In the overall reaction catalyzed by the mitochondrial respiratory chain, electrons move from NADH, succinate, or some other primary electron donor through flavoproteins, ubiquinone, iron-sulfur proteins, and cytochromes (nearly all of which are embedded in the inner membrane), and finally to O2. The sequence in which the carriers act has been deduced in several ways.
First, their standard reduction potentials have been determined experimentally One expects the carriers to function in order of increasing reduction potential, because electrons tend to flow spontaneously from carriers of lower E'0 to carriers of higher E0. The order of carriers deduced by this method is NADH, UQ, cytochrome b, cytochrome cl, cytochrome c, cytochrome a + a3.
Second, when the entire chain of carriers is reduced experimentally by providing an electron source but no electron acceptor (no O2), and then O2 is suddenly introduced into the system, the rate at which each electron carrier becomes oxidized (measured spectroscopically) shows, in favorable cases, the order in which the carriers function (Fig. 18-6). The carrier nearest O2 (at the end of the chain) gives up its electrons first, the second carrier from the end is oxidized next, and so on. Such experiments have confirmed the sequence deduced from standard reduction potentials.
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Third, agents that inhibit the flow of electrons through the chain
have been used in combination with measurement of the degree of oxidation of
each carrier (Fig. 18-7). In the presence of O2 and an
electron donor, carriers that function before the inhibited step are expected to
become fully reduced, and those that function after the block should be
completely oxidized. By using several inhibitors that block earlier or later in
the chain, the entire sequence has been deduced; it is the same as predicted
from the first two approaches.
Fourth, gentle treatment of the inner mitochondrial membrane with
detergents allows the resolution of four electron-carrier complexes, each
representing a fraction of the entire respiratory chain (Fig. 18-8). Each of the
four separated complexes has its own unique composition (Table 18-3), and each
is capable of catalyzing electron transfer through a portion of the chain.
Complexes I and II catalyze electron transfer to ubiquinone from two different
electron donors: NADH (Complex I) and succinate (Complex II). Complex III
carries electrons from ubiquinone to cytochrome c, and Complex IV completes the
sequence by transferring electrons from cytochrome c to
O2 (Fig. 18-8).
NADH + H+ + UQ
in which oxidized ubiquinone (UQ) accepts a hydride ion (two electrons and
one proton) from NADH and a proton from the solvent water in the matrix. The
enzyme complex first transfers a pair of reducing equivalents from NADH to its
prosthetic group, FMN (Fig. 18-9). The complex also contains seven Fe-S centers
of at least two different types, through which electrons pass on their way from
FMN to ubiquinone. Amytal (a barbiturate drug), rotenone (a plant product
commonly used as an insecticide), and the antibiotic piericidin A all inhibit
electron flow from these Fe-S centers to ubiquinone (Table 18-4).Ubiquinol (UQH2, the fully reduced form; Fig. 18-2) diffuses in the membrane from Complex I to Complex III, where it is oxidized to UQ. The flow of electrons through Complex I to ubiquinone to Complex III is accompanied by the movement of protons from the mitochondrial matrix to the outer (cytosolic) side of the inner mitochondrial membrane (the intermembrane space), as described below. Complex II Succinate to Ubiquinone We encountered Complex II in Chapter 15 under a different name: succinate dehydrogenase; it is the only membrane-bound enzyme in the citric acid cycle (p. 457). Although smaller and simpler than Complex I, it contains two types of prosthetic groups and at least four different proteins (Table 18-3). One protein has a covalently bound FAD and an Fe-S center with four Fe atoms; a second iron-sulfur protein is also present. Electrons are believed to pass from succinate to FAD, then through the Fe-S centers to ubiquinone. Other substrates for mitochondrial dehydrogenases also pass electrons into the respiratory chain at the level of ubiquinone, but not through Complex II. The first step in the β oxidation of fatty acyl-CoA, catalyzed by the flavoprotein acyl-CoA dehydrogenase (p. 486), involves transfer of electrons from the substrate to the FAD of the dehydrogenase, then to electron-transferring flavoprotein (ETFP), which inturn passes its electrons to ETFP-ubiquinone oxidoreductase (Fig. 18-9). This reductase, an iron-sulfur protein that also contains a bound flavin nucleotide, passes electrons into the respiratory chain by reducing ubiquinone in the inner mitochondrial membrane. In Chapter 16 we noted that glycerol released in the degradation of triacylglycerols is phosphorylated, then converted into dihydroxyacetone phosphate by glycerol-3-phosphate dehydrogenase (see Fig. 16-4). This enzyme is a flavoprotein located on the outer face of the inner mitochondrial membrane, and like succinate dehydrogenase and acyl-CoA dehydrogenase it channels electrons into the respiratory chain by reducing ubiquinone (Fig. 18-9). The important role of glycerol-3-phosphate dehydrogenase in shuttling reducing equivalents from cytosolic NADH into the mitochondrial matrix is described later (see Fig. 18-26).
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