THE MITOCHONDRION

I) MITOCHONDRIAL STRUCTURE Mitochondria are the energy power plants of the cell. They appear as spherical or elongate bodies 0.5P in diameter and 1-2P in length. The basic structure of the mitochondrion is that of a double membraned, multicompartmented organelle. (Figs. 1, 2) A) The Outer Membrane. The outer membrane forms a smooth boundary for the mitochondrion. It is remarkable only for its high content of the protein mitochondrial porin. This protein forms a beta barrel or large pore through the membrane. The pore is sufficiently large that substances up to about 5000 MW can pass while most macromolecules are blocked. B) The Intermembrane Space. Between the outer and inner mitochondrial membranes there is a thin compartment referred to as the intermembrane space. Since the outer membrane is relatively porous the metabolite content of this space roughly matches that of the cytosol. However, the protein content is significantly different. The compartment has few proteins, the primary one being the electron transporter cytochrome-c. C) The Inner Membrane The inner mitochondrial membrane has several distinguishing characteristics. 1) It is folded into cristae to increase is surface area. The greater the energy needs of the cell the higher the number of cristae. 2) The inner membrane is not smooth like most cellular membranes. Rather a large number of particles or projections are seen extending from its inner surface. 3) It has an unusual lipid composition (including cardiolipin) that is particularly impermeable to many substances including H+. 4) It has a high protein content including diverse transporters, electron carriers and enzymatic proteins. D) The Mitochondrial Matrix The mitochondrial matrix is the central fluid compartment of the mitochondrion. It is densely packed with protein and particles. The enzymes and metabolites of the citric acid cycle and beta-oxidation are housed here. In addition the DNA and ribosomes that make the mitochondrion a semi-autonomous organelle reside here. THE ELECTRON TRANSORT CHAIN The electron transport chain receives high-energy electrons from the oxidative reactions of catabolism and passes them, along with H+, to molecular oxygen forming water. This is accomplished in a series of small potentially energy yielding steps. (Fig. 3) The steps are coupled to the generation of an electrochemical, energy storing, gradient that is subsequently used to produce ATP. The electron transport chain consists of four large protein complexes (Fig. 4) embedded in the inner mitochondrial membrane and two mobile electron shuttles. The electron path moves through several steps. A) Complex I or NADH Dehydrogenase Complex I consists of over 20 subunit proteins embedded or associated with the inner membrane. Its flavin mononucleotide (FMN) containing subunit receives a pair of electrons from NADH. In the process of this transfer the NADH releases its H+ into the matrix. The FMN now passes the electrons one at a time through a series of FeS groups

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to a molecule of Coenzyme Q (CoQ) held in a binding pocket at the protein/lipid interface near the matrix side of the complex. (Fig. 4) The movement of electrons through the complex releases energy that alters the structure of the complex. This change allows the complex to pump 4 H+ from the matrix to the intermembrane space. Complex II or Succinate Dehydrogenase Complex II is actually an enzyme complex of the citric acid cycle that is associated with the membrane. Here as Succinate is oxidized its electrons and H+ are transferred to FAD creating FADH2. Again this complex releases the H+ to the matrix and passes the electrons via FeS groups to CoQ. In this case, however, there is insufficient release of energy to promote the transport of H+ across the membrane. Complex III or CoQ/Cyt-c Oxoreductase or Cytochrome bc1 and the Q-Cycle CoQ is a mobile electron carrier. It is highly hydrophobic and is able to dissolve and move within the lipids of the inner membrane. On receiving electrons from complex I or II, however, it becomes charged and trapped in the binding pocket. Its high level of charge allows it to absorb 2 H+ from the matrix creating CoQH2. This molecule is again hydrophobic and able to flow in the membrane. Complex III is composed of 11 protein subunits three of which are critical to electron transport. This complex has a binding pocket for CoQH2 near the intermembrane space side of the molecule. When CoQH2 docks here it releases its 2H+ into the intermembrane space. It now transfers one of its electrons through a FeS and cytochrome c1 group to the next mobile carrier Cytochrome-c. Its second electron is recycled to the matrix side of complex III where it is transferred to a second waiting CoQ molecule. After and additional leg of Q-cycle/Complex III activity this second CoQ can absorb 2H+ from the matrix and carry them to the intermembrane side. (Fig. 5) Overall the energy released in this process is sufficient to allow CoQ to shuttle 4H+ from the matrix to the intermembrane space for every pair of electrons transported along the chain. Complex IV or Cytochrome Oxidase (Figs. 6, 7) The electrons moving through complex III are passed one at a time to Cytochrome-c. This small protein is a mobile electron carrier. It is trapped in the thin compartment of the intermembrane space between the inner and outer membranes. It is able to move side to side but not away from the surface of the inner membrane. Thus it is ideally positioned to move electrons from complex III to complex IV. Complex IV is composed of 13 protein subunits, two of which are critical to its electron transport function. Subunit 2 is the cytochrome-c docking center. In addition it transfers electrons one at a time to the binuclear (Cu and Fe containing) reaction center of subunit 1. This subunit is responsible for: 1) Binding molecular oxygen 2) Transferring electrons to the oxygen creating reactive oxygen species. 3) Absorbing H+ from the matrix and uniting them with the reactive oxygen to form water. 4) Using the energy released in the process to pump 2H+ from the matrix to the intermembrane space for every pair of electrons transported. The Proton-Motive Force A primary function of the electron transport chain is to move protons (H+) from the matrix to the intermembrane space side of the inner membrane. When the pair of electrons originates from NADH, 4H+ are transported by Complex I, 4H+ by Qcycle/Complex III and 2H+ by Complex IV for a total of 10H+ transported. In contrast

when the pair come from FADH2 only 6H+ are transported since Complex II does not function in H+ movement. The movement of H+ from the matrix to the intermembrane space decreases its concentration in the matrix and increases it in the intermembrane space, effectively setting up a concentration gradient. In addition, since the transported component is charged the intermembrane space becomes relatively more positive and the matrix relatively more negative i.e. an electrical gradient is also produced. This electrochemical gradient for H+ has been called the proton-motive force. This gradient represents stored or potential energy. Movement of H+ down the gradient will liberate energy that can be used to do work such as the synthesis of ATP. III) ATP SYNTHASE A) Identification. The chemiosmotic hypothesis of Mitchell proposed that the H+ ion gradient was the driving force behind ATP synthesis. Further, it was suggested that the projections on the inner membrane might be the enzyme itself. This conclusion was drawn from the observation that dislodging the particle halted ATP production. Indeed the free particle was found to bind and hydrolyze ATP and therefore was designated coupling factor 1 (F1). Further since ATP synthesis could also be blocked when the chemical oligomycin bound to the membrane it was concluded that an additional portion of the synthase was within the membrane. Since this inhibitor blocked H+ flow through the membrane it was concluded that this portion of the synthase acted like a channel and it was designated the oligomycin sensitive factor or Fo. Thus the general name of this enzyme has come to be the FoF1 ATP Synthase (F-Type Pump). Confirmation of Mitchells hypothesis and the identity of the synthase were obtained in cell free systems reconstituted from a known H+ pump and the isolated particulate synthase. B) Synthase Structure/Function (Figs. 8, 9) The isolated, lollypop shaped F1 unit is composed of 3D3E plus one each J, G and H protein subunits. The Fo unit is composed of three types of proteins 1 a subunit, 2 b subunits and 12 c subunits. The overall structure of the synthase complex might be visualized as a mushroom growing from a thick base-plate embedded in the membrane (Fig 8). The 12 c subunits form a ring within the lipid of the inner membrane of the mitochondria. The a subunit is attached to the outer surface of the c-ring helping to form a H+ channel through the membrane between the a and c subunits. The two b subunits attach to the a and project out of the membrane into the matrix where they grip and hold the D/E headpiece in place with the help of the G subunit. The b/G combination is referred to as the stator pole since they hold the base-plate and headpiece in a relatively rigid orientation. Within the headpiece the D and E subunits are quite similar. Both bind adenine nucleotides. However, it is the E subunit that has catalytic activity while the D subunit is believed to act only in a modification or regulatory fashion. These subunits form an DEDEDE ring. The JH subunit combination runs as a pole from the center of the DE ring to the center of the c-ring in the membrane. This rod is referred to as the rotor since it has been shown to spin or rotate during enzymatic activity. The presently accepted model for the production of ATP by this enzyme complex is an updated version of Boyers Binding Change Mechanism. (Fig. 9, 10)

1) H+ is pulled down its electrochemical (concentration and charge) gradient from the intermembrane space to the matrix. In order to make this journey across the membrane the H+ must travel through a channel between the a and c subunits. As it does so it causes the c-ring to rotate relative to the position of the a subunit. 2) As the c-ring turns it moves the attached JH (rotor) unit causing it to rotate as well. This movement has now been demonstrated experimentally. A projection, or bend in the rotor unit sequentially contacts the E catalytic subunits of the headpiece causing them to change shape promoting the synthesis reaction. 3) The E subunits have three main conformations; a loose conformation that loosely binds the substrates, ADP and Pi, a tight or closed conformation in which ATP is synthesized and an open conformation that releases the ATP. Each of the three E subunits sequentially moves through the three conformations driven by the influence of the contact with the JH rotor. Since the evidence indicates that most of the energy needed for ATP production is used to release the product you could think of the rotor as prying open the catalytic site to allow the ATP to leave. Although the rotor contacts only one E subunit at a time it influences all three since they act cooperatively, with one E subunit in each of the three conformations at any one time. Thus as the rotor makes one complete turn 3 ATPs will be produced, one from each E subunit. C) ATP Yield (Fig. 12) Original theoretical predictions and chemical measurements suggested that 3 H+ moving down the electrochemical gradient would cause a 1/3 turn (120o) of the synthase rotor and the production of one ATP. This was consistent with the idea that electrons from one NADH would pump 10 H+ to the intermembrane space and produce 3 ATPs. (Or one FADH2 pumping 6H+ and producing 2 ATP). Further it matched the idea that 36 ATP could be produced from the complete oxidation of a single glucose molecule. However, some recent data suggest that the actual, useful ATP yield may be less than originally believed. First, from the structure of the synthase it appears that 4 rather than 3 H+ may be needed to move the rotor 120o. Second, once ATP is produced it is trapped in the mitochondrial matrix. It may require some additional expenditure of the H+ gradient to transport the ATP to the cytosol where it will actually be used.