H ATP ratio

The value of x = 2 was proposed by Mitchell [113], but recently the values have been estimated as x = 3 [13,15] in mitochondria [116], submitochondrial particles [117], E. coli [118,119] and chloroplasts [120]. Details of the thermodynamics of this reaction will be discussed in another chapter. The possibility of loose coupling of H -flux and ATP synthesis will be discussed in the last part of Section 4. 

proteoliposomes can be reconstituted by the cholate dialysis method [123] or another method [124]. The resultant reconstituted FqF may have two different orientations in liposomes, but since the membranes are not permeable to ATP, 

The roles of lipids in proton translocation can also be studied with FgFj proteoliposomes. Fluidity and acidity of the phospholipids are required for proton translocation [129,130]. 

Direct evidence for the chemiosmotic theory [1] is proton motive ATP synthesis in purified FgFj proteoliposomes on applying [38,131]. Jagendorf and Uribe [132] first demonstrated ATP synthesis in subchloroplast particles that had been loaded with a weak acid (pH 4.5) and were then transferred to alkaline media at pH values 

Analysis of ATP synthesis driven by an ion gradient, as described in the previous section, has the disadvantage that time resolution is poor and the energy components 

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Mitome, N., Suzuki, T., Hayashi, S., and Yoshida, M. (2004). Thermophilic ATP synthase has a decamer c-ring Indication of noninteger 10 3 H+/ATP ratio and permissive elastic coupling. Proc. Natl. Acad. Sci. USA 101, 12159-12164. 

The activated membrane-bound ATPase is functionally coupled to proton movements. Thus, a transmembrane pH gradient (acid inside) of a magnitude similar to that observed during light-induced coupled electron flow is developed during ATP hydrolysis. ATP hydrolysis is stimulated, while the coupled proton transport is inhibited, by the addition of uncouplers, indicating that the rate of ATP hydrolysis is also partially limited by the electrochemical gradient which it creates. Nevertheless, attempts to measure H /ATP ratios in this system yielded numbers much below the expected ratio of 3. 

ATPs. This value Is consistent with experimental data on proton flux during ATP synthesis, providing Indirect support for the model coupling proton movement to c-ring rotation depicted in Figure 8-24. The Fq from chloroplasts contains 14 c subunits per ring, and movement of 14 protons would be needed for synthesis of three ATPs. Why these otherwise similar FqFi complexes have evolved to have different H ATP ratios in not clear. 

The H /ATP ratio (the number of protons ejected per ATP hydrolysed) was originally also determined by a pulse method and found to be 2 [118]. Again, later experiments indicated a higher value of 3 or 4 [119], As discussed in Section If (i), in the case of ATP synthesis or hydrolysis the transport of substrates and products may be accompanied by net movement of a proton across the membrane, so that more protons are involved than those transported via the ATPase complex itself. H /ATP ratios have also been calculated via equations containing thermodynamic parameters a stoichiometry of around 2.5-3 was deduced [120-122]. 

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]. 

The importance of the H /ATP ratio, i.e. the number of which have to be translocated across the ATP-synthase for each ATP molecule being synthesized or hydrolysed, has its origin in the treefold aspect of... 

In this paper two complete independent experimental approaches, namely the kinetic approach realizing H flux measurements and the energetic approach realizing transmembrane ApH measurements, prove the H /ATP ratio at the chloroplastic ATP-synthase to be four. 

If additionally the H/e ratio is determined, the proton flux is calculable from the e--flow and the H/e ratio and may be compared to the rate of ATP hydrolysis. The results presented in the Table and Fig. 1 clearly demonstrate a H /ATP ratio of four, irrespective of the change of the rate of ATP hydrolysis produced by addition of different uncouplers, addition of the ATPase inhibitor tentoxin or by the use of different ATP concentrations. 

Examples of finding out the equilibrium state at the ATP-synthase are given in Fig. 3. If such equilibrium state data are analysed it comes out that Ig [ATP/ (ADP.P)] correlates linearly to ApH (Fig. 4). From the slope a H /ATP ratio of four is read out. The intersection at the ordinate gives a AGp° of 31,2 kJ/mol in full agreement with the value determined by Rosing and Slater [7]. 

With respect to mechanistic consequences of the H /ATP ratio of four we refer to a recent coupling model described in [11]. With respect to the overall process of photosynthesis we have to accept that on the basis of the H /ATP ratio of four, taken together with the probable H/e ratio of 2.5 (see [1]), linear electron transport alone does not produce enough ATP to drive CO2 reduction. 

The value n = 4 means that the H /ATP ratio is four. This agrees with our own very recent results of direct H /ATP determination [1]. 

Amount of phosphorylation-coupled protons translocated through the membrane as a function of the amount of synthesized ATP. The slope of the curve gives the H /ATP ratio. 

The H/e ratio determines the number of H which are available at the ATPase for the production of ATP. H/ATP ratios of 3 reported in the literature are mostly based on an assumed H/e ratio of 2 and any change of it would change the H/ATP ratio to the same extent. 

Uncoupled conditions resemble those of activated ATP synthesis and therefore an increased H/e ratio of approx. 2.5 should also be observed in the case of activated ATP synthesis (supposed extreme high light intensity of 1000 Wm-2 is excluded). This means that H/ATP ratios of 3 which have been based on H/e values of 2 should be corrected... 

In the Mitchell hypothesis, electron transfer is coupled to transmembrane movement of protons, and this transport process results in the creation of an electrochemical potential (proton motive force) at the outside of the membrane. When protons return, they do so through a proton channel in the membrane that leads to the H -ATPase, where synthesis is accomplished. Mitchell (1976) has elaborated on the concept of proticity, i.e., proton flow. One key feature of the chemiosmotic theory is the expectation that H /e = H" /ATP, the value of 2 for both ratios being determined experimentally. Reevaluation of experimental data led Brand and Lehninger (1977) to propose a modification of the chemiosmotic theory which accommodated H" /ATP ratios greater than 2 and H" /e = H" /ATP. Stoichiometric considerations of proton translocation have been reviewed by Papa (1976). 

Our previous analysis (McCarty, Portis, 1976 Davenport, 1983) of ATP synthesis and hydrolysis in relation to proton fluxes provided evidence that the common link between electron transport and ATP synthesis in the steady state is a proton activity gradient (ApH). From this analysis, we also derived a maximal phosphorylation efficiency (P/c2)max close to 4/3. The (P/c2)niax equivalent to 2 (H+/e ) / (H+/ATP), where H+/e is the ratio of protons appearing in the thylakoid interior to electrons transferred and H+/ATP is the ratio of protons translocated to ATP synthesized. Thus, if the (H+/e ) is 2, (H" /ATP) is three. (H+/ATP) ratios of three have been obtained by other methods (Avron, 1978 Hand-garter, Good, 1982). 

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