OXIDATIVE PHOSPHORYLATION 19.1 Electron-Transfer Reactions in Mitochondria:- Electrons Pass through a Series of Membrane-Bound Carriers
The mitochondrial respiratory chain consists of a series of sequentially acting electron carriers, most of which are integral proteins with prosthetic groups capable of accepting and donating either one or two electrons. Three types of electron transfers occur in oxidative phosphorylation: (1) direct transfer of electrons, as in the reduction of Fe3+ to Fe2+; (2) transfer as a hydro gen atom (H++ e-) and (3) transfer as a hydride ion (:H-), which bears two electrons. The term reducing equivalent is used to designate a single electron equivalent transferred in an oxidation-reduction reaction.
In addition to NAD and flavoproteins, three other types of electron-carrying molecules function in the respiratory chain: a hydrophobic quinone (ubiquinone) and two different types of iron-containing proteins (cytochromes and iron-sulfur proteins). Ubiquinone (also called coenzyme Q, or simply Q) is a lipid-soluble benzoquinone with a long isoprenoid side chain (Fig. 19–2). The closely related compounds plastoquinone (of plant chloroplasts) and menaquinone (of bacteria) play roles analogous to that of ubiquinone, carrying electrons in membrane-associated electron-transfer chains. Ubiquinone can accept one electron to become the semi quinone radical (QH) or two electrons to form ubiquinol (QH2) (Fig. 19–2) and, like flavoprotein carriers, it can act at the junction between a two-electron donor and a one-electron acceptor. Because ubiquinone is both small and hydrophobic, it is freely diffusible within the lipid bilayer of the inner mitochondrial membrane and can shuttle reducing equivalents between other, less mobile electron carriers in the membrane. And because it carries both electrons and protons, it plays a central role in coupling electron flow to proton movement. The cytochromes are proteins with characteristic strong absorption of visible light, due to their iron containing heme prosthetic groups (Fig. 19–3). Mitochondria contain three classes of cytochromes, designated a, b, and c, which are distinguished by differences in their light-absorption spectra. Each type of cytochrome in its reduced (Fe2+) state has three absorption bands in the visible range (Fig. 19–4). The longest wavelength band is near 600 nm in type α cytochromes,
FIGURE 19–2 Ubiquinone (Q, or coenzyme Q). Complete reduction of ubiquinone requires two electrons and two protons, and occurs in two steps through the semiquinone radical intermediate.
near 560 nm in type b, and near 550 nm in type c. To distinguish among closely related cytochromes of one type, the exact absorption maximum is sometimes used in the names, as in cytochrome b562. The heme cofactors of a and b cytochromes are tightly, but not covalently, bound to their associated proteins; the hemes of c-type cytochromes are covalently attached through Cys residues (Fig. 19–3). As with the flavoproteins, the standard reduction potential of the heme iron atom of a cytochrome depends on its inter action with protein side chains and is therefore different for each cytochrome. The cytochromes of type a and b and some of type c are integral proteins of the inner mitochondrial membrane. One striking exception is the cytochrome c of mitochondria, a soluble protein that associates through electrostatic interactions with the outer surface of the inner membrane. We encountered cytochrome c in earlier discussions of protein structure (see Fig. 4–18). In iron-sulfur proteins, first discovered by Helmut Beinert, the iron is present not in heme but in association with inorganic sulfur atoms or with the sulfur atoms of Cys residues in the protein, or both. These iron-sulfur (Fe-S) centers range from simple structures with a single Fe atom coordinated to four Cys OSH groups to more com plex Fe-S centers with two or four Fe atoms (Fig. 19–5). Rieske iron-sulfur proteins (named after their dis coverer, John S. Rieske) are a variation on this theme, in which one Fe atom is coordinated to two His residues rather than two Cys residues. All iron-sulfur proteins participate in one-electron transfers in which one iron atom of the iron-sulfur cluster is oxidized or reduced. At least eight Fe-S proteins function in mitochondrial electron transfer. The reduction potential of Fe-S proteins varies from -0.65 V to +0.45 V, depending on the microenvironment of the iron within the protein.
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, and finally to O2. A look at the methods used to determine the sequence in which the carriers act is instructive, as the same general approaches have been used to study other electron-transfer chains, such as those of chloroplasts.
FIGURE 19–3 Prosthetic groups of cytochromes. Each group consists of four five-membered, nitrogen-containing rings in a cyclic structure called a porphyrin. The four nitrogen atoms are coordinated with a central Fe ion, either Fe2+ or Fe3+. Iron protoporphyrin IX is found in b-type cytochromes and in hemoglobin and myoglobin (see Fig. 4–17). Heme c is covalently bound to the protein of cytochrome c through thioether bonds to two Cys residues. Heme a, found in the a-type cytochromes, has a long isoprenoid tail attached to one of the five-membered rings. The conjugated double-bond system (shaded pink) of the porphyrin ring accounts for the absorption of visible light by these hemes.
FIGURE 19–4 Absorption spectra of cytochrome c (cyt c) in its oxidized (red) and reduced (blue) forms. Also labeled are the characteristicα ,β and γ bands of the reduced form
FIGURE 19–5 Iron-sulfur centers. The Fe-S centers of iron-sulfur proteins may be as simple as (a), with a single Fe ion surrounded by the S atoms of four Cys residues. Other centers include both inorganic and Cys S atoms, as in (b) 2Fe-2S or (c) 4Fe-4S centers. (d) The ferredoxin of the cyanobacterium Anabaena 7120 has one 2Fe-2S center (PDB ID 1FRD); Fe is red, inorganic S2 is yellow, and the S of Cys is orange. (Note that in these designations only the inorganic S atoms are counted. For example, in the 2Fe-2S center (b), each Fe ion is actually surrounded by four S atoms.) The exact standard reduction potential of the iron in these centers depends on the type of center and its interaction with the associated protein.
First, the standard reduction potentials of the in dividual electron carriers have been determined experimentally (Table 19–2). We would expect the carriers to function in order of increasing reduction potential, because electrons tend to flow spontaneously from carriers of lower E to carriers of higher E0. The order of carriers deduced by this method is NADH→ Q cytochrome b → cytochrome c1 → cytochrome c → cytochrome α → cytochrome α 3 → O2. Note, however, that the order of standard reduction potentials is not necessarily the same as the order of actual reduction potentials under cellular conditions, which depend on the concentration of reduced and oxidized forms (p. 510). A second method for determining the sequence
FIGURE 19–6 Method for determining the sequence of electron carriers. This method measures the effects of inhibitors of electron transfer on the oxidation state of each carrier. In the presence of an electron donor and O2, each inhibitor causes a characteristic pattern of oxidized/reduced carriers: those before the block become reduced (blue), and those after the block become oxidized (pink).
of electron carriers involves reducing the entire chain of carriers experimentally by providing an electron source but no electron acceptor (no O2). When O2 is suddenly introduced into the system, the rate at which each electron carrier becomes oxidized (measured spectroscopically) reveals the order in which the carriers function. 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. In a final confirmation, agents that inhibit the flow of electrons through the chain have been used in com bination with measurements of the degree of oxidation of each carrier. In the presence of O2 and an electron donor, carriers that function before the inhibited step become fully reduced, and those that function after this step are completely oxidized (Fig. 19–6). By using several inhibitors that block different steps in the chain, in vestigators have determined the entire sequence; it is the same as deduced in the first two approaches.
