Cytosolic NADH, oxidation

AFR has been postulated to be an electron acceptor for cytosolic NADH oxidation. The existence of such an enzyme system has been reported in soluble and endomembrane fractions (Hossain et al., 1984 Borracino et al., 1986 Arrigoni, 1994) and isolated plasma membrane vesicles from plants (Morre et al., 1986, 1987 Luster and Buckout, 1988, 1989 Serrano et al., 1994). A role for AFR in the mechanism for membrane energization, on the basis of the generation of a proton gradient and/or changes in membrane potential, has been suggested (Navas, 1991 ... 

Most of the NADH used in electron transport is produced in the mitochondrial matrix space, an appropriate site because NADH is oxidized by Complex I on the matrix side of the inner membrane. Furthermore, the inner mitochondrial membrane is impermeable to NADH. Recall, however, that NADH is produced in glycolysis by glyceraldehyde-3-P dehydrogenase in the cytosol. If this NADH were not oxidized to regenerate NAD, the glycolytic pathway would cease to function due to NAD limitation. Eukaryotic cells have a number of shuttle systems that harvest the electrons of cytosolic NADH for delivery to mitochondria without actually transporting NADH across the inner membrane (Figures 21.33 and 21.34). 

Another pathway is the L-glycerol 3-phosphate shuttle (Figure 11). Cytosolic dihydroxyacetone phosphate is reduced by NADFl to s.n-glycerol 3-phosphate, catalyzed by s,n-glycerol 3-phosphate dehydrogenase, and this is then oxidized by s,n-glycerol 3-phosphate ubiquinone oxidoreductase to dihydroxyacetone phosphate, which is a flavoprotein on the outer surface of the inner membrane. By this route electrons enter the respiratory chain.from cytosolic NADH at the level of complex III. Less well defined is the possibility that cytosolic NADH is oxidized by cytochrome bs reductase in the outer mitochondrial membrane and that electrons are transferred via cytochrome b5 in the endoplasmic reticulum to the respiratory chain at the level of cytochrome c (Fischer et al., 1985). 

The final reactions to be considered in the metabolism of ethanol in the liver are those involved in reoxidation of cytosolic NADH and in the reduction of NADP. The latter is achieved by the pentose phosphate pathway which has a high capacity in the liver (Chapter 6). The cytosolic NADH is reoxidised mainly by the mitochondrial electron transfer system, which means that substrate shuttles must be used to transport the hydrogen atoms into the mitochondria. The malate/aspartate is the main shuttle involved. Under some conditions, the rate of transfer of hydrogen atoms by the shuttle is less than the rate of NADH generation so that the redox state in the cytosolic compartment of the liver becomes highly reduced and the concentration of NAD severely decreased. This limits the rate of ethanol oxidation by alcohol dehydrogenase. 

Shuttle Systems Indirectly Convey Cytosolic NADH into Mitochondria for Oxidation... 

The mitochondrial inner membrane has no transport system for NAD+ or NADH. In animal cells, most of the NADH that must be oxidized by the respiratory chain is generated in the mitochondrial matrix by the TCA cycle or the oxidation of fatty acids. However, NADH also is generated by glycolysis in the cytosol. If 02 is available, it clearly is advantageous to reoxidize this NADH by the respiratory chain, rather than by the formation of lactate or ethanol as described in chapter 12. This is evident from the findings that approximately 2.5 molecules of ATP can be formed for each NADH oxidized in the mitochondria, whereas no ATP is made when NADH is oxidized by the cytosolic lactate dehydrogenase or alcohol dehydrogenase. 

In fact, the glyceraldehyde 3-phosphate dehydrogenase reaction also consumes NADH, equivalent to two molecules of NADH for each molecule of glucose synthesized. Since each cytosolic NADH would normally be used to generate approximately two ATP molecules via the glycerol 3-phosphate shuttle and oxidative phosphorylation (see Topic L2), this is equivalent to the input of another four ATPs per glucose synthesized. 

Cytosolic NADH cannot cross the inner mitochondrial membrane and enter mitochondria to be reoxidized. However, it can be reoxidized via the glycerol 3-phosphate shuttle. Cytosolic glycerol 3-phosphate dehydrogenase oxidizes the NADH and reduces dihydroxyacetone phosphate to glycerol 3-phosphate. 

Fig. 3.4 The glycolytic pathway produces NADH which under regular conditions is oxidized to NAD+ while reducing acetaldehyde (ACA) to ethanol (EtOH), thereby in turn reducing NAD+ in order to keep hexose catabolism running. The actual cytosolic NADH concentration is determined by the respective conversion rates of the enzymes involved in the oxidation and regeneration of the compound. If these enzymes convert additional non-natural substrates (xenobiotics, i.e. drugs), the conversion rate changes. As a consequence, the cytosolic NADH concentration differs from the natural condition. Furthermore, if a xenobiotic acts as an enzyme inhibitor, e.g. for ADH, then NAD+ regeneration is substantially affected, which eventually results in altered cytosolic NADH concentration. Therefore the presence of a xenobiotic in the cell is conceivably a perturbation factor. Under the conditions where glycolytic oscillations...

Citrin is an aspartate-glutamate antiporter that has a role both in the urea cycle and in the malate aspartate shuttle. It is necessary for the transport of aspartate produced in the mitochondria into the cytosol, where it is used by AS. Its role in the malate-aspartate shuttle is to transport cytosolic NADH reducing equivalents into the mitochondria, where they are used in oxidative phosphorylation. Defects in citrin cause citrullinemia type II. Patients manifest later-onset intermittent hyperammonemic encephalopathy as in HHH syndrome. 

The FADH2 enters the electron-transport chain at coenzyme Q, while the dihydroxyacetone phosphate can return to the cytoplasm. Although this shuttle is generally inefficient, in the sense that only two ATP molecules are produced per FADH2 molecule oxidized, compared with three for NADH oxidation, it provides a mechanism for regeneration of NAD+ in the cytosol. The presence of cytosolic NAD+ is essential for continued glycolysis (see Fig. 11-20). 

NADH-dehydrogenase Iron-Sulfur Cytosol/Mitochondria Oxidation of NADH to NAD+... 

A FIGURE 8-7 Summary of the aerobic oxidation of pyruvate and fatty acids in mitochondria. The outer membrane is freely permeable to all metabolites, but specific transport proteins (colored ovals) in the inner membrane are required to import pyruvate (yellow), ADP (green), and P (purple) into the matrix and to export ATP (green). NADH generated in the cytosol is not transported directly to the matrix because the inner membrane is impermeable to NAD and NADH instead, a shuttle system (red) transports electrons from cytosolic NADH to NAD in the matrix. O2 diffuses into the matrix and CO2 diffuses out. Stage 1 Fatty acyl groups are transferred from fatty acyl CoA and transported across the inner membrane via a special carrier (blue oval) and then reattached to CoA on the matrix side. 

For aerobic oxidation to continue, the NADH produced during glycolysis In the cytosol must be oxidized to NAD. As with NADH generated in the mitochondrial matrix, electrons from cytosolic NADH are ultimately transferred to O2 via the respiratory chain, concomitant with the generation of... 

In addition to NADH dehydrogenase, succinic dehydrogenase and other flavopro-teins in the inner mitochondrial membrane also pass electrons to CoQ (see Fig. 21.5). Succinate dehydrogenase is part of the TCA cycle. ETF-CoQ oxidore-ductase accepts electrons from ETF (electron transferring flavoprotein), which acquires them from fatty acid oxidation and other pathways. Both of these flavo-proteins have Fe-S centers. a-Glycerophosphate dehydrogenase is a flavoprotein that is part of a shuttle for reoxidizing cytosolic NADH. 

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 reason that shuttles are required for the oxidation of cytosolic NADH by the electron transport chain is that the inner mitochondrial membrane is impermeable... 

A more complex and more efficient shutde mechanism is the malate-aspartate shuttle, which has been found in mammalian kidney, liver, and heart. This shuttle uses the fact that malate can cross the mitochondrial membrane, while oxaloacetate cannot. The noteworthy point about this shuttle mechanism is that the transfer of electrons from NADH in the cytosol produces NADH in the mitochondrion. In the cytosol, oxaloacetate is reduced to malate by the cytosolic malate dehydrogenase, accompanied by the oxidation of cytosolic NADH to NAD+ (Figure 20.24). The malate then crosses the mitochondrial membrane. In the mitochondrion, the conversion of malate back to oxaloacetate is catalyzed by the mitochondrial malate dehydrogenase (one of the enzymes of the citric acid cycle). Oxaloacetate is converted to aspartate, which can also cross the mitochondrial membrane. Aspartate is converted to oxaloacetate in the cytosol, completing the cycle of reactions. 

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