Coupled phosphorylation

Figure 16-2. The citric acid cycle the major catabolic pathway for acetyl-CoA in aerobic organisms. Acetyl-CoA, the product of carbohydrate, protein, and lipid catabolism, is taken into the cycle, together with HjO, and oxidized to CO2 with the release of reducing equivalents (2H). Subsequent oxidation of 2H in the respiratory chain leads to coupled phosphorylation of ADP to ATP. For one turn of the cycle, 11 are generated via oxidative phosphorylation and one arises at substrate level from the conversion of succinyl-CoA to succinate.

The participation of the phycobiliproteins in the absorption ofphotokinetically active light has been demonstrated above. Peaks of around 565 and 615 nm in the action spectra indicate the involvement of C-phycoerythrin andC-phycocanin. These pigments transfer energy to the reaction center of PS II and suggest the participation of the non-cyclic electron transport and coupled phosphorylation. 

Reaction 15.12 would be catalyzed by the electron-transport chain, with coupled phosphorylation, and all the oxygen in the sulfite product would be derived from water (in contrast to the oxygenase, in which two thirds of the oxygen atoms in sulfite come from dioxygen). Overall, Equations 15.11 and 15.12 produce the same result as Equation 15.10. 

The acetyl-CoA produced from the oxidation of fatty acids can be oxidized to C02 and H20 by the citric acid cycle. The following equation represents the balance sheet for the second stage in the oxidation of palmitoyl-CoA, together with the coupled phosphorylations of the third stage ... 

States" of mitochondria and spectrophoto-metric observation. Chance and Williams defined five states of tightly coupled mitochondria60 163 of these, states 3 and 4 are most often mentioned. If no oxidizable substrate or ADP is added the mitochondria have a very low rate of oxygen uptake and are in state 1. If oxidizable substrate and ADP are added rapid 02 uptake is observed, the rate depending upon the rate of flow of electrons through the electron transport chain. This is state 3. As respiration occurs the coupled phosphorylation converts ADP into ATP, exhausting the ADP. Respiration slows to a very low value and the mitochondria are in state 4. If the substrate is present in excess, addition of more ADP will return the mitochondria to state 3. 

Tanford, C., Reynolds, J.A., Johnson, E.A. (1987). Sarcoplasmic reticulum calcium pump A model for Ca2+ binding and Ca2+-coupled phosphorylation. Proc. Natl. Acad. Sci. USA 84,7094-7098. 

Photoinduced electron transport and the coupled phosphorylation reactions as they are postulated to occur in chloroplasts are presented schematically in Figure 2. Not all investigators agree on the details of this scheme, and some even question the sequence of the intermediates. The numbers and locations of the phosphorylation sites also remain to be identified precisely. However, the scheme is a reasonable approximation based on available information. Reactions that occur in the light are represented by the open arrows and the solid arrows represent electron transfers that occur in the dark. 

Determination of the role of polyphosphate in transport-coupled phosphorylation in the yeast... 

The membrane-bound ATP synthetase couples phosphorylation to a proton gradient [90] which is generated by the cyclic electron transfer system (Fig. 3). This system includes the RC, a UQ pool [91], a Cyt bic complex [92,93], and a specialized Cyt c (E j = -fO.34 V) for transferring electrons to the oxidized primary donor (P-870 or P-970 ) of the RC. In some bacteria such as Chromatiurn vi-nosum and Rhodopseudomonas viridis this specialized Cyt c is bound to the RC in the membrane [93,94], whereas in other bacteria such as Rb. sphaeroides and Rhodospirillum rubrum this cytochrome is a periplasmic protein (Cyt C2) that binds to the membrane-bound RC [90]. 

Finally, transport can also be driven by the conversion of intracellular substrate to another chemical form. For example, in the case of nucleoside drugs, conversion to the corresponding nucleotides by appropriate kinases may be the limiting factor in cellular uptake and activation. The same principle applies to sulfation, glu euro nidation, prodrug activations, or other metabolic processes that provide a removal of the transported species from the transportable (free) internal pool. In some cases, transport is directly coupled to substrate modification, as in the uptake of sugars into bacterial cells by phosphoenolpyruvate (PEP)-coupled phosphorylation systems. 

Thus, it is not necessary for this microorganism to carry out electron transport-coupled phosphorylation during growth with lactate and sulfate. This is in contrast to the sulfate-reducing bacteria belonging to the genus Desulfovibrio, which do not have the acetate, PP phosphotransferase, and therefore appear to have to carry out electron transport-coupled phosphorylation in order to obtain a net yield of ATP. 

Before proceeding to the next topic, we look at another version of artificial phosphorylation by chloroplasts in the dark, i.e., not driven hyphotoinducedelectron transfer. This new type oftwo stage phosphorylation, called dark oxidation-reduction coupled phosphorylation, was reported by Selman and Psczolla and may be considered as a variant of the postillumination or the acid-base transition types discussed above. The authors found that ATP formation in chloroplasts in the dark can be achieved by an artificial, transmembrane redox reaction using ascorbate as the reductant for ferricyanide trapped inside the chloroplasts, provided it is mediated by a redox carrier such as DAD, DCIP or PMS that liberates protons during its oxidation, as illustrated in the scheme in Figure 13. 

In practice, a concentrated chloroplast sample (3 mg Chl/ml) is loaded or charged with a high concentration (100 mM) of ferricyanide by abrief sonication. Ferricyanide must be present during sonica-tion in order for the chloroplasts to be able to synthesize ATP in the dark. The sample is then diluted 15 fold with a buffer that contains ADP and Pj plus 10 mM ascorbate as the reductant and 0.4 mM DAD as the redox mediator. After incubation for two minutes at 20 °C in the dark, the reaction was quenched with HCIO4 and ATP analyzed. This dark redox-coupled phosphorylation has a yield of 70 nmoles ATP/mg Chi, amounting to about one-half to one-fourth of the amount usually obtained by acid-base transition. Ascorbate alone was not sufficient to catalyze ATP synthesis. As expected, the dark phosphorylation was also inhibited by uncouplers. 

Fig. 12. Model for the dark oxidation-reduction coupled phosphorylation in sonicated chloroplast vesicles. M formedlator Ascfor ascorbate subscripts and for oxidized and reduced states Fe Cy and Fe Cy for ferri- and ferrocyanide, respectively.

P-Type ATPases Couple Phosphorylation and Conformational Changes to Pump Calcium Ions Across Membranes... 

The substrate, glucose, is phosphorylated by the enzyme hexokinase in a coupled phosphorylation reaction. The source of the phosphoryl group is ATP. At first this reaction seems contrary to the overall purpose of catabolism, the production of ATP. The expenditure of ATP in these early reactions must be thought of as an "investment." The cell actually goes into energy "debt" in these early reactions, but this is absolutely necessary to get the pathway started. 

An increase in lipophilicity of 3-CIPC with the replacement of a hexyl for the isopropyl side chain (3-CHPC versus 3-CIPC) resulted in increased inhibition of electron transport and an even greater inhibition of the coupled phosphorylation (Table II). Increased reductive inhibitory potency of 3,4-DCIPC over 3-CIPC also is shown. 

The cytochrome system represents the final common path of oxidation and coupled phosphorylation in the eukaryotic cell. The components of this system [1-3] encompass the crystallizable cytochromes c, which have yielded much information to the protein chemist, as well as the matrix-bound cytochromes b, Ci, and a, which have revealed themselves largely to the spectroscopist. The cytochromes can react with one another and can undergo reversible changes in the proteins themselves [3]. In this sense, they resemble the subunits of a protein more than they do independent individuals, forming a constellation of apoproteins each with its characteristic heme prosthetic group. The complete system appears only in the mitochondrion, where the vectorial reactions in which the cytochromes participate are regulated in concert by phosphate acceptor. Induction and repression similarly involve not only particular cytochromes but the entire complex. This, in turn, is one aspect of the regulation of metalloporphyrins which is coordinated with the control of the hydrophobic matrices essential to their function. 

Pullman, M. E., Penefsky, H. and Racker, E. (1958) A soluble protein fraction required for coupling phosphorylation to oxidation in submitocbondrial fragments of beef heart mitochondria. Arch. 

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