6.6 Auxiliary shuttle systems for the oxidation of cytosolic NADH

In the chapter on glycolysis, it was mentioned that under aerobic conditions the NAD⁺ converted to NADH by glyceraldehyde-3-phosphate dehydrogenase is regenerated in the respiratory chain. However, NADH cannot pass the inner (in fact, not even the outer) mitochondrial membrane. So, how is its oxidation accomplished?

It turns out that NADH is actually re-oxidized (dehydrogenated) in the cytosol, and the hydrogen brought to the mitochondrion by other carriers. There are several shuttle systems that accomplish this, in a somewhat roundabout manner. These shuttles tie together several enzyme activities with specific transporters in the inner mitochondrial membrane (Figure 6.6-1).

One enzyme that regenerates cytosolic NAD⁺ is cytosolic malate dehydrogenase, which produces malate from oxaloacetate. Malate is then exchanged for α-ketoglutarate by a specific transporter and converted back to oxaloacetate in the mitochondrion by (mitochondrial) malate dehydrogenase. The question whether or not oxaloacetate can leave the mitochondrion in exchange for α-ketoglutarate is, in my opinion, not settled. If it can, it is possible to draw a pretty simple shuttle mechanism (Figure 6.6-1a). If it cannot, we have to use transamination (see section 12.2) as a workaround: Oxaloacetate is transaminated using mitochondrial glutamate and the resulting aspartate exchanged for cytosolic glutamate. In the cytosol, transamination is reversed, which closes the cycle. This textbook-approved cycle is known as the malate-aspartate shuttle (Figure 6.6-1b).

In the glycerolphosphate shuttle, the hydrogen is never actually transported to the mitochondrion. Dihydroxyacetonephosphate serves as the intermediate hydrogen acceptor and is reduced in the cytosol to glycerolphosphate by glycerolphosphate dehydrogenase. Glycerolphosphate traverses the outer and reaches the mitochondrial membrane, where it is converted back to dihydroxyacetonephosphate by a second dehydrogenase, which abstracts the electrons and feeds them into the respiratory chain at the level of ubiquinone. This is similar to the activity of succinate dehydrogenase, and as with the latter FAD is the coenzyme employed by the mitochondrial glycerolphosphate dehydrogenase.

The glycerolphosphate shuttle bypasses complex I in the respiratory chain and therefore induces ejection of probably four fewer protons from the cytosol. However, this deficit is partially compensated for by the two protons that stay behind in the cytosol (or more accurately, the intermembrane space) when the electrons get abstracted from glycerolphosphate (Figure 6.6-1c). While this shuttle is somewhat less energy-efficient than the malate-aspartate shuttle, it is more straightforward than the latter, since it avoids all substrate transport across the inner mitochondrial membrane. Remember that the free concentration of oxaloacetate is low; thus, the participation of oxaloacetate probably forms the bottleneck in the other cycles. It is interesting to note that the glycerolphosphate shuttle is highly active in insect muscle, which has an extremely high energy turnover during flight.