Proton chemiosmotic hypothesis

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. 

Peter Mitchell s chemiosmotic hypothesis revolutionized our thinking about the energy coupling that drives ATP synthesis by means of an electrochemical gradient. How much energy is stored in this electrochemical gradient For the transmembrane flow of protons across the inner membrane (from inside [matrix] to outside), we could write... 

In 1961, Peter Mitchell proposed a novel coupling mechanism involving a proton gradient across the inner mitochondrial membrane. In Mitchell s chemiosmotic hypothesis, protons are driven across the membrane from the matrix to the intermembrane... 

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... 

The chemiosmotic hypothesis had the great virtue of predicting the following consequences which could be tested (1) electron-transport driven proton pumps with defined stoichiometries and (2) a separate ATP synthase, which could be driven by a pH gradient or membrane potential. Mitchell s hypothesis was initially greeted with skepticism but it encouraged many people, including Mitchell and his associate Jennifer Moyle, to test these predictions, which were soon found to be correct.178... 

Oxidative phosphorylation is ATP synthesis linked to the oxidation of NADH and FADH2 by electron transport through the respiratory chain. This occurs via a mechanism originally proposed as the chemiosmotic hypothesis. Energy liberated by electron transport is used to pump H+ ions out of the mitochondrion to create an electrochemical proton (H+) gradient. The protons flow back into the mitochondrion through the ATP synthase located in the inner mitochondrial membrane, and this drives ATP synthesis. Approximately three ATP molecules are synthesized per NADH oxidized and approximately two ATPs are synthesized per FADH2 oxidized. 

Oxidative phosphorylation is the name given to the synthesis of ATP (phosphorylation) that occurs when NADH and FADH2 are oxidized (hence oxidative) by electron transport through the respiratory chain. Unlike substrate level phosphorylation (see Topics J3 and LI), it does not involve phosphorylated chemical intermediates. Rather, a very different mechanism was proposed by Peter Mitchell in 1961, the chemiosmotic hypothesis. This proposes that energy liberated by electron transport is used to create a proton gradient across the mitochondrial inner membrane and that it is this that is used to drive ATP synthesis. Thus the proton gradient couples electron transport and ATP synthesis, not a chemical intermediate. The evidence is overwhelming that this is indeed the way that oxidative phosphorylation works. The actual synthesis of ATP is carried out by an enzyme called ATP synthase located in the inner mitochondrial membrane (Fig. 3). 

Figure 17.2 The chemiosmotic hypothesis. Electrons from NADH and/or FADH2 are passed to Oz via the electron transport chain. In the process, protons are extruded into the mitochondrial intermembrane space. The proton gradient thus created causes the movement of protons back into mitochondria through a channel in the F ATPase. In the process, one molecule of ATP is formed frm ADP and phosphate for every two to three protons channeled back into the mitochondria. ATP moves into the intermembrane space and cytosol in exchange for ADP moving in the opposite direction. Phosphate is taken up in exchange for OH".

In the previous chapter we indicated that the components involved with electron flow are situated in the lamellar membranes of chloroplasts such that they lead to a vectorial or unidirectional movement of electrons and protons (see Fig. 5-19). We now return to this theme and focus on the gradients in H+ (protons) thus created. In the light, the difference in the chemical potential of H+ from the inside to the outside of a thylakoid acts as the energy source to drive photophosphorylation. This was first clearly recognized in the 1960s by Peter Mitchell, who received the 1978 Nobel Prize in chemistry for his enunciation of what has become known as the chemiosmotic hypothesis for interpreting the relationship among electron flow, proton movements, and ATP formation. 

An artificial system was created to elegantly demonstrate the basic principle of the chemiosmotic hypothesis. Synthetic vesicles containing bacteriorhodopsin, a purple-membrane protein from halobacteria that pumps protons when illuminated, and mitochondrial ATP synthase purified from beef heart were created (Figure 18.26). When the vesicles were exposed to light, ATP was formed. This key experiment clearly showed that the respiratory chain and ATP synthase are biochemically separate systems, linked only by a proton-motive force. 

Figure 18.25. Chemiosmotic Hypothesis. Electron transfer through the respiratory chain leads to the pumping of protons from the matrix to the cytosolic side of the inner mitochondrial membrane. The pH gradient and membrane potential constitute a proton-motive force that is used to drive ATP synthesis.

Figure 18.26. Testing the Chemiosmotic Hypothesis. ATP is synthesized when reconstituted membrane vesicles containing bacteriorhodopsin (a light-driven proton pump) and ATP synthase are illuminated. The orientation of ATP synthase in this reconstituted membrane is the reverse of that in the mitochondrion.

Figure 1. The major transmembrane photosynthetic reaction centers (RC) (top) and respiratory complexes (bottom) are composed of light (zigzag) activated chains (dark gray) of redox centers (open polygons) that create a transmembrane electric field and move protons (double arrows) to create a transmembrane proton gradient, fulfilling the requirements of Mitchell s chemiosmotic hypothesis. Diffusing substrates include ubiquinone (hexagon) and other sources of oxidants and reductants. PSI and PSII, photosystems I and II, respectively.

The ATP synthase is a reversible proton-translocating ATP ase The initial experiments which were important for the verification of the chemiosmotic hypothesis were those which showed that the complex was an autonomous proton pump when hydrolyzing ATP, and which showed that an artificial A/a could cause the ATP synthase to generate ATP. Thus, if a limiting amount of ATP is injected into an anaerobic mitochondrial incubation a net expulsion of protons is observed, followed by a decay which is accelerated by proton translocators [15]. Less complications arise if the experiment is repeated with inverted sub-mitochondrial... 

Two major ATP synthesizing reactions in living organisms are oxidative phosphorylation and photophosphorylation. Both reactions take place in H -ATPase (FqF,), which is driven by an electrochemical potential difference of protons across the biomembrane, as predicted by Mitchell [1]. In Racket s laboratory, ATPases related to oxidative phosphorylation were prepared, but their relationship to Mitchell s chemiosmotic hypothesis [1] was not described [2], Later, an insoluble ATPase (H -ATPase) was shown to translocate protons across the membrane when it was reconstituted into liposomes [3], H -ATPase was shown to be composed of a catalytic moiety called F, (coupling factor 1) [4], and a membrane moiety called Fq [5], which confers inhibitor sensitivity to F,. F was shown to be a proton channel, which translocates down an electrochemical potential gradient across the membrane when Fg is reconstituted into liposomes (Fig. 5.1) [6]. Thus, -ATPase was called FqFj or ATP synthetase. 

In this review, the chemiosmotic hypothesis [1] at the physiological level, i.e., ATP synthesis in FqF, driven by an electrochemical potential difference of protons (Fig. 5.1) is supported, while the hypothesis [1] at the level of molecular mechanism, i.e., the direct participation of the translocated protons in the dehydration of phosphate during ATP synthesis in F F, is excluded. The solid chemical and physical experiments on the purified Fj, Fq and FqF, and genetic analysis of the F F, established a new concept on the proton motive ATP synthesis. 

According to the chemiosmotic hypothesis, ejection of two or more protons occurs at each of three sites in complexes I, III, and IV. Thus, in the transfer of two reducing equivalents from NADH to oxygen, at least six protons... 

The anisotropic arrangement of the respiratory chain and the vectorial transport of protons are supported by experimental observations. The distribution of the redox carriers required by the chemiosmotic hypothesis is remarkably similar to that derived from studies on enzyme topology of the inner membrane. In respiring mitochondria, the intramembrane space is more acidic than the matrix space by about 1.4 pH units, and the transmembrane potential is about 0.180-0.220 V. Thus, the basic premise of the chemiosmotic hypothesis is... 

The answer is b. (Murray, pp 123-148. Scriver, pp 2367-2424. Sack, pp 159-115. Wilson, pp 287-3111) The chemiosmotic hypothesis of Mitchell describes the coupling of oxidative phosphorylation and electron transport. The movement of electrons along the electron transport chain allows protons to be pumped from the matrix of the mitochondria to the cytoplasmic side. The protons are pumped at three sites in the electron transport chain to produce a proton gradient. When protons flow back through proton channels of the asymmetrically oriented ATPase of the inner mitochondrial membrane, ATP is synthesized. 

Our understanding of oxidative phosphorylation is based on the chemiosmotic hypothesis, which proposes that the energy for ATP synthesis is provided by an electrochemical gradient across the inner mitochondrial membrane. This electrochemical gradient is generated by the components of the electron transport chain, which pump protons across the inner mitochondrial membrane as they sequentially accept and donate electrons (see Fig. 21.1). The final acceptor is O2, which is reduced to H2O. 

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