Electrons from cytoplasmic NADH enter mitochondria by shuttles
One function of the respiratory chain is to regenerate NAD+ for use in glycolysis. NADH generated by the citric acid cycle and fatty acid oxidation is already in the mitochondrial matrix, but how is cytoplasmic NADH reoxidized to NAD+ under aerobic conditions? NADH cannot simply pass into mitochondria for oxidation by the respiratory chain, because the inner mitochondrial membrane is impermeable to NADH and NAD+. The solution is that electrons from NADH, rather than NADH itself, are carried across the mitochondrial membrane. One of several means of introducing electrons from NADH into the electron-transport chain is the glycerol 3-phosphate shuttle (Figure 18.35). The first step in this shuttle is the transfer of a pair of electrons from NADH to dihydroxyacetone phosphate, a glycolytic intermediate, to form glycerol 3-phosphate. This reaction is catalyzed by a glycerol 3-phosphate dehydrogenase in the cytoplasm. Glycerol 3-phosphate is reoxidized to dihydroxyacetone phosphate on the outer surface of the inner mitochondrial membrane by a membrane-bound isozyme of glycerol 3-phosphate dehydrogenase. An electron pair from glycerol 3-phosphate is transferred to an FAD prosthetic group in this enzyme to form FADH2. This reaction also regenerates dihydroxyacetone phosphate.
Figure 18.35: Glycerol 3-phosphate shuttle. Electrons from NADH can enter the mitochondrial electron-transport chain by being used to reduce dihydroxyacetone phosphate to glycerol 3-phosphate. Glycerol 3-phosphate is reoxidized by electron transfer to an FAD prosthetic group in a membrane-bound glycerol 3-phosphate dehydrogenase. Subsequent electron transfer to Q to form QH2 allows these electrons to enter the electron-transport chain.
The reduced flavin transfers its electrons to the electron carrier Q, which then enters the respiratory chain as QH2. When cytoplasmic NADH transported by the glycerol 3-phosphate shuttle is oxidized by the respiratory chain, 1.5 rather than 2.5 molecules of ATP are formed. The yield is lower because FAD rather than NAD+ is the electron acceptor in mitochondrial glycerol 3-phosphate dehydrogenase. The use of FAD enables electrons from cytoplasmic NADH to be transported into mitochondria against an NADH concentration gradient. The price of this transport is one molecule of ATP per two electrons. This glycerol 3-phosphate shuttle is especially prominent in muscle and enables it to sustain a very high rate of oxidative phosphorylation. Indeed, some insects lack lactate dehydrogenase and are completely dependent on the glycerol 3-phosphate shuttle for the regeneration of cytoplasmic NAD+.
In the heart and liver, electrons from cytoplasmic NADH are brought into mitochondria by the malate–aspartate shuttle , which is mediated by two membrane carriers and four enzymes (Figure 18.36). Electrons are transferred from NADH in the cytoplasm to oxaloacetate, forming malate, which traverses the inner mitochondrial membrane in exchange for α-ketoglutarate and is then reoxidized by NAD+ in the matrix to form NADH in a reaction catalyzed by the citric acid cycle enzyme malate dehydrogenase. The resulting oxaloacetate does not readily cross the inner mitochondrial membrane and so a transamination reaction (Section 23.3) is needed to form aspartate, which can be transported to the cytoplasmic side in exchange for glutamate. Glutamate donates an amino group to oxaloacetate, forming aspartate and α-ketoglutarate. In the cytoplasm, aspartate is then deaminated to form oxaloacetate and the cycle is restarted.
Figure 18.36: Malate–aspartate shuttle.
The entry of ADP into mitochondria is coupled to the exit of ATP by ATP-ADP translocase
The major function of oxidative phosphorylation is to generate ATP from ADP. ATP and ADP do not diffuse freely across the inner mitochondrial membrane. How are these highly charged molecules moved across the inner membrane into the cytoplasm? A specific transport protein, ATP-ADP translocase, enables these molecules to transverse this permeability barrier.
Most important, the flows of ATP and ADP are coupled. ADP enters the mitochondrial matrix only if ATP exits, and vice versa.This process is carried out by the translocase, an antiporter:
ATP-ADP translocase is highly abundant, constituting about 15% of the protein in the inner mitochondrial membrane. The abundance is a manifestation of the fact that human beings exchange the equivalent of their weight in ATP each day. Despite the fact that mitochondria are the sites of ATP synthesis, both the cytoplasm and the nucleus have more ATP than the mitochondria, a testament to the efficiency of transport.
The 30-kDa translocase contains a single nucleotide-binding site that alternately faces the matrix and the cytoplasmic sides of the membrane (Figure 18.37). ATP and ADP bind to the translocase without Mg2+, and ATP has one more negative charge than that of ADP. Thus, in an actively respiring mitochondrion with a positive membrane potential, ATP transport out of the mitochondrial matrix and ADP transport into the matrix are favored. This ATP-ADP exchange is energetically expensive; about a quarter of the proton-motive force generated by the respiratory chain is consumed by this exchange process. The inhibition of the translocase leads to the subsequent inhibition of cellular respiration as well.
Figure 18.37: Mechanism of mitochondrial ATP-ADP translocase. The translocase catalyzes the coupled entry of ADP into the matrix and the exit of ATP from it. The binding of ADP (1) from the cytoplasm favors eversion of the transporter (2) to release ADP into the matrix (3). Subsequent binding of ATP from the matrix to the everted form (4) favors eversion back to the original conformation (5), releasing ATP into the cytoplasm (6).
Mitochondrial transporters for metabolites have a common tripartite structure
Examination of the amino acid sequence of the ATP-ADP translocase revealed that this protein consists of three tandem repeats of a 100-aminoacid module, each of which appears to have two transmembrane segments. This tripartite structure has recently been confirmed by the determination of the three-dimensional structure of this transporter (Figure 18.38). The transmembrane helices form a tepee-like structure with the nucleotide-binding site (marked by a bound inhibitor) lying in the center. Each of the three repeats adopts a similar structure.
Figure 18.38: 
Structure of mitochondrial transporters. The structure of the ATP-ADP translocase is shown. Notice that this structure comprises three similar units (shown in red, blue, and yellow) that come together to form a binding site, here occupied by atractyloside, an inhibitor of this transporter. Other members of the mitochondrial transporter family adopt similar tripartite structures.
[Drawn from 1OKC.pdb.]
ATP-ADP translocase is but one of many mitochondrial transporters for ions and charged metabolites (Figure 18.39). The phosphate carrier, which works in concert with ATP-ADP translocase, mediates the electroneutral exchange of H2PO4− for OH−. The combined action of these two transporters leads to the exchange of cytoplasmic ADP and Pi for matrix ATP at the cost of the influx of one H+ (owing to the transport of one OH− out of the matrix). These two transporters, which provide ATP synthase with its substrates, are associated with the synthase to form a large complex called the ATP synthasome.
Figure 18.39: Mitochondrial transporters. Transporters (also called carriers) are transmembrane proteins that carry specific ions and charged metabolites across the inner mitochondrial membrane.
Other homologous carriers also are present in the inner mitochondrial membrane. The dicarboxylate carrier enables malate, succinate, and fumarate to be exported from the mitochondrial matrix in exchange for Pi. The tricarboxylate carrier exchanges citrate and H+ for malate. In all, more than 40 such carriers are encoded in the human genome. A different type of carrier is responsible for pyruvate transport into the mitochondria. Pyruvate in the cytoplasm enters the mitochondrial membrane by way of a heterodimer composed of two small transmembrane proteins.
