Upstream electron carriers

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 use of respiratory inhibitors to determine the order of the electron transport chain depends on determining the relative amounts of oxidized and reduced forms of the various electron carriers in intact mitochondria. The logic of the experiment can be seen from the analogy of the blocked pipe. In this case, the reduced form of the carrier upstream (reduced carrier 2) accumulates because it cannot pass electrons farther in the chain. Likewise, the oxidized form of the carrier downstream (oxidized carrier 3) also accumulates because the supply of electrons that it could accept has been cut off (Figure 20.20). By use of careful techniques, intact mitochondria can be isolated from cells and can carry out electron transport if an oxidizable substrate is available. If electron transport in mitochondria occurs in the presence and absence of a respiratory inhibitor, different relative amounts of oxidized and reduced forms of the electron carriers will be present. 

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