Recall that when glucose is catabolised aerobically in a eukaryotic cell, glycolysis gives rise to two molecules of NADH per glucose in the cytosol, while the catabolism of pyruvate generates another eight molecules of NADH in the matrix of the mitochondrion.

This spatial distinction is important because the inner membrane of the mitochondrion does not have a carrier protein for NADH or NAD+, so NADH generated in the cytosol cannot enter the mitochondrion to deliver its electrons to complex I of the ETS. Instead, the electrons and H+ ions are passed inward by one of several electron shuttle systems that differ in the number of ATP molecules formed per NADH molecule oxidised.

An electron shuttle system consists of one or more electron carriers that can be reversibly reduced, with transport proteins present in the membrane for both the oxidised and the reduced forms of the carrier. For electron movement into the mitochondrion, cytosolic NADH passes its electrons and proton to a carrier molecule in the cytosol as it is oxidised to NAD+ that can again be used as an electron acceptor in glycolysis or other cytosolic processes. Meanwhile, the reduced form of the carrier is transported into the mitochondrion, where it is oxidised by a mitochondrial enzyme while producing either NADH or FADH2 in the mitochondrion. The difference in ATP yield among eukaryotic cells comes about because the oxidation of the carrier molecule within the mitochondrion may involve the transfer of electrons to either NAD+ or FAD, depending on the cell type (2 ATP/glucose fewer for transfer to mitochondrial FAD).

In liver, kidney and heart cells, electrons from cytosolic NADH are transferred into the mitochondrion by means of the malate-aspartate shuttle, a complex mechanism that results in the generation of NADH from NAD+ in the matrix. Electrons derived from cytosolic NADH therefore pass through all three proton-pumping complexes of the transport system and are therefore capable of generating three molecules of ATP per molecule of coenzyme.

In contrast, in skeletal muscle, brain and other tissues, electrons are delivered from cytosolic NADH to the mitochondrial respiratory complexes by means of a shuttle mechanism that uses FAD rather than NAD+ as the mitochondrial electron acceptor. This mechanism is called the glycerol phosphate shuttle and involves cytosolic and mitochondrial forms of the enzyme glycerol-3-phosphate dehydrogenase (G3PDH), which reversibly oxidises glycerol-3-phosphate (glycerol-3-P) to dihydroxy-acetone phosphate (DHAP). The cytosolic form of the enzyme transfers electrons from NADH to DHAP, reducing it to glycerol-3-P.

The glycerol-3-P then passes into the mitochondrion, where it is reoxidised to DHAP by an FAD-dependent G3PDH located on the outer face of the inner membrane. Because this membrane-bound enzyme uses FAD instead of NAD+ as its electron acceptor, the electrons are transferred directly to coenzyme Q. As a result, these electrons bypass the first energy-conserving site of the ETS, generating only two molecules of ATP instead of three, and reducing the maximum theoretical yield by one ATP per cytosolic NADH and therefore by two ATP molecules per molecule of glucose (from 38 to 36).

Yields of 36 or 38 ATP molecules per molecule of glucose are possible only if we assume that the energy of the electrochemical proton gradient is used solely to drive ATP synthesis. Such an assumption may be useful for the calculation of maximum possible yields, but it is not otherwise realistic because the pmf of the proton gradient provides the driving force not only for ATP synthesis but also for other energy-requiring reactions and processes. For example, some of the energy of the proton gradient is used to drive the transport of various metabolites and ions across the membrane and to drive the import of proteins into mitochondria.

Depending on the relative concentrations of pyruvate, fatty acids, amino acids and TCA-cycle intermediates in the cytosol and the mitochondrial matrix, variable amounts of energy may be needed to ensure that the mitochondrion has adequate supplies of oxidisable substrates and TCA-cycle intermediates. Moreover, the inward transport of phosphate ions needed for ATP synthesis is accompanied by the concomitant outward movement of hydroxyl ions, which are neutralised by protons in the inter-membrane space, thereby also drawing on the proton gradient.

The writer is Associate Professor, Head, Department of Botany, Ananda Mohan College, Kolkata, and also fellow, Botanical Society of Bengal.