Experiment 14 Electron Transport

Mitchell s chemiosmotic hypothesis. The ratio of protons transported per pair of electrons passed through the chain—the so-called HV2 e ratio—has been an object of great interest for many years. Nevertheless, the ratio has remained extremely difficult to determine. The consensus estimate for the electron transport pathway from succinate to Og is 6 H /2 e. The ratio for Complex I by itself remains uncertain, but recent best estimates place it as high as 4 H /2 e. On the basis of this value, the stoichiometry of transport for the pathway from NADH to O2 is 10 H /2 e. Although this is the value assumed in Figure 21.21, it is important to realize that this represents a consensus drawn from many experiments. 

Engelhardt s experiments in 1930 led to the notion that ATP is synthesized as the result of electron transport, and, by 1940, Severo Ochoa had carried out a measurement of the P/O ratio, the number of molecules of ATP generated per atom of oxygen consumed in the electron transport chain. Because two electrons are transferred down the chain per oxygen atom reduced, the P/O ratio also reflects the ratio of ATPs synthesized per pair of electrons consumed. After many tedious and careful measurements, scientists decided that the P/O ratio was 3 for NADH oxidation and 2 for succinate (that is, [FADHg]) oxidation. Electron flow and ATP synthesis are very tightly coupled in the sense that, in normal mitochondria, neither occurs without the other. 

When Mitchell first described his chemiosmotic hypothesis in 1961, little evidence existed to support it, and it was met with considerable skepticism by the scientific community. Eventually, however, considerable evidence accumulated to support this model. It is now clear that the electron transport chain generates a proton gradient, and careful measurements have shown that ATP is synthesized when a pH gradient is applied to mitochondria that cannot carry out electron transport. Even more relevant is a simple but crucial experiment reported in 1974 by Efraim Racker and Walther Stoeckenius, which provided specific confirmation of the Mitchell hypothesis. In this experiment, the bovine mitochondrial ATP synthasereconstituted in simple lipid vesicles with bac-teriorhodopsin, a light-driven proton pump from Halobaeterium halobium. As shown in Eigure 21.28, upon illumination, bacteriorhodopsin pumped protons... 

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. 

Allelopathic inhibition of mineral uptake results from alteration of cellular membrane functions in plant roots. Evidence that allelochemicals alter mineral absorption comes from studies showing changes in mineral concentration in plants that were grown in association with other plants, with debris from other plants, with leachates from other plants, or with specific allelochemicals. More conclusive experiments have shown that specific allelochemicals (phenolic acids and flavonoids) inhibit mineral absorption by excised plant roots. The physiological mechanism of action of these allelochemicals involves the disruption of normal membrane functions in plant cells. These allelochemicals can depolarize the electrical potential difference across membranes, a primary driving force for active absorption of mineral ions. Allelochemicals can also decrease the ATP content of cells by inhibiting electron transport and oxidative phosphorylation, which are two functions of mitochondrial membranes. In addition, allelochemicals can alter the permeability of membranes to mineral ions. Thus, lipophilic allelochemicals can alter mineral absorption by several mechanisms as the chemicals partition into or move through cellular membranes. Which mechanism predominates may depend upon the particular allelochemical, its concentration, and environmental conditions (especially pH). 

These experiments show that, as in the case of chlorophyll a and the carotenoids, the energy absorbed by the phycobiliproteins is utilized via the photosynthetic apparatus furthermore, they provide evidence that photophobic responses in blue-green algae are caused by sudden changes in the steady state of the photosynthetic electron transport, especially the non-cyclic one. 

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],... 

Such an experimental characterization is a necessary step to carry out a detailed comparison of emission properties as measured experimentally with the corresponding quantities as calculated by numerical models capable of describing transport and energy deposition of fast electrons in matter and consequent emission of characteristic X-ray emission. A possible modeling approach of fast electron transport experiments is given here, where the above results on Ka imaging were interpreted using the hybrid code PETRA [53] to... 

The NADPH oxidase is in fact a multicomponent enzyme system that constitutes an electron transport chain from NADPH to O2. The components of this oxidase complex are now almost completely defined, and experiments performed primarily with CGD neutrophils have helped to identify these major constituents. 

Pretreatment with the Type I substrate, ethylmorphine, resulted in 100% mortality in both rats and mice, and aminopyrine pretreatment resulted in 100% and 64% mortality in rats and mice, respectively, exposed to disulfoton (Pawar and Fawade 1978). Nickel chloride, cobalt chloride, or cycloheximide decreased the levels of cytochrome bs, cytochrome c reductase, and total heme in rats (Fawade and Pawar 1983). These electron transport components were further decreased in rats pretreated with these inhibitors and given a single dose of disulfoton. Data from this study suggests an additive effect, since disulfoton also decreases the activities of these components. Evidence of an additive effect between disulfoton and these metabolic inhibitors was suggested by the decrease in ethylmorphine N-demethylase and acetanilide hydroxylase activities when rats were given an inhibitor followed by disulfoton. In another experiment, these inhibitors decreased the activity of delta-aminolevulinic acid synthetase, but this decrease was reversed when disulfoton was administered. 

In summary, methanophenazine (10) is the first phenazine whose involvement in the electron transport of biologic systems could be established. The experiments indicate that its role in the energy metabolism of methanogens corresponds to that of ubiquinones in mitochondria and bacteria. 

What should be emphasized is that the redox potentials measured for 10 and 22 allow for both the reduction of 10 to dihydro-10 via F420H2 and H2, and the oxidation of dihydro-10 to 10 by 22. This finding, supported by electrochemical experiments, also strongly corroborates the hypothesis that 10 plays a prominent role as an electron carrier in the electron transport system of methanogens. 

Fig. 2.7 Schematic representation of electrochemical processes (reduction) for organic solids able to experience coupled proton transport/electron transport processes...

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