Electron transport chain antimycin

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. 

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. 

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. 

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 ... 

The addition of NADH to membranes from S. acidocaldarius (DSM 639) results in the reduction of cytochromes. Antimycin A does not affect cytochrome reduction, while the absence of complete reduction in the presence of cyanide suggests the presence of a branched electron transport chain [65]. 

A short electron transport chain involving flavocytochrome b2, cytochrome c, and cytochrome c oxidase allows yeast to respire on L-lactate even if the main electron transport chain is blocked, for example, by antimycin (3). The topological arrangement of this respiratory pathway is shown diagrammatically in Fig. 2. 

The answer is c. (Murray, pp 123-148. Scriver, pp 2367-2424. Sack, pp 159-175. Wilson, pp 287-317.) The electron transport chain shown contains three proton pumps linked by two mobile electron carriers. At each of these three sites (NADH-Q reductase, cytochrome reductase, and cytochrome oxidase) the transfer of electrons down the chain powers the pumping of protons across the inner mitochondrial membrane. The blockage of electron transfers by specific point inhibitors leads to a buildup of highly reduced carriers behind the block because of the inability to transfer electrons across the block. In the scheme shown, rotenone blocks step A, antimycin A blocks step B, and carbon monoxide (as well as cyanide and azide) blocks step E. Therefore a carbon monoxide inhibition leads to a highly reduced state of all of the carriers of the chain. Puromycin and chloramphenicol are inhibitors of protein synthesis and have no direct effect upon the electron transport chain. 

The electron transport chains of mitochondria and chloroplasts are similar. In mitochondria, antimycin A inhibits electron transfer from cytochrome b to coenzyme Q in the Q cycle. By analogy, it can be argued that antimycin A inhibits electron flow from plastoquinone to cytochrome b-f. A Q cycle may also operate in chloroplasts. 

On the basis of inhibition studies with antimycin, it has been postulated that coenzyme Q occupies, in the electron transport chain, a position intermediate between the succinate dehydrogenase flavoprotein and cytochrome c in the succinoxidase system, and between cytochrome b and cytochrome c in the NADH oxidase system. 

When they further observed that the normal nucleus contains a high proportion of mono-, di-, and trinucleotides of adenine, they claimed to have provided direct proof of their theory by demonstrating that the mono-or dinucleotides in the nucleus may be converted to ATP when oxygen is present. (The nucleotides can be extracted from the nucleus with acetate buffer at pH 5.1.) This conversion certainly suggested the existence of an intranuclear process of oxidative phosphorylation. As in mitochondria, oxidative phosphorylation in the nucleus is inhibited by uncouplers or agents blocking the electron transport chain. Nuclear oxidative phosphorylation is blocked by cyanide, azide, and antimycin A, or by dinitrophenol but, in contrast to mitochondria, it is resistant to Janus green, methylene blue, carbon monoxide, Dicumarol, and calcium. 

Isoectane extracts only a small fraction of the vitamin E associated with the NADH cytochrome c reductase system. Added lipids could restore the activity of the system because they fulfill nonspecific functions proper to many lipids, including vitamin E. Therefore, added lipids could spare the vitamin E remaining in the isoectane-extracted system for participation in the electron transport chain. This possibility is in keeping with observations made in Mason s laboratory, in which the vitamin E content of the NADH cytochrome c reductase system was completely extracted in that case the activity of the system is restored by a-tocopherol, and apparently by a-tocopherol only. Unfortunately, activity is not restored in all the preparations tested, probably because many uncontrollable factors can influence the preparations. In these experiments, antimycin A acts as a competitive inhibitor of a-tocopherol. Therefore, it appears that vitamin E is in some way involved in electron transport between cytochrome b and cytochrome c. Furthermore, among a number of lipids tested, only tocopherol can prevent antimycin A inhibition of NADH cytochrome c reductase. 

Certain compounds inhibit the activity of the respiratory chain by blocking the transfer of electrons at certain points. Rotenone and amytal inhibit electron transfer through Complex I. Antimycin A inhibits at the level of Complex III. Cytochrome oxidase activity is inhibited by carbon monoxide, cyanide and hydrogen sulphide. The prevention of electron transport by cyanide which is very rapidly absorbed is responsible for the high toxicity of this compound. 

Antimycin A inhibits the cycle associated with electron transport to Fd/02 but has no effect on cyclic photophosphorylation associated with NADP reduction (Table 2). Antimycin A has been reported to inhibit the PSI cycle during NADP reduction (Amon, Chain, 1977), but we were unable to repeat this result under a wide variety of reaction conditions. Heparin has been found by us to be a partial competitive inhibitor of Fd reactions at the Fd binding site on FNR (data not shown) this inhibition is presumed to result from the ionic similarity between negatively charged Fd and heparin. Table 2 shows that heparin selectively inhibits the cycle associated with oxygen reduction, and has no effect on non-cyclic photophosphorylation (H2O—> Mev). The cycle associated with NADP reduction is only affected at much higher heparin concentrations. 

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