (Logan, 2006). Isolated mitochondria usually occur in one of two morphologic states, condensed or orthodox. The first one is characterized by contracted, very dense matrix and wide cristae. In the second state matrix is expanded and cristae compartments are more compact. Osmotic and metabolic changes in mitochondria are responsible for these alterations. It is believed that mitochondrial inner

formation of defects in mitochondria, as well as in a whole cell (Edeas et al., 2010a).

**3.1 Mitochondrial structure and biogenesis** 

synthase complex (Complex V).

Fig. 3. Mitochondria structure and components.

Mitochondrial ATP production involves three main steps: a) the enzymatic "combustion" of acetyl in tricarbolxylic acid cycle (TCA), b) the electron transport chain activity and c) ATP synthase action. Energy released during this cycle is used to reduce the electron carriers NAD+ to NADH and FAD2+ to FADH (Duchen, 2004). Electrons from NADH and FADH2 are transferred to the respiratory chain - a coupled enzyme systems composed of four complexes (Complex I - IV). Complex I (NADH dehydrogenase) is the major entrance point of electrons to respiratory chain and is composed of two domains. One domain, localized in withe membrane, is involved in proton translocation across the bilayer, and the other, matrix-exposed domain, is responsible for oxidation of NADH. FADH2 is the donor of electrons to succinate dehydrogenase (Complex II) which is the second entrance point of electrons to the ETC. Electrons from both complexes are transferred on mobile intermediate – ubiquinone, which is converted to reduced form - ubiquinol. The flow of electrons from ubiquinol is directed through the Complex III, also known as ubiquinol-cytochrome c reductase, to another carrier - cytochrome c, which transfers electrons to Complex IV cytochrome c oxidase. Finally, at the very end of the respiratory chain, Complex IV reduces the oxygen to water in sequential four-electron transfer (Adam-Vizi & Chinopoulos, 2006). The oxidation of NADH and FADH2 provides the energy to transport protons from mitochondrial matrix into the intermembrane space by the proton pumps (Complexes I, III, IV). The difference in the proton concentration, and thus the difference in the electric charge across the inner mitochondrial membrane creates the electrochemical potential gradient, also called an electrochemical proton gradient or a 'proton-motive force', which is mainly expressed as a mitochondrial transmembrane potential (Nazaret, 2008). The structure of mitochondrial electron transport and the scheme showing ATP production by mitochondria was introduced in Fig. 4.

Energy needed to phosphorylate ADP by ATP synthase comes from the entry of protons back into the matrix through the proton channel of this complex. This process is called oxidative phosphorylation (Frey & Mannella, 2000). ATP is then transported to the cytoplasm by the adenine nucleotide translocase (ANT). However, there are several mechanisms that may lead to the loss of mitochondrial potential, including an inhibition of respiration, failure in substrate supply and uncoupling mechanisms that cause proton leak across the membrane.

Fig. 4. The general mechanism leading to oxidative phosphorylation is as follows: highenergy electrons (marked as e- ) derived from NADH and FADH2, are moving along the respiratory chain composed of four protein complexes (Complex I - IV) and two additional electrons carriers: ubiquinone (coenzyme Q, **Q**), a small molecule freely moving in the inner mitochondrial membrane layer, and cytochrome c (**Cyt c**), localized in the intermembrane space attached to the inner membrane. Part of the energy released in this process is used up in the action of proton pumps transporting protons (H+) from matrix to the intermembrane space. Across the inner membrane electrochemical gradient of protons is formed. Protons tend to return to the mitochondrial matrix and restore alignment of H+ concentration on both sides of the membrane. When they pass back through transmembrane protein complex – ATP synthase - the energy of their movement is used for the synthesis of ATP from ADP and inorganic phosphate (**Pi**).

### **3.3 Free radical generation by mitochondria**

Oxidative metabolism and ATP synthesis are closely associated with ROS generation in mitochondria. These organelles consume 80–90% of cell's oxygen during oxidative phosphorylation. The electron transport chain is the main source of ROS in functioning mitochondria. Approximately 0.2–2% of the oxygen taken up by a cell is converted by mitochondria to ROS. Superoxide (O2•−) is the main product of these transformations, and it is then converted to hydrogen peroxide (H2O2) by spontaneous dismutation or by superoxide dismutase (SOD). Glutathione peroxidase or catalase, in turn, convert hydrogen peroxide into water. If this change does not occur, in the presence of divalent cations H2O2 can undergo Fenton's reaction to produce even more harmful hydroxyl radical (•OH). Oxygen can be reduced to superoxide in one-electron step, theoretically, at each step of the respiratory chain, but in reality two major sites of superoxide generation are Complex I and Complex III (Paradies et al., 2010, as cited in Murphy, 2009). There is a considerable experimental support for two mechanisms of ROS production by complex I. The first one is the production of ROS as a consequence of so-called reverse electron transfer (RET) in the mitochondrial respiratory chain. RET is a set of redox reactions in the mitochondrial ETC that allows electrons to ow from coenzyme Q to NAD+ instead to oxygen. The other one takes place under normal conditions, whereas most of the energy from the creation of mitochondrial potential difference is used to generate ATP through ATP synthase. This process causes collapse of the proton gradient. The amplitude of the electrochemical proton gradient regulates the flow of electrons through the ETC. When the electrochemical potential gradient is high, for instance under conditions of high glucose concentrations, the life of electron transport intermediates that are involved in superoxide formation, such as ubisemiquinone, is prolonged. The reason of such condition is that the activities of of ETC proton pumps depend on the proton gradient across the inner membrane and the membrane itself – two components of proton-motive force (Duchen, 2004).
