Figure 18.9
.Sequence of Electron Carriers in the Respiratory Chain
 |
| Oxidant or reductant | ||||||
|---|---|---|---|---|---|---|
| Enzyme complex | Mass (kd) | Subunits | Prosthetic group | Matrix side | Membrane core | Cytosolic side |
| NADH-Q oxidoreductase | 880 | ≥ 34 | FMN | NADH | Q | |
| Fe-S | ||||||
| Succinate-Q reductase | 140 | 4 | FAD | Succinate | Q | |
| Fe-S | ||||||
| Q-cytochrome c oxidoreductase | 250 | 10 | Heme bH | Q | Cytochrome c | |
| Heme bL | ||||||
| Heme c1 | ||||||
| Fe-S | ||||||
| Cytochrome c oxidase | 160 | 10 | Heme a | Cytochrome c | ||
| Heme a3 | ||||||
| CuA and CuB | ||||||
Sources: J. W. DePierre and L. Ernster, Annu. Rev. Biochem. 46(1977):215; Y. Hatefi, Annu Rev. Biochem. 54(1985);1015; and J. E. Walker, Q. Rev. Biophys. 25(1992):253.
and Table 18.2). Electron flow within these transmembrane complexes leads to the transport of protons across the inner mitochondrial membrane. Electrons are carried from NADH-Q oxidoreductase to Q-cytochrome c oxidoreductase, the second complex of the chain, by the reduced form of coenzyme Q (Q), also known as ubiquinone because it is a ubiquitous quinone in biological systems. Ubiquinone is a hydrophobic quinone that diffuses rapidly within the inner mitochondrial membrane. Ubiquinone also carries electrons from FADH2, generated in succinate dehydrogenase in the citric acid cycle, to Q-cytochrome c oxidoreductase, generated through succinate-Q reductase. Cytochrome c, a small, soluble protein, shuttles electrons from Q-cytochrome c oxidoreductase to cytochrome c oxidase, the final component in the chain and the one that catalyzes the reduction of O2. NADH-Q oxidoreductase, succinate-Q reductase, Q-cytochrome c oxidoreductase, and cytochrome c oxidase are also called Complex I, II, III, and IV, respectively. Succinate-Q reductase (Complex II), in contrast with the other complexes, does not pump protons.
The reduction of ubiquinone (Q) to ubiquinol (QH2) proceeds through a semiquinone anion intermediate (Q•-).
). In the fully oxidized state (Q), coenzyme Q has two keto groups. The addition of one electron and one proton results in the semiquinone form (QH·). The semiquinone form is relatively easily deprotonated to form a semiquinone radical anion (Q·-). The addition of a second electron and proton generates ubiquinol (QH2), the fully reduced form of coenzyme Q, which holds its protons more tightly. Thus, for quinones, electron-transfer reactions are coupled to proton binding and release, a property that is key to transmembrane proton transport.
The structure, determined by electron microscopy at 22-Å resolution, consists of a membrane-spanning part and a long arm that extends into the matrix. NADH is oxidized in the arm, and the electrons are transferred to reduce Q in the membrane. [After N. Grigorieff, J. Mol. Biol. 277(1998):1033–1048.]
). NADH-Q oxidoreductase is L-shaped, with a horizontal arm lying in the membrane and a vertical arm that projects into the matrix. Although a detailed understanding of the mechanism is likely to require higher-resolution structural information, some aspects of the mechanism have been established.
The reaction catalyzed by this enzyme appears to be
The reduction of flavin mononucleotide (FMN) to FMNH2 proceeds through a semiquinone intermediate.
). The electron acceptor of FMN, the isoalloxazine ring, is identical with that of FAD. Electrons are then transferred from FMNH2 to a series of iron-sulfur clusters, the second type of prosthetic group in NADH-Q oxidoreductase.
(A) A single iron ion bound by four cysteine residues. (B) 2Fe-2S cluster with iron ions bridged by sulfide ions. (C) 4Fe-4S cluster. Each of these clusters can undergo oxidation-reduction reactions.
). In the simplest kind, a single iron ion is tetrahedrally coordinated to the sulfhydryl groups of four cysteine residues of the protein. A second kind, denoted by 2Fe-2S, contains two iron ions and two inorganic sulfides. Such clusters are usually coordinated by four cysteine residues, although exceptions exist, as we shall see when we consider Q-cytochrome c oxidoreductase. A third type, designated 4Fe-4S, contains four iron ions, four inorganic sulfides, and four cysteine residues. We encountered a variation of this type of cluster in aconitase in Section 17.1.4. NADH-Q oxidoreductase contains both 2Fe-2S and 4Fe-4S clusters. Iron ions in these Fe-S complexes cycle between Fe2+ (reduced) or Fe3+(oxidized) states. Unlike quinones and flavins, iron-sulfur clusters generally undergo oxidation-reduction reactions without releasing or binding protons.
The reduction of a quinone (Q) to QH2 in an appropriate site can result in the uptake of two protons from the mitochondrial matrix.
). The pair of electrons on bound QH2 are transferred to a 4Fe-4S center and the protons are released on the cytosolic side. Finally, these elections are transferred to a mobile Q in the hydrophobic core of the membrane, resulting in the uptake of two additional protons from the matrix. The challenge is to delineate the binding events and conformational changes induced by these electron transfers and learn how the uptake and release of protons from the appropriate sides of the membrane is facilitated.
The citric acid cycle enzyme succinate dehydrogenase, which generates FADH2 with the oxidation of succinate to fumarate (Section 17.1.8), is part of the succinate-Q reductase complex (Complex II), an integral membrane protein of the inner mitochondrial membrane. FADH2 does not leave the complex. Rather, its electrons are transferred to Fe-S centers and then to Q for entry into the electron-transport chain. Two other enzymes that we will encounter later, glycerol phosphate dehydrogenase (Section 18.5.1) and fatty acyl CoA dehydrogenase (Section 22.2.4), likewise transfer their highpotential electrons from FADH2 to Q to form ubiquinol (QH2), the reduced state of ubiquinone. The succinate-Q reductase complex and other enzymes that transfer electrons from FADH2 to Q, in contrast with NADH-Q oxidoreductase, do not transport protons. Consequently, less ATP is formed from the oxidation of FADH2 than from NADH.
The second of the three proton pumps in the respiratory chain is Q-cytochrome c oxidoreductase (also known as Complex III and cytochrome reductase). A cytochrome is an electron-transferring protein that contains a heme prosthetic group. The iron ion of a cytochrome alternates between a reduced ferrous (+2) state and an oxidized ferric (+3) state during electron transport. The function of Q-cytochrome c oxidoreductase is to catalyze the transfer of electrons from QH2 to oxidized cytochrome c (cyt c), a water-soluble protein, and concomitantly pump protons out of the mitochondrial matrix.
This enzyme is a homodimer with 11 distinct polypeptide chains. The major prosthetic groups, three hemes and a 2Fe-2S cluster, mediate the electron-transfer reactions between quinones in the membrane and cytochrome c in the intermembrane space.
A heme group is covalently attached to a protein through thioether linkages formed by the addition of sulfhydryl groups of cysteine residues to vinyl groups on protoporphyrin.
). Q-cytochrome c oxidoreductase itself contains a total of three hemes, contained within two cytochrome subunits: two b-type hemes, termed heme bL (L for low affinity) and heme bH (H for high affinity), within cytochrome b, and one c-type heme within cytochrome c1. The prosthetic group of the heme in cytochromes b, c1, and c is iron- protoporphyrin IX, the same heme as in myoglobin and hemoglobin (Section 10.2.1). The hemes in cytochromes c and c1, in contrast with those in cytochrome b, are covalently attached to the protein (Figure 18.16). The linkages are thioethers formed by the addition of the sulfhydryl groups of two cysteine residues to the vinyl groups of the heme. Because of these groups, this enzyme is also known as cytochrome bc1. In addition to the hemes, the enzyme also contains an iron-sulfur protein with an 2Fe-2S center. This center, termed the Rieske center, is unusual in that one of the iron ions is coordinated by two histidine residues rather than two cysteine residues. This coordination stabilizes the center in its reduced form, raising its reduction potential. Finally, Q-cytochrome c oxidoreductase contains two distinct binding sites for ubiquinone termed Qo and Qi, with the Qi site lying closer to the inside of the matrix.
The two electrons of a bound QH2 are transferred, one to cytochrome c and the other to a bound Q to form the semiquinone Q•-. The newly formed Q dissociates and is replaced by a second QH2, which also gives up its electrons, one to a second molecule of cytochrome c and the other to reduce Q•- to QH2. This second electron transfer results in the uptake of two protons from the matrix. Prosthetic groups are shown in their oxidized forms in blue and in their reduced forms in red.
). The Q cycle also facilitates the switch from the two-electron carrier ubiquinol to the one-electron carrier cytochrome c. The cycle begins as ubiquinol (QH2) binds in the Qo site. Ubiquinol transfers its electrons, one at a time. One electron flows first to the Rieske 2Fe-2S cluster, then to cytochrome c1, and finally to a molecule of oxidized cytochrome c, converting it into its reduced form. The reduced cytochrome c molecule is free to diffuse away from the enzyme. The second electron is transferred first to cytochrome bL, then to cytochrome bH, and finally to an oxidized uniquinone bound in the Qi site. This quinone (Q) molecule is reduced to a semiquinone anion (Q · -). Importantly, as the QH2 in the Qo site is oxidized to Q, its protons are released to the cytosolic side of the membrane. This Q molecule in the Qo site is free to diffuse out into the ubiquinone pool.
At this point, Q · - resides in the Qi site. A second molecule of QH2 binds to the Qo site and reacts in the same way as the first. One of its electrons is transferred through the Rieske center and cytochrome c1 to reduce a second molecule of cytochrome c. The other electron goes through cytochromes bL and bH to Q · - bound in the Qi site. On the addition of the second electron, this quinone radical anion takes up two protons from the matrix side to form QH2. The removal of these two protons from the matrix contributes to the formation of the proton gradient. At the end of the Q cycle, two molecules of QH2 are oxidized to form two molecules of Q, and one molecule of Q is reduced to QH2, two molecules of cytochrome c are reduced, four protons are released on the cytoplasmic side, and two protons are removed from the mitochondrial matrix.
The final stage of the electron-transport chain is the oxidation of the reduced cytochrome c generated by Complex III, which is coupled to the reduction of O2 to two molecules of H2O. This reaction is catalyzed by cytochrome c oxidase (Complex IV). The four-electron reduction of oxygen directly to water without the release of intermediates poses a challenge. Nevertheless, this reaction is quite thermodynamically favorable. From the reduction potentials in Table 18.1, the standard free-energy change for this reaction is calculated to be ΔG°´ = -55.4 kcal mol-1 (-231.8 kJ mol-1). As much of this free energy as possible must be captured in the form of a proton gradient for subsequent use in ATP synthesis.
This enzyme consists of 13 polypeptide chains. The major prosthetic groups include CuA/CuA, heme a, and heme a3-CuB. Heme a3-CuB is the site of the reduction of oxygen to water. CO(bb) is a carbonyl group of the peptide backbone.
). It consists of 13 subunits, of which 3 (called subunits I, II, and III) are encoded by the mitochondrial genome. Cytochrome c oxidase contains two heme A groups and three copper ions, arranged as two copper centers, designated A and B. One center, CuA/CuA, contains two copper ions linked by two bridging cysteine residues. This center initially accepts electrons from reduced cytochrome c. The remaining copper ion, CuB, is coordinated by three histidine residues, one of which is modified by covalent linkage to a tyrosine residue. Heme A differs from the heme in cytochrome c and c1 in three ways: (1) a formyl group replaces a methyl group, (2) a C15 hydrocarbon chain replaces one of the vinyl groups, and (3) the heme is not covalently attached to the protein.
The two heme A molecules, termed heme a and heme a3, have distinct properties because they are located in different environments within cytochrome c oxidase. Heme a functions to carry electrons from CuA/CuA, whereas heme a3 passes electrons to CuB, to which it is directly adjacent. Together, heme a3 and CuB form the active center at which O2 is reduced to H2O.
The cycle begins with all prosthetic groups in their oxidized forms (shown in blue). Reduced cytochrome c introduces an electron that reduces CuB. A second reduced cytochrome c then reduces the iron in heme a3. This Fe2+ center then binds oxygen. Two electrons are transferred to the bound oxygen to form peroxide, which bridges between the iron and CuB. The introduction of an additional electron by a third molecule of reduced cytochrome c cleaves the O-O bond and results in the uptake of a proton from the matrix. The introduction of a final electron and three more protons generates two molecules of H2O, which are released from the enzyme to regenerate the initial state. The four protons found in the water molecules come from the matrix.
The oxygen bound to heme a3 is reduced to peroxide by the presence of CuB.
). One molecule of reduced cytochrome c transfers an electron, initially to CuA/CuA. From there, the electron moves to heme a, then to heme a3, and finally to CuB, which is reduced from the Cu2+ (cupric) form to the Cu+ (cuprous) form. A second molecule of cytochrome c introduces a second electron that flows down the same path, stopping at heme a3, which is reduced to the Fe2+ form. Recall that the iron in hemoglobin is in the Fe2+ form when it binds oxygen (Section 10.2.1). Thus, at this stage, cytochrome c oxidase is poised to bind oxygen and does so. The proximity of CuB in its reduced form to the heme a3-oxygen complex allows the oxygen to be reduced to peroxide (O22-), which forms a bridge between the Fe3+ in heme a3 and CuB2+ (Figure 18.20). The addition of a third electron from cytochrome c as well as a proton results in the cleavage of the O-O bond, yielding a ferryl group, Fe4+ = O, at heme a3 and CuB2+-OH. The addition of the final electron from cytochrome c and a second proton reduces the ferryl group to Fe3+-OH. Reaction with two additional protons allows the release of two molecules of water and resets the enzyme to its initial, fully oxidized form.
This reaction can be summarized as
Four “chemical” protons are taken up from the matrix side to reduce one molecule of O2 to two molecules of H2O. Four additional “pumped” protons are transported out of the matrix and released on the cytosolic side in the course of the reaction. The pumped protons double the efficiency of free-energy storage in the form of a proton gradient for this final step in the electron-transport chain.
). The details of how these protons are transported through the protein is still under study. However, two effects contribute to the mechanism. First, charge neutrality tends to be maintained in the interior of proteins. Thus, the addition of an electron to a site inside a protein tends to favor the binding of a proton to a nearby site. Second, conformational changes take place, particularly around the heme a3-CuB center, in the course of the reaction cycle, and these changes must be used to allow protons to enter the protein exclusively from the matrix side and to exit exclusively to the cytosolic side. Thus, the overall process catalyzed by cytochrome c oxidase is
As discussed earlier, molecular oxygen is an ideal terminal electron acceptor, because its high affinity for electrons provides a large thermodynamic driving force. However, danger lurks in the reduction of O2. The transfer of four electrons leads to safe products (two molecules of H2O), but partial reduction generates hazardous compounds. In particular, the transfer of a single electron to O2 forms superoxide anion, whereas the transfer of two electrons yields peroxide.
These compounds and, particularly, their reaction products can be quite harmful to a variety of cell components. The strategy for the safe reduction of O2 is clear from the discussion of the reaction cycle: the catalyst does not release partly reduced intermediates. Cytochrome c oxidase meets this crucial criterion by holding O2 tightly between Fe and Cu ions.
Although cytochrome c oxidase and other proteins that reduce O2 are remarkably successful in not releasing intermediates, small amounts of superoxide anion and hydrogen peroxide are unavoidably formed. Superoxide, hydrogen peroxide, and species that can be generated from them such as OH· are collectively referred to as reactive oxygen species or ROS.
What are the cellular defense strategies against oxidative damage by ROS? Chief among them is the enzyme superoxide dismutase. This enzyme scavenges superoxide radicals by catalyzing the conversion of two of these radicals into hydrogen peroxide and molecular oxygen.
A reaction in which a single reactant is converted into two different products.
The oxidized form of superoxide dismutase (Mox) reacts with one superoxide ion to form O2 and generate the reduced form of the enzyme (Mred). The reduced form then reacts with a second superoxide and two protons to form hydrogen peroxide and regenerate the oxidized form of the enzyme.
). The oxidized form of the enzyme is reduced by superoxide to form oxygen. The reduced form of the enzyme, formed in this reaction, then reacts with a second superoxide ion to form peroxide, which takes up two protons along the reaction path to yield hydrogen peroxide.
The hydrogen peroxide formed by superoxide dismutase and by other processes is scavenged by catalase, a ubiquitous heme protein that catalyzes the dismutation of hydrogen peroxide into water and molecular oxygen.
Superoxide dismutase and catalase are remarkably efficient, performing their reactions at or near the diffusion-limited rate (Section 8.4.2). Other cellular defenses against oxidative damage include the antioxidant vitamins, vitamins E and C. Because it is lipophilic, vitamin E is especially useful in protecting membranes from lipid peroxidation.
| Atherogenesis |
| Emphysema; bronchitis |
| Parkinson disease |
| Duchenne muscular dystrophy |
| Cervical cancer |
| Alcoholic liver disease |
| Diabetes |
| Acute renal failure |
| Down syndrome |
| Retrolental fibroplasia |
| Cerebrovascular disorders |
| Ischemia; reperfusion injury |
Source: After D.B. Marks, A.D. Marks, and C.M. Smith, Basic Medical Biochemistry: A Clinical Approach (Williams & Wilkins, 1996, p. 331).
The side chains are shown for the 21 conserved amino acids and the heme.
Cytochrome c is present in all organisms having mitochondrial respiratory chains: plants, animals, and eukaryotic microorganisms. This electron carrier evolved more than 1.5 billion years ago, before the divergence of plants and animals. Its function has been conserved throughout this period, as evidenced by the fact that the cytochrome c of any eukaryotic species reacts in vitro with the cytochrome c oxidase of any other species tested thus far. Finally, some prokaryotic cytochromes, such as cytochrome c2 from a photosynthetic bacterium and cytochrome c 550 from a denitrifying bacterium, closely resemble cytochrome c from tuna heart mitochondria (Figure 18.23). This evidence attests that the structural and functional characteristics of cytochrome c present an efficient evolutionary solution to electron transfer.
Branch lengths are proportional to the number of amino acid changes that are believed to have occurred. This drawing is an adaptation of the work of Walter M. Fitch and Emanuel Margoliash.
).
