Mitochondria Mitochondrial membrane, inner

Energy-linked transhydrogenase, a protein in the inner mitochondrial membrane, couples the passage of protons down the electrochemical gradient from outside to inside the mitochondrion with the transfer of H from intramitochondrial NADH to NADPH for intramitochondrial enzymes such as glutamate dehydrogenase and hydroxylases involved in steroid synthesis. 

Mitochondrial DNA is inherited maternally. What makes mitochondrial diseases particularly interesting from a genetic point of view is that the mitochondrion has its own DNA (mtDNA) and its own transcription and translation processes. The mtDNA encodes only 13 polypeptides nuclear DNA (nDNA) controls the synthesis of 90-95% of all mitochondrial proteins. All known mito-chondrially encoded polypeptides are located in the inner mitochondrial membrane as subunits of the respiratory chain complexes (Fig. 42-3), including seven subunits of complex I the apoprotein of cytochrome b the three larger subunits of cytochrome c oxidase, also termed complex IV and two subunits of ATPase, also termed complex V. 

Mitochondria are distinct organelles with two membranes. The outer membrane limits the organelle and the inner membrane is thrown into folds or shelves that project inward and are called cristae mitochondriales. The uptake of most mitochondrion-selective dyes is dependent on the mitochondrial membrane potential. Conventional fluorescent stains for mitochondria, such as rhodamine and tetramethylrosamine, are readily sequestered by functioning mitochondria. They are, however, subsequently washed out of the cells once the mitochondrion s membrane potential is lost. This characteristic limits their use in experiments in which cells must be treated with aldehyde-based fixatives or other agents that affect the energetic state of the mitochondria. To overcome this limitation, the research... 

What do I mean by a proton concentration gradient Simply, there is a higher concentration of protons in the space between the inner and outer membranes of the mitochondrion than in the mitochondrial interior. The gradient is formed from the energy released in the transfer of electrons down the electron transport chain. Put another way, the released energy is employed to pump protons across the inner mitochondrial membrane into the intermembrane space. 

Figure 9.13 Examples of mitochondrial transport systems for anions. 0 The anb port system transfers malate into but oxo-glutarate out of the mitochondrion. The symport system transfers both pyruvate and protons into the mitochondrion across the inner membrane. Both transport processes are electroneutral.

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. 

The oxaloacetate is then transported from mitochondrion into the cytosol but not directly, since there is no transporter for oxaloacetate in the mitochondrial membrane. This problem is solved by conversion of oxaloacetate to aspartate, by transamination, and it is the aspartate that is transported across the inner mitochondrial membrane to the cytosol, where oxaloacetate is regenerated from aspartate by a cytosolic aminotransferase enzyme. 

In addition to the processes described above, there still remains one further process which, at least in some cells or tissues, is required prior to the utilisation of ATP in the cytosol that is, the transport of energy within the cytosol, via a shuttle. The transport of ATP out and ADP into the mitochondrion, via the translocase, results in a high ATP/ ADP concentration ratio in the cytosol. However, a high ratio means that the actual concentration of ADP in the cytosol is low, which could result in slow diffusion of ADP from a site of ATP utilisation back to the inner mitochondrial membrane. If sufficiently slow, it could limit the rate of ATP generation. To overcome this, a process exists that transports energy within the cytosol, not by diffusion of ATP and ADP, but by the diffusion of phosphocreatine and creatine, a process known as the phosphocreatine/creatine shuttle. The reactions involved in the shuttle in muscle help to explain the significance of the process. They are ... 

The tricarboxylic acid cycle not only takes up acetyl CoA from fatty acid degradation, but also supplies the material for the biosynthesis of fatty acids and isoprenoids. Acetyl CoA, which is formed in the matrix space of mitochondria by pyruvate dehydrogenase (see p. 134), is not capable of passing through the inner mitochondrial membrane. The acetyl residue is therefore condensed with oxaloacetate by mitochondrial citrate synthase to form citrate. This then leaves the mitochondria by antiport with malate (right see p. 212). In the cytoplasm, it is cleaved again by ATP-dependent citrate lyase [4] into acetyl-CoA and oxaloacetate. The oxaloacetate formed is reduced by a cytoplasmic malate dehydrogenase to malate [2], which then returns to the mitochondrion via the antiport already mentioned. Alternatively, the malate can be oxidized by malic enzyme" [5], with decarboxylation, to pyruvate. The NADPH+H formed in this process is also used for fatty acid biosynthesis. 

Malate leaves the mitochondrion through a specific transporter in the inner mitochondrial membrane (see Fig. 19-27), and in the cytosol it is reoxidized to oxaloacetate, with the production of cytosolic NADH ... 

What are the molar concentrations of the electron carriers in mitochondrial membranes In one experiment, cytochrome b was found in rat liver mitochondria to the extent of 0.28 pmol/g of protein. If we take a total mitochondrion as about 22% protein, the average concentration of the cytochrome would be 0.06 mM. Since all the cytochromes are concentrated in the inner membranes, which may account for 10% or less of the volume of the mitochondrion, the concentration of cytochromes may approach 1 mM in these membranes. This is sufficient to ensure rapid reactions with substrates. 

Acyl-CoA is not transported across the inner membrane of the mitochondrion. Instead, the acyl-CoA reacts with carnitine to yield the acyl-carnitine derivative. This reaction is catalyzed by carnitine acyltransferase I, which is located on the outer mitochondrial membrane. The acyl-carnitine is transported across the inner membrane by a specific carrier protein. Once inside the matrix of the mitochondrion, the acyl-carnitine is converted back to its acyl-CoA... 

This reaction is catalyzed by carnitine acyltransferase I on the outer membrane (fig. 18.21). A protein carrier in the inner mitochondrial membrane transfers the acyl-carnitine derivatives across the membrane. Once inside the mitochondria, the reaction is reversed by carnitine acyltransferase II to yield a fatty acyl-CoA (see fig. 18.21). Thus, at least two distinct pools of acyl-CoA occur in the cell, one in the cytosol and the other in the mitochondrion. 

Oxaloacetate, the product of the first step in gluconeogenesis, must leave the mitochondrion and enter the cytosol where the subsequent enzyme steps take place. Since the inner mitochondrial membrane is impermeable to oxaloacetate, it is converted to malate by mitochondrial malate dehydrogenase. This leaves the mitochondrion and is converted back to oxaloacetate in the cytosol by cytoplasmic malate dehydrogenase. 

Pyruvate carboxylase is a mitochondrial matrix enzyme whereas the other enzymes of gluconeogenesis are located outside the mitochondrion. Thus oxaloacetate, produced by pyruvate carboxylase, needs to exit the mitochondrion. However the inner mitochondrial membrane is not permeable to this compound. Thus oxaloacetate is converted to malate inside the mitochondrion... 

Oxidative phosphorylation is ATP synthesis linked to the oxidation of NADH and FADH2 by electron transport through the respiratory chain. This occurs via a mechanism originally proposed as the chemiosmotic hypothesis. Energy liberated by electron transport is used to pump H+ ions out of the mitochondrion to create an electrochemical proton (H+) gradient. The protons flow back into the mitochondrion through the ATP synthase located in the inner mitochondrial membrane, and this drives ATP synthesis. Approximately three ATP molecules are synthesized per NADH oxidized and approximately two ATPs are synthesized per FADH2 oxidized. 

A similar shuttle, the malate-aspartate shuttle, operates in heart and liver (Fig. 6). Oxaloacetate in the cytosol is converted to malate by cytoplasmic malate dehydrogenase, reoxidizing NADH to NAD+ in the process. The malate enters the mitochondrion via a malate-a-ketoglutarate carrier in the inner mitochondrial membrane. In the matrix the malate is reoxidized to oxaloacetate by NAD+ to form NADH. Oxaloacetate does not easily cross the inner mitochondrial membrane and so is transaminated to form aspartate which then exits from the mitochondrion... 

Answer The transport of fatty acid molecules into mitochondria requires a shuttle system involving a fatty acyl-carnitine intermediate. Fatty acids are first converted to fatty acyl-CoA molecules in the cytosol (by the action of acyl-CoA synthetases) then, at the outer mitochondrial membrane, the fatty acyl group is transferred to carnitine (by the action of carnitine acyl-transferase I). After transport of fatty acyl-carnitine through the inner membrane, the fatty acyl group is transferred to mitochondrial CoA. The cytosolic and mitochondrial pools of CoA are thus kept separate, and no labeled CoA from the cytosolic pool enters the mitochondrion. 

Answer Malate dehydrogenase catalyzes the conversion of malate to oxaloacetate in the citric acid cycle, which takes place in the mitochondrion, and also plays a key role in the transport of reducing equivalents across the inner mitochondrial membrane via the malate-aspartate shuttle (Fig. 19-29). This shuttle requires the presence of malate dehydrogenase in the cytosol and the mitochondrial matrix. 

Answer NADH produced in the cytosol cannot cross the inner mitochondrial membrane, but must be oxidized if glycolysis is to continue. Reducing equivalents from NADH enter the mitochondrion by way of the malate-aspartate shuttle. NADH reduces oxaloacetate to form malate and NAD+, and the malate is transported into the mitochondrion. Cytosolic oxidation of glucose can continue, and the malate is converted back to oxaloacetate and NADH in the mitochondrion (see Fig. 19-29). 

Answer Pyruvate dehydrogenase is located in the mitochondrion, and glyceraldehyde 3-phosphate dehydrogenase in the cytosol. Because the mitochondrial and cytosolic pools of NAD are separated by the inner mitochondrial membrane, the enzymes do not compete for the same NAD pool. However, reducing equivalents are transferred from one nicotinamide coenzyme pool to the other via shuttle mechanisms (see Problem 21). 

Fig. 3. The rate-limiting step of steroidogenesis under ACTH regulation. The transfer of cholesterol (C) from the outer to the inner mitochondrial membrane under ACTH regulation (step 3) makes cholesterol available to cytochrome /M50scc for conversion to pregnenolone (step 4), which diffuses out of the mitochondrion (step 5). Because of its insolubility in aqueous media, cholesterol must be transported to mitochondria, probably by SCP2, from a precursor pool (step 2). Here, cholesterol in the precursor pool is shown as being formed from cholesterol esters (CE) by cholesterol ester hydrolase (CEH) (step 1) other possible pathways are shown in Figs. 4 and 6. From Ref. 14.

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. 

Figure 17.1. Mitochondrion. The double membrane of the mitochondrion is evident in this electron micrograph. The numerous invaginations of the inner mitochondrial membrane are called cristae. The oxidative decarboxylation of pyruvate and the sequence of reactions in the citric acid cycle take place within the matrix. [(Left) Omikron/Photo Researchers.]...

All three respiratory complexes are typical integral membrane proteins that span the inner mitochondrial membrane. Each consists of several different subunits, the exact number of which is still under debate. The genes of some subunits of cytochrome oxidase and the />c, complex are in mitochondrial DNA (mtDNA). These proteins are synthesised inside the mitochondrion. However, most proteins of these complexes, as well as cytochrome c, are synthesised on cytoplasmic ribosomes and coded by the nuclear genome. This raises intriguing questions of how the latter are imported into the mitochondrion and inserted into the mitochondrial membrane, as well as of how mitochondrial and cytoplasmic transcription and translation are synchronised [3-5]. 

---