Electron transport chain inhibitors

Identifying the inhibition. You are asked to determine whether a chemical is an electron-transport-chain inhibitor or an inhibitor of ATP synthase. Design an experiment to determine this. 

The initial sensors and subsequent signal transduction systems of hypoxic vasoconstriction (HPV) remain an area of intense investigation (21,22). At the cellular level, pulmonary artery smooth muscle (PASM) contraction depends on an increase in cytosolic calcium from the extracellular space as well as release from intracellular stores, and membrane depolarization due to closure of K+ channels. Many argue that the mitochondria is a primary oxygen sensor such that electron transport chain inhibitors can specifically inhibit HPV and/or prevent the hypoxia-specific response. Reactive oxygen species are also implicated in HPV. Two different models are proposed one describes an increase in mitochondrial ROS mediated via increased intracellular calcium release, and the second describes a decrease in mitochondrial ROS mediated via inhibition of the Kv channel (Figure 8.2). 

The egg and the embryonic cell are well endowed with bioenergetic pathways. The multiple-enzyme systems involved in glycolysis, the hexose monophosphate shunt, the Krebs cycle, the electron transport chain, and oxidative phosphorylation have all been found in the vertebrate embryo. In the embryonic and in the mature cell, oxidation through the Krebs cycle, electron transport, and coupling of oxidation and phosphorylation occur in mitochondria. The chemical energy provided by these pathways is needed for normal development because if either glycolysis, Krebs cycle, or electron transport chain inhibitors are administered in vivo or added to explanted chick or sea urchin embryos, embryonic development is arrested. 

Glycolysis and the citric acid cycle (to be discussed in Chapter 20) are coupled via phosphofructokinase, because citrate, an intermediate in the citric acid cycle, is an allosteric inhibitor of phosphofructokinase. When the citric acid cycle reaches saturation, glycolysis (which feeds the citric acid cycle under aerobic conditions) slows down. The citric acid cycle directs electrons into the electron transport chain (for the purpose of ATP synthesis in oxidative phosphorylation) and also provides precursor molecules for biosynthetic pathways. Inhibition of glycolysis by citrate ensures that glucose will not be committed to these activities if the citric acid cycle is already saturated. 

Flutolanil is an inhibitor of succinate dehydrogenase complex (Complex II), in the mitochondrial respiratory electron transport chain. ... 

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. 

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. 

The key enzyme hydrogenase catalyses the reversible reduction of protons to molecular hydrogen. Inhibitor experiments indicate that the ferredoxin PetF functions as natural electron donor linking the hydrogenase to the photosynthetic electron transport chain [Florin et al., 2001],... 

This type of effect can occur in all tissues and is caused by a metabolic inhibitor such as azide or cyanide, which inhibits the electron transport chain. Inhibition of one or more of the enzymes of the tricarboxylic acid cycle such as that caused by fluoroacetate (Fig. 6.7) also results in inhibition of cellular respiration (for more details of cyanide and fluoroacetate see chap. 7). 

Hollingworth, R.M., Ahammadsahib, K.I., Gadelhak, G., and McLaughlin, J.L. Inhibitors of complex I in the mitochondrial electron transport chain with activities as pesticides. Biochem. Soc. Trans., 22, 230, 1994. 

Blocking electron transfer by any one of these inhibitors stops electron flow from substrate to oxygen because the reactions of the electron transport chain are tightly coupled like meshed gears. 

Site-specific inhibitors Site-specific inhibitors of electron transport have been identified and are illustrated in Figure 6.10. These compounds prevent the passage of electrons by binding to a component of the chain, blocking the oxidation/reduction reaction. Therefore, all electron carriers before the block are fully reduced, whereas those located after the block are oxidized. [Note Because electron transport and oxidative phosphorylation are tightly coupled, site-specific inhibition of the electron transport chain also inhibits ATP synthesis.]... 

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. 

In soybean seedlings in-vivo supply of cadmium and lead was reported to stimulate the respiration rate (Lee et al., 1976a, b) this effect was ascribed to a demand for ATP production through oxidative phosphorylation because photophosphorylation was reduced. In vitro, several metal ions were effective inhibitors of the mitochondrial respiratory electron transport chain (Kleiner, 1974 Koeppe, 1977) Koeppe (1977) considered the inhibition of the electron transfer at the terminal NADH-oxidase to be specific for cadmium. 

In-vitro approach Data are available in abundance concerning metal effects on isolated chloroplasts (for a review, see Clijsters and Van Assche, 1985). All the metals studied were found to be potential inhibitors of photosystem 2 (PS 2) photosystem 1 (PS 1) was reported to be less sensitive. From the in-vitro experiments, at least two potential metal-sensitive sites can be derived in the photosynthetic electron transport chain the water-splitting enzyme at the oxidising side of PS 2, and the NADPH-oxido-reductase (an enzyme with functional SH-groups) at the reducing side of PS 1 (Clijsters and Van Assche, 1985). Moreover, in vitro, non cyclic photophosphorylation was very sensitive to lead (Hampp et al., 1973 b) and mercury (Honeycutt and Korgmann, 1972). Both cyclic and non-cyclic photophosphorylation were proven to be inhibited by excess of copper (Uribe and Stark, 1982) and cadmium (Lucero et al, 1976). 

Uncouplers. Uncouplers dissociate electron transport from photophosphorylation. Both noncyclic and cyclic phosphorylation are inhibited, but electron transport reactions are either unaffected or stimulated. Because uncouplers relieve the inhibition of electron transport imposed by energy transfer inhibitors, they are considered to act at a site closer to the electron transport chain than the site of phosphate uptake. In Figure 2, they are shown (site 2) as dissipating some form of conserved energy represented as on the noncyclic and cyclic ATP-gener-ating pathways. Perfluidone is the only herbicide identified to date that functions as a pure uncoupler at pH 8.0 (2). Compounds that uncouple photophosphorylation also uncouple mitochondrial oxidative phosphorylation. 

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. 

Thcnoyltrifluoroacetone Fig. 14-3 Sites of action of inhibitors of the electron-transport chain. 

The absence of ADP is acting, in effect, as an inhibitor of electron transport, for reasons discussed in Prob. 14.6 below. Hence, by application of the crossover theorem (Chap. 10), there are large differences in the reduction of sites of the electron-transport-chain between NAD and coenzyme Q, between cytochrome b and cytochrome c, and between cytochrome c and cytochrome a. Therefore, the absence of ADP must be inhibiting electron transport at these points in fact, these are the sites of proton extrusion leading to ATP synthesis during electron transport. 

Tischer and Strotmann ( 7), the binding constant corresponds to the inhibition constant, i. e. the I,. value (the concentration necessary for 50% inhibition of photosynthetic electron transport), provided the I. value is extrapolated to zero chlorophyll concentration. The value of 527 molecules of chlorophyll per molecule of bound inhibitor indicates that roughly one molecule of herbicide binds per electron transport chain, because about 400-600 molecules of chlorophyll are considered to be associated with each electron transport chain. 

The relative and absolute configuration of Stigmatellin A, one of the most potent inhibitors of the electron transport chain, was determined via alkylation of diethyl ketone SAMP hydrazone. ... 

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