Dnp

Valinomycin Thermogenin

Inhibition of ATP-ADP exchange Atractyloside

Inhibit cytochrome oxidase

Blocks electron transfer from cytochrome b to cytochrome c1

Prevent electron transfer from Fe-S center to ubiquinone

Competes with QB for binding site in PSII Inhibits F1

Inhibit Fo and CFo

Blocks proton flow through Fo and CFo Hydrophobic proton carriers K+ ionophore

In brown fat, forms proton-conducting pores in inner mitochondrial membrane Inhibits adenine nucleotide translocase

*DCMU is 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCCD, dicyclohexylcarbodiimide; FCCP cyanide-p-trifluoromethoxyplienylliydrazone; DNP 2,4-dinitrophenol.

Substrate binding site

Substrate binding site

Periplasm (p side)

The heme b of Complex II is apparently not in the direct path of electron transfer; it may serve instead to reduce the frequency with which electrons "leak" out of the system, moving from succinate to molecular oxygen to produce the reactive oxygen species (ROS) hydrogen peroxide (H2O2) and the superoxide radical (°O2) described in Section 19.5. Humans with point mutations in Complex II subunits near heme b or the quinone-binding site suffer from hereditary para-ganglioma. This inherited condition is characterized by benign tumors of the head and neck, commonly in the carotid body, an organ that senses O2 levels in the blood. These mutations result in greater production of ROS and perhaps greater tissue damage during succinate oxidation. ■

Other substrates for mitochondrial dehydrogenases pass electrons into the respiratory chain at the level of ubiquinone, but not through Complex II. The first step in the 3 oxidation of fatty acyl-CoA, catalyzed by the

FIGURE 19-10 Structure of Complex II (succinate dehydrogenase) of E. coli (PDB ID 1NEK). The enzyme has two transmembrane sub-units, C (green) and D (blue); the cytoplasmic extensions contain sub-units B (orange) and A (purple). Just behind the FAD in subunit A (gold) is the binding site for succinate (occupied in this crystal structure by the inhibitor oxaloacetate, green). Subunit B has three sets of Fe-S centers (yellow and red); ubiquinone (yellow) is bound to subunit C; and heme b (purple) is sandwiched between subunits C and D. A cardi-olipin molecule is so tightly bound to subunit C that it shows up in the crystal structure (gray spacefilling). Electrons move (blue arrows) from succinate to FAD, then through the three Fe-S centers to ubiquinone. The heme b is not on the main path of electron transfer but protects against the formation of reactive oxygen species (ROS) by electrons that go astray.

flavoprotein acyl-CoA dehydrogenase (see Fig. 17-8), involves transfer of electrons from the substrate to the FAD of the dehydrogenase, then to electron-transferring flavoprotein (ETF), which in turn passes its electrons to ETF: ubiquinone oxidoreductase (Fig. 19-8). This enzyme transfers electrons into the respiratory chain by reducing ubiquinone. Glycerol 3-phosphate, formed either from glycerol released by triacylglycerol breakdown or by the reduction of dihydroxyacetone phosphate from glycolysis, is oxidized by glycerol 3-phosphate dehydrogenase (see Fig. 17-4). This enzyme is a flavo-protein located on the outer face of the inner mito-chondrial membrane, and like succinate dehydrogenase and acyl-CoA dehydrogenase it channels electrons into the respiratory chain by reducing ubiquinone (Fig. 19-8). The important role of glycerol 3-phosphate de-hydrogenase in shuttling reducing equivalents from cytosolic NADH into the mitochondrial matrix is described in Section 19.2 (see Fig. 19-28). The effect of each of these electron-transferring enzymes is to contribute to the pool of reduced ubiquinone. QH2 from all these reactions is reoxidized by Complex III.

Complex III: Ubiquinone to Cytochrome c The next respiratory complex, Complex III, also called cytochrome bc1 complex or ubiquinone:cytochrome c oxidore-

ductase, couples the transfer of electrons from ubiquinol (QH2) to cytochrome c with the vectorial transport of protons from the matrix to the intermembrane space. The determination of the complete structure of this huge complex (Fig. 19-11) and of Complex IV (below) by x-ray crystallography, achieved between 1995 and 1998, were landmarks in the study of mito-chondrial electron transfer, providing the structural framework to integrate the many biochemical observations on the functions of the respiratory complexes.

Based on the structure of Complex III and detailed biochemical studies of the redox reactions, a reasonable model has been proposed for the passage of electrons

FIGURE 19-11 Cytochrome bc1 complex (Complex III). The complex is a dimer of identical monomers, each with 11 different sub-units. (a) Structure of a monomer. The functional core is three sub-units: cytochrome b (green) with its two hemes (bH and bL, light red); the Rieske iron-sulfur protein (purple) with its 2Fe-2S centers (yellow); and cytochrome c1 (blue) with its heme (red) (PDB ID 1BGY). (b) The dimeric functional unit. Cytochrome c1 and the Rieske iron-sulfur protein project from the P surface and can interact with cytochrome c (not part of the functional complex) in the intermembrane space. The complex has two distinct binding sites for ubiquinone, QN and QP, which correspond to the sites of inhibition by two drugs that block oxidative phosphorylation. Antimycin A, which blocks electron flow from heme bH to Q, binds at QN, close to heme bH on the N (matrix) side of the membrane. Myxothiazol, which prevents electron flow from

QH2 to the Rieske iron-sulfur protein, binds at QP, near the 2Fe-2S center and heme bL on the P side. The dimeric structure is essential to the function of Complex III. The interface between monomers forms two pockets, each containing a QP site from one monomer and a QN site from the other. The ubiquinone intermediates move within these sheltered pockets.

Complex III crystallizes in two distinct conformations (not shown). In one, the Rieske Fe-S center is close to its electron acceptor, the heme of cytochrome c1, but relatively distant from cytochrome b and the QH2-binding site at which the Rieske Fe-S center receives electrons. In the other, the Fe-S center has moved away from cytochrome c1 and toward cytochrome b. The Rieske protein is thought to oscillate between these two conformations as it is reduced, then oxidized.

FIGURE 19-11 Cytochrome bc1 complex (Complex III). The complex is a dimer of identical monomers, each with 11 different sub-units. (a) Structure of a monomer. The functional core is three sub-units: cytochrome b (green) with its two hemes (bH and bL, light red); the Rieske iron-sulfur protein (purple) with its 2Fe-2S centers (yellow); and cytochrome c1 (blue) with its heme (red) (PDB ID 1BGY). (b) The dimeric functional unit. Cytochrome c1 and the Rieske iron-sulfur protein project from the P surface and can interact with cytochrome c (not part of the functional complex) in the intermembrane space. The complex has two distinct binding sites for ubiquinone, QN and QP, which correspond to the sites of inhibition by two drugs that block oxidative phosphorylation. Antimycin A, which blocks electron flow from heme bH to Q, binds at QN, close to heme bH on the N (matrix) side of the membrane. Myxothiazol, which prevents electron flow from and protons through the complex. The net equation for the redox reactions of this Q cycle (Fig. 19-12) is

The Q cycle accommodates the switch between the two-electron carrier ubiquinone and the one-electron carri-ers—cytochromes b562, b566, c1, and c—and explains the measured stoichiometry of four protons translocated per pair of electrons passing through the Complex III to cytochrome c. Although the path of electrons through this segment of the respiratory chain is complicated, the net effect of the transfer is simple: QH2 is oxidized to Q and two molecules of cytochrome c are reduced.

Cytochrome c (see Fig. 4-18) is a soluble protein of the intermembrane space. After its single heme accepts an electron from Complex III, cytochrome c moves to Complex IV to donate the electron to a binuclear copper center.

QH2 to the Rieske iron-sulfur protein, binds at QP, near the 2Fe-2S center and heme bL on the P side. The dimeric structure is essential to the function of Complex III. The interface between monomers forms two pockets, each containing a QP site from one monomer and a QN site from the other. The ubiquinone intermediates move within these sheltered pockets.

Complex III crystallizes in two distinct conformations (not shown). In one, the Rieske Fe-S center is close to its electron acceptor, the heme of cytochrome c1, but relatively distant from cytochrome b and the QH2-binding site at which the Rieske Fe-S center receives electrons. In the other, the Fe-S center has moved away from cytochrome c1 and toward cytochrome b. The Rieske protein is thought to oscillate between these two conformations as it is reduced, then oxidized.

Complex IV: Cytochrome c to O2 In the final step of the respiratory chain, Complex IV, also called cytochrome oxidase, carries electrons from cytochrome c to molecular oxygen, reducing it to H2O. Complex IV is a large enzyme (13 subunits; Mr 204,000) of the inner mito-chondrial membrane. Bacteria contain a form that is much simpler, with only three or four subunits, but still capable of catalyzing both electron transfer and proton pumping. Comparison of the mitochondrial and bacterial complexes suggests that three subunits are critical to the function (Fig. 19-13).

Mitochondrial subunit II contains two Cu ions com-plexed with the —SH groups of two Cys residues in a binuclear center (CuA; Fig. 19-13b) that resembles the 2Fe-2S centers of iron-sulfur proteins. Subunit I contains two heme groups, designated a and a3, and another copper ion (CuB). Heme a3 and CuB form a second binuclear center that accepts electrons from heme a and transfers them to O2 bound to heme a3.

Oxidation of first QH2

Oxidation of second QH2

Intermembrane I space (p side)

J)QQ

Intermembrane I space (p side)

■jolxdoo

Matrix (n side)

■jolxdoo

Matrix (n side)

Net equation:

QH2 + 2 cyt c1 (oxidized) + 2H+ -> Q + 2 cyt c1 (reduced) + 4H+

FIGURE 19-12 The Q cycle. The path of electrons through Complex III is shown by blue arrows. On the P side of the membrane, two molecules of QH2 are oxidized to Q near the p side, releasing two protons per Q (four protons in all) into the intermembrane space. Each

Electron transfer through Complex IV is from cytochrome c to the CuA center, to heme a, to the heme a3-CuB center, and finally to O2 (Fig. 19-14). For every four electrons passing through this complex, the enzyme consumes four "substrate" H+ from the matrix (N side) in converting O2 to 2H2O. It also uses the energy of this redox reaction to pump one proton outward into the intermembrane space (P side) for each electron that passes through, adding to the electrochemical potential produced by redox-driven proton transport through Complexes I and III. The overall reaction catalyzed by Complex IV is

This four-electron reduction of O2 involves redox centers that carry only one electron at a time, and it must occur without the release of incompletely reduced intermediates such as hydrogen peroxide or hydroxyl free radicals—very reactive species that would damage cellular components. The intermediates remain tightly

QH2 donates one electron (via the Rieske Fe-S center) to cytochrome c1, and one electron (via cytochrome b) to a molecule of Q near the n side, reducing it in two steps to QH2. This reduction also uses two protons per Q, which are taken up from the matrix.

bound to the complex until completely converted to water.

The Energy of Electron Transfer Is Efficiently Conserved in a Proton Gradient

The transfer of two electrons from NADH through the respiratory chain to molecular oxygen can be written as

This net reaction is highly exergonic. For the redox pair NAD+/NADH, E'° is -0.320 V, and for the pair O2/H2O, E'° is 0.816 V. The AE'° for this reaction is therefore 1.14 V, and the standard free-energy change (see Eqn 13-6, p. 510) is

= -2(96.5 kJ/V • mol)(1.14 V) = -220 kJ/mol (of NADH)

This standard free-energy change is based on the assumption of equal concentrations (1 M) of NADH and

FIGURE 19-13 Critical subunits of cytochrome oxidase (Complex IV). The bovine complex is shown here (PDB ID 1OCC). (a) The core of Complex IV, with three subunits. Subunit I (yellow) has two heme groups, a and a3 (red), and a copper ion, CuB (green sphere). Heme a3 and CuB form a binuclear Fe-Cu center. Subunit II (blue) contains two Cu ions (green spheres) complexed with the —SH groups of two Cys residues in a binuclear center, CuA, that resembles the 2Fe-2S centers of iron-sulfur proteins. This binuclear center and the cytochrome

FIGURE 19-13 Critical subunits of cytochrome oxidase (Complex IV). The bovine complex is shown here (PDB ID 1OCC). (a) The core of Complex IV, with three subunits. Subunit I (yellow) has two heme groups, a and a3 (red), and a copper ion, CuB (green sphere). Heme a3 and CuB form a binuclear Fe-Cu center. Subunit II (blue) contains two Cu ions (green spheres) complexed with the —SH groups of two Cys residues in a binuclear center, CuA, that resembles the 2Fe-2S centers of iron-sulfur proteins. This binuclear center and the cytochrome c-binding site are located in a domain of subunit II that protrudes from the P side of the inner membrane (into the intermembrane space). Subunit III (green) seems to be essential for Complex IV function, but its role is not well understood. (b) The binuclear center of CuA. The Cu ions (green spheres) share electrons equally. When the center is reduced they have the formal charges Cu1+Cu1+; when oxidized, Cu15+Cu15 + . Ligands around the Cu ions include two His (dark blue), two Cys (yellow), an Asp (red), and Met (orange) residues.

NAD + . In actively respiring mitochondria, the actions of many dehydrogenases keep the actual [NADH]/[NAD+] ratio well above unity, and the real free-energy change for the reaction shown in Equation 19-5 is therefore substantially greater (more negative) than —220 kJ/mol. A similar calculation for the oxidation of succinate shows that electron transfer from succinate (E'° for fumarate/succinate = 0.031 V) to O2 has a smaller, but still negative, standard free-energy change of about — 150 kJ/mol.

FIGURE 19-14 Path of electrons through Complex IV. The three proteins critical to electron flow are subunits I, II, and III. The larger green structure includes the other ten proteins in the complex. Electron transfer through Complex IV begins when two molecules of reduced cytochrome c (top) each donate an electron to the binuclear center CuA. From here electrons pass through heme a to the Fe-Cu center (cytochrome a3 and CuB). Oxygen now binds to heme a3 and is reduced to its peroxy derivative (Of—) by two electrons from the Fe-Cu center. Delivery of two more electrons from cytochrome c (making four electrons in all) converts the O|— to two molecules of water, with consumption of four "substrate" protons from the matrix. At the same time, four more protons are pumped from the matrix by an as yet unknown mechanism.

Intermembrane space (p side)

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