Many Shuttles Allow Movement Across the Mitochondrial Membranes

II. Transducing and Storing Energy
18. Oxidative Phosphorylation
18.5. Many Shuttles Allow Movement Across the Mitochondrial Membranes
The inner mitochondrial membrane must be impermeable to most molecules, yet much exchange has to take place
between the cytosol and the mitochondria. This exchange is mediated by an array of membrane-spanning transporter
proteins (Section 13.4).
18.5.1. Electrons from Cytosolic NADH Enter Mitochondria by Shuttles
Recall that the glycolytic pathway generates NADH in the cytosol in the oxidation of glyceraldehyde 3-phosphate, and
NAD+ must be regenerated for glycolysis to continue. How is cytosolic NADH reoxidized 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 18.37). 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 cytosol. 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 a FAD prosthetic
group in this enzyme to form FADH2. This reaction also regenerates dihydroxyacetone phosphate.
The reduced flavin transfers its electrons to the electron carrier Q, which then enters the respiratory chain as QH2. When
cytosolic NADH transported by the glycerol 3-phosphate shuttle is oxidized by the respiratory chain, 1.5 rather than 2.5
ATP are formed. The yield is lower because FAD rather than NAD+ is the electron acceptor in mitochondrial glycerol 3phosphate dehydrogenase. The use of FAD enables electrons from cytosolic 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 3phosphate shuttle for the regeneration of cytosolic NAD+.
In the heart and liver, electrons from cytosolic NADH are brought into mitochondria by the malate-aspartate shuttle,
which is mediated by two membrane carriers and four enzymes (Figure 18.38). Electrons are transferred from NADH in
the cytosol to oxaloacetate, forming malate, which traverses the inner mitochondrial membrane 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 23.3.1) is needed to form aspartate, which can be transported to the cytosolic side. Mitochondrial glutamate
donates an amino group, forming aspartate and α -ketoglutarate. In the cytoplasm, aspartate is then deaminated to form
oxaloacetate and the cycle is restarted. This shuttle, in contrast with the glycerol 3-phosphate shuttle, is readily
reversible. Consequently, NADH can be brought into mitochondria by the malate- aspartate shuttle only if the NADH/
NAD+ ratio is higher in the cytosol than in the mitochondrial matrix. This versatile shuttle also facilitates the exchange
of key intermediates between mitochondria and the cytosol.
18.5.2. The Entry of ADP into Mitochondria Is Coupled to the Exit of ATP by ATPADP Translocase
The major function of oxidative phosphorylation is to generate ATP from ADP. However, 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 cytosol? A specific transport protein, ATP-ADP translocase (also called adenine nucleotide
translocase or ANT), enables these molecules to traverse 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. The reaction catalyzed by the
translocase, which acts as an antiporter, is
ATP-ADP translocase is highly abundant, constituting about 14% of the protein in the inner mitochondrial membrane.
The translocase, a dimer of identical 30-kd subunits, contains a single nucleotide-binding site that alternately faces the
matrix and cytosolic sides of the membrane (Figure 18.39). ATP and ADP (both devoid of Mg2+) are bound with nearly
the same affinity. In the presence of a positive membrane potential (as would be the case for an actively respiring
mitochondrion), the rate of binding-site eversion from the matrix to the cytosolic side is more rapid for ATP than for
ADP because ATP has one more negative charge. Hence, ATP is transported out of the matrix about 30 times as rapidly
as is ADP, which leads to a higher phosphoryl transfer potential on the cytosolic side than on the matrix side. The
translocase does not evert at an appreciable rate unless a molecule of ADP is bound at the open, cytosolic site, which
then everts to the mitochondrial matrix side. This feature ensures that the entry of ADP into the matrix is precisely
coupled to the exit of ATP. The other side of the coin is that the membrane potential and hence the proton-motive force
are decreased by the exchange of ATP for ADP, which results in a net transfer of one negative charge out of the matrix.
ATP-ADP exchange is energetically expensive; about a quarter of the energy yield from electron transfer by the
respiratory chain is consumed to regenerate the membrane potential that is tapped by this exchange process. The
inhibition of this process leads to the subsequent inhibition of cellular respiration as well (Section 18.6.3).
18.5.3. Mitochondrial Transporters for Metabolites Have a Common Tripartite Motif
ATP-ADP translocase is but one of many mitochondrial transporters for ions and charged metabolites (Figure 18.40).
For historical reasons, these transmembrane proteins are sometimes called carriers. Recall that some of them function as
symporters and others as antiporters (Section 13.4). The phosphate carrier, which works in concert with ATP-ADP
translocase, mediates the electroneutral exchange of H2PO4 - for OH- (or, indistinguishably, the electroneutral symport
of H2PO4 - and H+). The combined action of these two transporters leads to the exchange of cytosolic ADP and Pi for
matrix ATP at the cost of an influx of one H+. The dicarboxylate carrier enables malate, succinate, and fumarate to be
exported from mitochondria in exchange for Pi. The tricarboxylate carrier transports citrate and H+ in exchange for
malate. Pyruvate in the cytosol enters the mitochondrial matrix in exchange for OH- (or together with H+) by means of
the pyruvate carrier. These mitochondrial transporters and more than five others have a common structural motif. They
are constructed from three tandem repeats of a 100-residue module, each containing two putative transmembrane
segments (Figure 18.41).
II. Transducing and Storing Energy
18. Oxidative Phosphorylation
18.5. Many Shuttles Allow Movement Across the Mitochondrial Membranes
Figure 18.37. 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.
II. Transducing and Storing Energy
18. Oxidative Phosphorylation
18.5. Many Shuttles Allow Movement Across the Mitochondrial Membranes
Figure 18.38. Malate-Aspartate Shuttle.
II. Transducing and Storing Energy
18. Oxidative Phosphorylation
18.5. Many Shuttles Allow Movement Across the Mitochondrial Membranes
Figure 18.39. Mechanism of Mitochondrial ATP-ADP Translocase. The translocase catalyzes the coupled entry of
ADP and exit of ATP into and from the matrix. The reaction cycle is driven by membrane potential. The actual
conformational change corresponding to eversion of the binding site could be quite small.
II. Transducing and Storing Energy
18. Oxidative Phosphorylation
18.5. Many Shuttles Allow Movement Across the Mitochondrial Membranes
Figure 18.40. Mitochondrial Transporters. Transporters (also called carriers) are transmembrane proteins that move
ions and charged metabolites across the inner mitochondrial membrane.
II. Transducing and Storing Energy
18. Oxidative Phosphorylation
18.5. Many Shuttles Allow Movement Across the Mitochondrial Membranes
Figure 18.41. Structure of Mitochondrial Transporters. Many mitochondrial transporters consist of three similar 100residue units. These proteins contain six putative membrane-spanning segments. [After J. E. Walker. Curr. Opin. Struct.
Biol. 2(1992):519.]