Mitochondria substrate transport

The control of the respiration process and ATP synthesis shifts as the metabolic state of the mitochondria changes. In an isolated mitochondrion, control over the respiration process in state 4 is mainly due to the proton leak through the mitochondrial inner membrane. This type of control decreases from state 4 to state 3, while the control by the adenine nucleotide and the dicarboxylate carriers, cytochrome oxidase, increases. ATP utilizing reactions and transport activities also increase. Therefore, in state 3, most of the control is due to respiratory chain and substrate transport. 

Under aerobic conditions, the hydrogen atoms of NtUDH are oxidised within the mitochondrion pyruvate is also oxidised in the mitochondrion (Figure 9.15). However, NADH cannot be transported across the inner mitochondrial membrane, and neither can the hydrogen atoms themselves. This problem is overcome by means of a substrate shuttle. In principle, this involves a reaction between NADH and an oxidised substrate to produce a reduced product in the cytosol, followed by transport of the reduced product into the mitochondrion, where it is oxidised to produce hydrogen atoms or electrons, for entry into the electron transfer chain. Finally, the oxidised compound is transported back into the cytosol. The principle of the shuttle is shown in Figure 9.16. 

Oxidizible substrates from glycolysis, fatty acid or protein catabolism enter the mitochondrion in the form of acetyl-CoA, or as other intermediaries of the Krebs cycle, which resides within the mitochondrial matrix. Reducing equivalents in the form of NADH and FADH pass electrons to complex I (NADH-ubiquinone oxidore-ductase) or complex II (succinate dehydrogenase) of the electron transport chain, respectively. Electrons pass from complex I and II to complex III (ubiquinol-cyto-chrome c oxidoreductase) and then to complex IV (cytochrome c oxidase) which accumulates four electrons and then tetravalently reduces O2 to water. Protons are pumped into the inner membrane space at complexes I, II and IV and then diffuse down their concentration gradient through complex V (FoFi-ATPase), where their potential energy is captured in the form of ATP. In this way, ATP formation is coupled to electron transport and the formation of water, a process termed oxidative phosphorylation (OXPHOS). 

Current estimates are that three protons move into the matrix through the ATP-synthase for each ATP that is synthesized. We see below that one additional proton enters the mitochondrion in connection with the uptake of ADP and Pi and export of ATP, giving a total of four protons per ATP. How does this stoichiometry relate to the P-to-O ratio When mitochondria respire and form ATP at a constant rate, protons must return to the matrix at a rate that just balances the proton efflux driven by the electron-transport reactions. Suppose that 10 protons are pumped out for each pair of electrons that traverse the respiratory chain from NADH to 02, and 4 protons move back in for each ATP molecule that is synthesized. Because the rates of proton efflux and influx must balance, 2.5 molecules of ATP (10/4) should be formed for each pair of electrons that go to 02. The P-to-O ratio thus is given by the ratio of the proton stoichiometries. If oxidation of succinate extrudes six protons per pair of electrons, the P-to-O ratio for this substrate is 6/4, or 1.5. These ratios agree with the measured P-to-O ratios for the two substrates. 

Almost all cells have an active transport system to maintain nonequilibrium concentration levels of substrates. For example, in the mitochondrion, hydrogen ions are pumped into the intermembrane space of the organelle as part of producing ATP. Active transport concentrates ions, minerals, and nutrients inside the cell that are in low concentration... 

Inside the inner membrane of a mitochondrion is a viscous region known as the matrix (Fig. 1-9). Enzymes of the tricarboxylic acid (TCA) cycle (also known as the citric acid cycle and the Krebs cycle), as well as others, are located there. For substrates to be catabolized by the TCA cycle, they must cross two membranes to pass from the cytosol to the inside of a mitochondrion. Often the slowest or rate-limiting step in the oxidation of such substrates is their entry into the mitochondrial matrix. Because the inner mitochondrial membrane is highly impermeable to most molecules, transport across the membrane using a carrier or transporter (Chapter 3, Section 3.4A) is generally invoked to explain how various substances get into the matrix. These carriers, situated in the inner membrane, might shuttle important substrates from the lumen between the outer and the inner mitochondrial membranes to the matrix. Because of the inner membrane, important ions and substrates in the mitochondrial matrix do not leak out. Such permeability barriers between various subcellular compartments improve the overall efficiency of a cell. 

The bulk of the energy demands of the cell are met within the mitochondria by the production of ATP during the oxidation of substrates by way of the hydrogen transport line (see Chapter 7). When the enzymes and carriers of this system are studied in isolation, they are found to be capable of extremely rapid reactions, yet if the intact mitochondrion is presented with substrates such as pyruvic acid it is found that the rate of pyruvic acid oxidation reaches a maximum which is considerably below the maximum velocities shown by the individual carriers. As increasing the amount of pyruvic acid does not alter this oxidation rate, it is clear that the mitochondrion must contain its own built-in control system to limit the rate at which it burns fuel. We can isolate some of the elements in this control system if we draw a schematic flowsheet of the operations involved in oxidation (Figure 25). 

When the levels of ADP and (p) within the mitochondria rise again, oxidation can proceed smoothly and rapidly. It therefore follows that the rate of oxidation of substrate through the electron transport chain is controlled by the concentrations of ADP and inorganic phosphate present in the mitochondrion. When there is plenty of ADP and phosphate, oxidation proceeds smoothly and rapidly. When the concentrations are low, oxidation is correspondingly slow. 

The urea cycle converts ammonium ions into urea, which is less toxic. The sources of the atoms are shown in color and the intracellular locations of the reactions are indicated. Citrulline, formed in the reaction between ornithine and carbamoyl phosphate, is transported out of the mitochondrion and into the cytoplasm. Ornithine, a substrate for the formation of citrulline, is transported from the cytoplasm into the mitochondrion. 

Malate is then transported into the mitochondrion where it is reoxidized to oxaloacetate. Mitochondrial NAD is reduced in the process. These electrons are then used in oxidative phosphorylation to produce three ATP per NADH. Thus the energy yield of glycolysis in heart and liver cells is two ATP, produced by substrate level phosphorylation, plus six ATP (three ATP per NADH), produced by oxidative phosphorylation. This gives an energy yield of eight ATP per glucose. 

Overall, each NADH donates two electrons, equivalent to the reduction of V2 of an O2 molecule. A generally (but not universally) accepted estimate of the stoichiometry of ATP synthesis is that four protons are pumped at complex I, four protons at complex III, and two at complex IV. With four protons translocated for each ATP synthesized, an estimated 2.5 ATPs are formed for each NADH oxidized and 1.5 ATPs for each of the other FAD(2H)-containing flavoproteins that donate electrons to CoQ. (This calculation neglects proton requirements for the transport of phosphate and substrates from the cytosol, as well as the basal proton leak.) Thus, only approximately 30% of the energy available from NADH and FAD(2H) oxidation by O2 is used for ATP synthesis. Some of the remaining energy in the electrochemical potential is used for the transport of anions and Ca into the mitochondrion. The remainder of the energy is released as heat. Consequently, the electron transport chain is also our major source of heat. 

The most likely deficiency is a lack of 2,4-dienoyl CoA reductase, an enzyme that is essential for the degradation of unsaturated fatty acids with double bonds at even-numbered carbons. Such fatty acids include linoleate (9-ds,12-ds 18 2). Four rounds of oxidation of linoleoyl CoA generate a 10-carbon acyl CoA that contains a trans-A and a cis-A double bond. This intermediate is a substrate for the reductase, which converts the 2,4-dienoyl CoA to ds-A -enoyl CoA. A dehciency of 2,4-dienoyl reductase leads to an accumulation of trans-A, ds-A -decadienoyl CoA molecules in the mitochondrion. The observation that carnitine derivatives of the 2,4-dienoyl CoA are found in blood and urine provides evidence that these molecules accumulate in the mitochondrion and are then attached to carnitine. Formation of carnitine decadienoate allows the acyl molecules to be transported across the inner mitochondrial membrane into the cytosol, and then into the circulation. 

Fig. 13.1.1. Schematic overview of mitochondrial oxidative phosphorylation. A part of the mitochondrion is represented, showing the outer mitochondrial membrane (OMM), inner mitochondrial membrane (IMM) and crista (an invagination of the inner membrane). Substrates for oxidation enter the mitochondrion through specific carrier proteins, e.g., the pyruvate transporter, (PyrT). Reducing equivalents from fatty acyl CoA dehydrogenases, pyruvate dehydrogenase and the TCA cycle are delivered to the electron transport chain through NADH, succinate ubiquinol oxidoreductase (SQO), electron transfer flavoprotein (ETF) and its ubiquinol-...

The conservation of energy from electron transport requires not only the synthesis of ATP within the mitochondrion, but also its export to the cytoplasm as well as the import of substrates for oxidation and phosphorylation. Only the proteins responsible for synthesis of ATP and exchange of ATP for ADP across the inner mitochondrial membrane will be considered in any detail here, as they are known targets for antibiotics and pesticides. However, numerous other mitochondrial transporters identified in plants, fungi and animals may provide future opportunities for useful chemical intervention [110, 111]. 

Mitochondrial P-oxidation of long-chain fatty acids is the major source of energy production in man. The mitochondrial inner membrane is impermeable to long chain fatty acids or their CoA esters whereas acylcamitines are transported. Three different gene products are involved in this carnitine dependent transport shuttle carnitine palmi-toyl transferase I (CPT I), carnitine acyl-camitine carrier (CAC) and carnitine palmitoyl transferase II (CPT II). The first enzyme (CPT I) converts fatty acyl-CoA esters to their carnitine esters which are subsequently translocated across the mitochondrial inner membrane in exchange for free carnitine by the action of the carnitine acyl-camitine carrier (CAC). Once inside the mitochondrion, CPT II reconverts the carnitine ester back to the CoA ester which can then serve as a substrate for the P-oxidation spiral. 

If the mitochondrion is a principal site of the PDC, hence the formation of acetyl-CoA, then the plant cell must employ a transport system which can export this highly reactive substrate from the matrix of the mitochondrion to the appropriate site where it is utilized for fatty acid biosynthesis. However, this problem may not be serious, since in seeds that contain high levels of lipid at maturity the proplastid appears to be the principal site of the conversion of pyruvate to acetyl-CoA by the PDC and of its efficient utilization for the biosynthesis of palmitoyl-and/or stearoyl-ACP. In the chloroplast, evidence for the PDC is not clear, although indirect evidence does suggest PDC activity (Murphy and Leech, 1978) in these organelles. Since acetate moves freely across the chloroplast membrane and since acetyl-CoA synthetase occurs in the chloroplast stroma (Jacobson and Stumpf, 1972), it is possible that the primary source of acetyl-CoA derives from its synthesis at a site other than the chloroplast, its transport into the chloroplast as the undissociated acid and/or free anion, and the conversion of acetate back to acetyl-CoA by the stroma acetyl-CoA synthetase. Further work is necessary to clarify this important point. It is difficult to explain the function of acetyl-... 

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