Electron Antimycin

Thenoyltrifluoroacetone and carboxin and its derivatives specifically block Complex II, the succinate-UQ reductase. Antimycin, an antibiotic produced by Streptomyees griseus inhibits the UQ-cytochrome c reductase by blocking electron transfer between bn and coenzyme Q in the Q site. Myxothiazol inhibits the same complex by acting at the site. 

Although only two protons are pumped out of the matrix, two others from the matrix are consumed in the formation of H2O. There is therefore a net translocation of four positive charges out of the matrix which is equivalent to the extrusion of four protons. If four protons are required by the chemiosmotic mechanism to convert cytosolic ADP + Pj to ATP, then 0.5 mol ATP is made for the oxidation of one mol of ubiquinol and one mol ATP for the oxidation of 2 mols of reduced cytochrome c. These stoichiometries were obtained experimentally when ubiquinol was oxidized when complexes I, II, and IV were inhibited by rotenone, malonate, and cyanide, respectively, and when reduced cytochrome c was oxidized with complex III inhibited by antimycin (Hinkle et al., 1991). (In these experiments, of course, no protons were liberated in the matrix by substrate oxidation.) However, in the scheme illustrated in Figure 6, with the flow of two electrons through the complete electron transport chain from substrate to oxygen, it also appears valid to say that four protons are extmded by complex I, four by complex III, and two by complex 1. 

Antimycin Electron transfer from cyt b to cyt c. All intermediates before and including cyt a will be in the reduced state all intermediates after and including cyt Cj will be in the oxidized state. Blocks at site II. 

It is important that mitochondrial oxygen radical production depends on the type of mitochondria. Recently, Michelakis et al. [78] demonstrated that hypoxia and the proximal inhibitors of electron transport chain (rotenone and antimycin) decreased mitochondrial oxygen radical production by pulmonary arteries and enhanced it in renal arteries. This difference is probably explained by a lower expression of the proximal components of electron transport chain and a greater expression of mitochondrial MnSOD in pulmonary arteries compared to renal arteries. 

Figure 13.12 The protonmotive Q cycle. Electron transfer reactions are numbered and circled. Dashed arrows designate movement of ubiquinol or ubiquinone between centres N and P and of the ISP between cytochrome b and cytochrome c,. Solid black bars indicate sites of inhibition by antimycin, UHDTB and stigmatellin. (From Hunte et al., 2003. Copyright 2003, with permission from Elsevier.)...

This was confirmed by Keilin and Hartree using antimycin A as an inhibitor. The antibiotic blocked the reduction of cytochrome cx by NADH or succinate but did not block the reduction of cytochrome b. This site-specific inhibition brought antimycin A into popular use by biochemists in the analysis of electron transfer and oxidative phosphorylation. 

Between 1945 and 1960 the links between succinate, its dehydrogenase, NADH, and the cytochrome chain were aggressively reexamined by Slater, Chance, and David Green s groups. Slater used the well-established inhibitor approach (CO, CN", azide to block cytochrome oxidase, and BAL, antimycin A, and amytal to stop electron transfer from succinate or NADH to cytochrome c) to show... 

Oxidative phosphorylation DNP, potassium cyanide Antimycin A Sodium azide Formaldehyde Uncouples the oxidative phosphorylation from electron transport Acts at cytochrome oxidase B 7 Decreases the mitochondrial membrane potential 105 101,102 93,101,102... 

Only a few of the many known cytochrome bci complex inhibitors will be mentioned in this chapter. Cytochrome bci inhibitors that bind at the Qo site (p side, near the intermembrane space) include myxothiazol (MYX) and stigmatellin (SMA) (see Figure 7.29). MYX and SMA inhibit electron transfer from ubiquinol (QH2) to cytochrome c (outside the bci complex) or to other domains of the bci complex. Antimycin A (see Figure 7.29), a fungicide, binds... 

Antimycin A Blocks electron transfer from cytochrome b to cytochrome c2... 

FIGURE 19-11 Cytochrome be, complex (Complex III). The complex is a dimer of identical monomers, each with 11 different subunits. (a) Structure of a monomer. The functional core is three subunits cytochrome b (green) with its two hemes (bH and foL, light red) the Rieske iron-sulfur protein (purple) with its 2Fe-2S centers (yellow) and cytochrome ci (blue) with its heme (red) (PDB ID 1BGY). (b) The dimeric functional unit. Cytochrome c, 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... 

Effect of Rotenone and Antimycin A on Electron Transfer Rotenone, a toxic natural product from plants, strongly inhibits NADH dehydrogenase of insect and fish mitochondria. Antimycin A, a toxic antibiotic, strongly inhibits the oxidation of ubiquinol. 

This scheme was supported and refined by examining the effects of specific inhibitors of individual steps in the electron-transport chain. If CO or CN was added in the presence of a reducing substrate and 02, all of the electron carriers became more reduced. This fits the idea that these inhibitors act at the end of the respiratory chain, preventing the transfer of electrons from cytochrome to 02. If amytal (a barbiturate) or rotenone (a plant toxin long used as a fish poison) was added instead, NAD+ and the flavin in NADH dehydrogenase were reduced, but the carriers downstream became oxidized. The antibiotic antimycin caused NAD+, flavins, and the b cytochromes to become more reduced, but cytochromes c, cx, a, and a3 all became more oxidized. The situation here is analogous to the construction of a dam across a stream When the gates are closed, the water level rises upstream from the dam, and falls downstream. The observation that antimycin did not inhibit reduction of UQ showed that the quinone fits into the chain upstream of cytochromes c, t i, a, and a3. 

As shown in figure 14.11, the sites at which UQH2 and UQH undergo oxidation face the intermembrane space, whereas the UQ reduction site is on the matrix side. UQ and UQH2 evidently diffuse through the membrane from one site to the other. Antimycin blocks electron transfer from cytochrome bH to UQ at the reduction site this inhibitor was particularly helpful in clarifying the steps of the Q cycle. 

Rotenone and amytal inhibit electron transport at NADH dehydrogenase, antimycin A inhibits the cytochrome bci complex, and cyanide (CN ), azide (N3 ) and carbon monoxide (CO) all inhibit cytochrome oxidase. 

Detection of the components of an electron transport system, involving cytochromes, is based on (a) identification of the various cytochromes by absorption and difference spectroscopy at room and liquid-nitrogen temperatures, and (b) the use of specific inhibitors, such as antimycin A and cyanide, for the various... 

The action of some inhibitors is indicated in Figure 17.4. It is sometimes difficult to pinpoint exactly where an inhibitor may act, however, because our knowledge of the composition and function of the four complexes is far from complete. Complex I inhibitors, such as rotenone, piericidin A, and the barbiturates, are believed to inhibit the transfer of elctrons from the Fe-S centers to UQ. In complex III, antimycin appears to inhibit the reduction of UQ by cytochrome b. Myxothiazol and 2,3-dimercaptopropanol (BAL) inhibit the transfer of electrons from UQH2 to Rieske s protein, because they destroy the Fe-S centers. The action of cyanide and azide on complex IV is also unclear, but it is believed that these substances combine with the Fe3+ moiety of the a3 heme prosthetic group. 

The sites of action of some of the commonly used inhibitors of the electron-transport chain are shown in Fig. 14-3. These sites have been established by application of the crossover theorem (Chap. 10). For example, the fungus-derived antibiotic antimycin A causes an increase in the level of reduced cytochrome b and a decrease in the level of reduced cytochrome C] (i.e., an increase in the level of oxidized cytochrome c() thus, it is inferred that antimycin A interacts with complex III. 

Fe3+ in iron-sulfur proteins has an electron spin resonance (esr) signal, while Fe2+ does not. Assume that you have a preparation of mitochondria that are able to synthesize ATP via oxidation of NADH supplies of rotenone, antimycin A, and KCN and access to an esr spectrometer. How could you establish which of the complexes of the electron-transport chain contain iron-sulfur proteins ... 

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