Electron transport chain rotenone

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. 

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. 

In the presence of the inhibitor rotenone (to prevent the oxidation of NADH by the electron transport chain), succinate can be metabolized only to fumarate, producing an FADH2 in the process. 

In the absence of rotenone, the NADH that is made from the conversion of succinate to oxaloacetate can be oxidized by the electron transport chain. The metabolism of succinate then becomes... 

Rotenone inhibits the transfer of electrons from NADH into the electron transport chain. The oxidation of substrates that generate NADH is, therefore, blocked. However, substrates that are oxidized to generate FADH2 (such as succinate or a-glycerol phosphate) can still be oxidized and still generate ATP. Because NADH oxidation is blocked, the NADH pool becomes more reduced in the presence of rotenone since there s nowhere to transfer the electrons. 

Furthermore, Marshall et al. developed the extractable MBF tracer 7 -[ F] fluoro-6, 7 -dihydrorotenone (p F]FDHR) [72]. p F]FDHR is a derivative of the neutral and lipophilic lead compound rotenone that binds to the complex I of the mitochondrial electron transport chain [73-76]. It was prepared from 7 -tosyl-oxy-6, 7 -dihydroroten-12-ol (DHR-ol-OTos) in two steps. After nucleophilic substitution of DHR-ol-OTos with p F]fluoride, the intermediate was oxidized with manganese dioxide to yield the target compound [ F]FDHR (Fig. 11). 

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. 

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 ... 

Rotenone inhibits the electron transport chain by blocking transport between the flavoprotein and ubiquinone. The oxidation of pyruvate in rat mitochondria is virtually completely blocked by rotenone in vitro < 1 pmol 1 concentration). 

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. 

Do respiratory inhibitors have a connection with respiratory complexes Many of the workings of the electron transport chain have been elucidated by experiments using respiratory inhibitors. These inhibitors specifically block the transfer of electrons at specific points in the respiratory complexes. Fixamples are GO and CN", both of which block the hnal step of the electron transport chain, and rotenone, which blocks the transfer of electrons from NADH reductase to coenzyme Q. When such a blockage occurs, it causes electrons to pile up behind the block, giving a reduced carrier that cannot be oxidized. By noting which carriers become trapped in a reduced state and which ones are trapped in an oxidized state, we can establish the hnk between carriers. 

A block at the last complex in the electron transport chain (ETC) prevents the reduction of oxygen and causes all the electron transport complexes to become fully saturated (reduced) with electrons. As with rotenone (Example 10.14), a lack of electron/proton movement along the chain means that NADH is not reoxidized and fuel oxidation ceases. Similarly, the lack of electron transport activity means that the proton gradient soon dissipates and there is no driving force for ATP synthesis. ATP levels falls rapidly and the cell dies. 

Figure 2 Effects of inhibitors of the mitochondrial electron transport chain on outward K" " current in neonatal rat chromaffin cells. Representative traces for a voltage step to +30 mV are shown on the left, for a variety of inhibitor concentrations as indicated in (a) cyanide, (b) 2,4-dinitrophenol (DNP), and (c) rotenone. The corresponding I-V relation for each cell is shown on the right. Note that both cyanide and DNP had no effect, whereas rotenone caused a dose-dependent inhibition of outward K+ current.

The development by Chance of a dual wavelength spectrophotometer permitted easy observation of the state of oxidation or reduction of a given carrier within mitochondria.60 This technique, together with the study of specific inhibitors (some of which are indicated in Fig. 18-5 and Table 18-4), allowed some electron transport sequences to be assigned. For example, blockage with rotenone and amytal prevented reduction of the cytochrome system by NADH but allowed reduction by succinate and by other substrates having their own flavoprotein components in the chain. Artificial electron acceptors, some of which are shown in Table 18-5,... 

Palmer et al. (91) have suggested that in addition to the above site, rotenone and piericidin A also inhibit electron transport immediately on the substrate side of cytochrome Ci. This view has not been accepted by others. Teeter et al. (9 ) have shown that secondary effects of rotenone and piericidin can be observed at other regions of the respiratory chain when high concentrations of the inhibitors are used, as by necessity did Palmer et al. in their EPR experiments. 

Oxidative phosphorylation is susceptible to inhibition at all stages of the process. Specific inhibitors of electron transport were invaluable in revealing the sequence of electron carriers in the respiratory chain. For example, rotenone and amytal block electron transfer in NADH-Q oxidoreductase and thereby prevent the utilization of NADH as a substrate (Figure 18.43). In contrast, electron flow resulting from the oxidation of succinate is unimpaired, because these electrons enter through QH2, beyond the block. AntimycinA interferes with electron flow from cytochrome h Q-cytochrome c... 

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