Intermediates in oxidative phosphorylation

Not the papers that reported the discovery and characterization of the phosphohistidine, but the ones that said we thought it was an intermediate in oxidative phosphorylation. That was a learning experience. But I perhaps tend to move ahead to the next possibility more readily than many scientists do. If you want to finish off a research project, make sure that everything seems right in that area, document everything, and so forth, you may never get to the next important aspect. 

The implications of the different stoichiometries for the overall P/0 ratios are obvious. If the proton gradient is the only and necessary intermediate in oxidative phosphorylation, the theoretical P/0 ratio can be calculated from the H " /O and H /ATP ratios. The original experiments agreed with the long-accepted P/O ratios of 3, 2 and 1 for NAD-linked substrates, succinate and cytochrome c, respectively. The more recent findings may not always agree with these accepted values [123]. 

An intense new absorption band occurs in a number of nickel-quinone complexes such as pOCVn). This has been characterized as a charge transfer band. It might be noted that an iron complex of coenzyme Q (XXVni) has been proposed " as an intermediate in oxidative phosphoryl-... 

A logical starting point for such studies is to look for phosphorylated derivatives of any of the components that might serve as intermediates in oxidative phosphorylation, such as phosphorylated derivatives of the elements of the electron transport chain, whether they be proteins or low molecular weight cofactors. 

Again, a phosphorylated protein may be an intermediate therefore, Boyer s [135] discovery of a phospho-histidine compound that may act as an intermediate in oxidative phosphorylation was most intriguing. The compound is either 1- or 3-phosphohistidine. It is not known which of the two isomers is the natural compound, but only one of them is active. The phosphorylated histidine residue was found to be part of a polypeptide sequence, but little information is available on the protein that contains the phosphorylated histidine, although that protein has been obtained in a pure form. 

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. 

It was also mentioned [22] that in the recent decade it was observed that /ZH is not only conjugation intermediate of oxidation phosphorylation, but also hard currency in membranes of mitochondria, chloroplasts and bacteria. .. It is clearly determined that /ZH can be used for implementing all types of work in the living systems chemical, osmotic, mechanical and heat production . 

Zhao et al. (36) found that some citric acid metabolites are decreased in acute ischemia and acute myocardial disease. The citric acid plays an important role in oxidative phosphorylation and ATP production in the cardiomyo-cytes in which levels of citric acid cycle intermediates are supplied by glycolysis and (1-oxidation of fatty acids. 

Additional studies [212,218,219,242,243] to quantitate the role of the adenine nucleotide translocator in the control of mitochondrial respiration have been performed utilizing inhibitor titrations with carboxyatractyloside. The results indicated that in State 4 (no ADP), no control was exerted by the translocator. However, as the rate of respiration was increased up to State 3 (excess ADP), the control strength of the carrier increased to a maximum value of 30%, at 80% of State 3 respiration. These studies indicate that the adenine nucleotide translocator cannot be considered to be the only rate-controlling step in oxidative phosphorylation. However, they do provide experimental support for a controlling role for the carrier at intermediate to maximal levels of respiration. An important corollary of these studies is that the reaction rate may be altered by a change in substrate concentration (elasticity). It is also clear that to confirm these studies quantitatively, they must be extended to intact cells. Although such studies have been more difficult, the results are compatible with the conclusion reached by Tager et al. [212]. 

The addition of ATP to anaerobic or terminally inhibited mitochondria or submitochondrial particles containing succinate Eo = 0.03 V at pH 7) induces reduction of cjdiochrome bj 16,17,65 see also 6 6). The original concept of the possible mechanism of this phenomenon described by Wilson and Dutton 19) was that the Eo of cytochrome f T changes because of the formation of a high energy derivative which is the primary intermediate for site 2 energy conservation reaction in oxidative phosphorylation. However, there has been another possible mechanism presented in which ATP can induce reduction of cytochrome bx by the decrease in the effective redox potential Ek) of the cytochrome because of reversed electron flow 57) or of the abolition of an accessibility barrier between the substrate and the cytochrome 58). The former explanation would be favored by the chemical hypothesis of oxidative phosphorylation, while the latter is favorable for the chemiosmotic hypothesis. 

DR Sanadi, DM Gibson, P Ayengar, L Quellet. Evidence for a new intermediate in the phosphorylation coupled to a-ketoglutarate oxidation. Biochim Biophys Acta 13 146-148, 1954. 

These conjectures were soon supported by experimental evidence obtained with mitochondrial preparations, first by Chance and Hollunger and later by many others, " who demonstrated that reversed electron transfer could involve not only flavin-coupled systems but also NAD-, NADP, and cytochrome-linked reactions. It was subsequently realized that ATP itself might not be directly involved in the energy transfer, and the notion of a high-energy intermediate of oxidative phosphorylation was invoked... 

From inhibitor studies at various intermediate steps of electron transfer and from other considerations, such as the substrate specificity of tocopherol-responsive respiratory decline, it seemed likely that the site of the main metabolic block leading to respiratory decline was not in the electron transfer chain proper, or in oxidative phosphorylation, as suspected earlier, but in certain dehydrogenase systems which connect the carboxylic acid cycle wdth the cytochrome chain. Using a-ketoglutarate as the substrate. 

Research in the field of intermediates of oxidative phosphorylation has developed in two main directions (1) Some investigators attacked the problem directly and attempted either to reconstruct in vitro the enzyme system responsible for the coupling of electron transport and phosphorylation, or to isolate intermediates (2) others have approached the problem by studying elementary reactions suspected to participate in oxidative phosphorylation. So much data has been gathered on all the aspects of oxidative phosphorylation that it would be unrealistic to attempt to cover the subject comprehensively. We will consider only a few representative experiments in the hope of illustrating the amplitude and complexity of the problem. 

It is highly probable that the new compound plays a role in oxidative phosphorylation. It is not formed in the absence of succinate, and it can yield ATP from ADP in the presence of magnesium. Furthermore, the formation of the intermediate is inhibited by dinitro-phenol. Oligomycin has no effect on the formation of the intermediate but blocks the formation of ATP from the phosphorylated derivative. Amytal has an opposite effect it blocks the formation of the phosphorylated derivative but does not affect the transphosphorylation of ADP. 

Changes in oxidative phosphorylation seem to be more closely related to the primary site of action of the thyroid hormones. Thyroxine uncouples oxidative phosphorylation in mitochondria and causes the energy released during oxidation of the Krebs cycle intermediates to be less efficiently used for ATP synthesis. A direct effect of thyroxine on oxidative phosphorylation would conveniently explain many metabolic effects of thyroxine, but it would leave unexplained the beneficial effects of small amounts of thyroxine. A direct effect on oxidative phosphorylation is also inconsistent with the fact that uncoupling agents are unable to correct hypothyroidism. 

The similarity of the four quinones is conspicuous. It has been proposed frequently that they participate in oxidative phosphorylation of the respiratory chain. They could do this in two w ays Either they are additional redox systems in the transport of hydrogens (or electrons) or they are intermediate carriers of the energy-rich phosphate. These roles have already been discussed m Chapt. X-4. It should be mentioned that ubiquinone is found in relative abundance in the mitochondria, while the other quinones have not been found there with equal certainty. Plastoquinone presumably is involved in the electron-transport system of chloroplasts connected with photosynthesis (cf. Chapt. XVI-2). 

A dynamic model has been proposed for the synthesis of ATP during oxidative phosphorylation in which ADP and inorganic phosphate combine directly to give a quinquecovalent intermediate. While such a model may be valuable in drawing attention to the possible participation of quinquecovalent intermediates in ATP synthesis, no mention is made of the role of oxidation in this reaction. 

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