Mitochondria proton pump

Homeostatic mechanisms also allow animals to control their intracellular pH very strictly. In humans for example, blood pH (usually taken as a reliable but indirect measure of cellular pH) is 7.4 0.04. At 37 °C cytosolic pH is actually slightly lower at about 7.0 but different compartments within the eukaryotic cells may have quite different pH, for example, lysosomes have an internal pH of about 5 the inside of a mitochondrion is more alkaline than the outside whilst the inside of a phagosome in a white blood cell is more acidic than its surrounding cytosol, both situations arising due to proton pumping across a membrane. 

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.

During acidosis, the cells of the renal tubule can respond by inserting two proteins into the apical region of the plasma membrane. (The apical part is that region that is exposed to the developing urine.) The two proteins are H,K-ATPase and H+-ATPase. H,K-ATPase, and the enzymes that act in concert with it, is better known as a component of the parietal cell where it creates stomach acid. The other proton pump of the renal tubule, which is H" -ATPase, is closely related to FoFiH" -ATPase of the mitochondrial membrane. Hence, anyone who imderstands how protons are pumped out of the mitochondrion and how stomach acid is made will clearly understand how the renal tubule can shuttle protons to the lumen of the renal tubule and into the developing urine. 

FIGURE 4.1 Schematics of the mitochondrion. Electron transport chain, proton pumps, and ATP synthase are components of oxidative phosphorylation. Major complexes of electron transport chain are NADH dehydrogenase, bcl complex, and cytochrome c oxidase. The quinone, Q, and cytochrome c are intermediates that shuttle electrons between the complexes. Each of the three complexes is a proton pump. Cytochrome c oxidase is the terminal complex of the electron transport chain. 

Four protein complexes, three ofthem function as proton pumps, are embedded in the inner mitochondrial membrane and constitute essential components of the electron transport chain. Every complex consists of a different set of proteins with a variety of redox-active prosthetic groups. AU in all, through oxidation of NADH, a proton gradient between the matrix and the intermembrane space is created, which eventually drives the ATP synthase-complex. The correspondingly released electrons are consumed in the reduction of oxygen to water. Both, the NADH oxidation and the oxygen reduction, as well as the ATP synthesis, take place in the matrix of the mitochondrion (Fig. 8.14). 

By consuming four protons from the interior of the mitochondrion and four electrons from the exterior, a proton-electrochemical potential gradient is formed across the membrane, providing fuel for ATP synthesis and other processes. This process is commonly referred to as proton pumping. 

What do I mean by a proton concentration gradient Simply, there is a higher concentration of protons in the space between the inner and outer membranes of the mitochondrion than in the mitochondrial interior. The gradient is formed from the energy released in the transfer of electrons down the electron transport chain. Put another way, the released energy is employed to pump protons across the inner mitochondrial membrane into the intermembrane space. 

Oxidizible substrates from glycolysis, fatty acid or protein catabolism enter the mitochondrion in the form of acetyl-CoA, or as other intermediaries of the Krebs cycle, which resides within the mitochondrial matrix. Reducing equivalents in the form of NADH and FADH pass electrons to complex I (NADH-ubiquinone oxidore-ductase) or complex II (succinate dehydrogenase) of the electron transport chain, respectively. Electrons pass from complex I and II to complex III (ubiquinol-cyto-chrome c oxidoreductase) and then to complex IV (cytochrome c oxidase) which accumulates four electrons and then tetravalently reduces O2 to water. Protons are pumped into the inner membrane space at complexes I, II and IV and then diffuse down their concentration gradient through complex V (FoFi-ATPase), where their potential energy is captured in the form of ATP. In this way, ATP formation is coupled to electron transport and the formation of water, a process termed oxidative phosphorylation (OXPHOS). 

Current estimates are that three protons move into the matrix through the ATP-synthase for each ATP that is synthesized. We see below that one additional proton enters the mitochondrion in connection with the uptake of ADP and Pi and export of ATP, giving a total of four protons per ATP. How does this stoichiometry relate to the P-to-O ratio When mitochondria respire and form ATP at a constant rate, protons must return to the matrix at a rate that just balances the proton efflux driven by the electron-transport reactions. Suppose that 10 protons are pumped out for each pair of electrons that traverse the respiratory chain from NADH to 02, and 4 protons move back in for each ATP molecule that is synthesized. Because the rates of proton efflux and influx must balance, 2.5 molecules of ATP (10/4) should be formed for each pair of electrons that go to 02. The P-to-O ratio thus is given by the ratio of the proton stoichiometries. If oxidation of succinate extrudes six protons per pair of electrons, the P-to-O ratio for this substrate is 6/4, or 1.5. These ratios agree with the measured P-to-O ratios for the two substrates. 

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. 

Fig. 17.6. The vectorial pumping of calcium ions and protons across the mitochondrion membranes. A schematic enlargement of the inner (cristae) membrane is shown to indicate the existence of protein-based electron (e ) and proton (H+) conduction pathways (from Ref. 26 with permission).

Moore then explained how mitochondria are biological fuel cells. The oxygen reduction taking place in a mitochondrion is exactly the same as in a standard fuel cell. Using several enzymes and only earth-abundant elements, the mitochondrion converts electrochemical potential to biochemical work with efficiency greater than 90 percent. This is a steady-state process in which protons are pumped across the membrane to maintain its electrical potential. If... 

Briefly, a series of enzymes located on the inner membrane of the mitochondrion pump protons from the matrix into the inner membrane space, generating an... 

Electrons in the iron-sulfur clusters of NADH-Q oxidoreduetase are shuttled to coenzyme Q. The flow of two electrons from NADH to coenzyme Q through NADH-Q oxidoreduetase leads to the pumping offour hydrogen ions out of the matrix of the mitochondrion. The details of this process remain the subject of active investigation. However, the coupled electron- proton transfer reactions of Q are crucial. NADH binds to a site on the vertical arm and transfers its electrons to FMN. These electrons flow within the vertical unit to three 4Fe-4S centers and then to a bound Q. The reduction of Q to... 

The inconsistency between experiment and prediction must lead to the rejection of the model used to describe the system. In the case of oxidative phosphorylation this has led to a refined model, in which the chemiosmotic coupling is visualized as taking place within units of one (or a few) respiratory chain(s) plus ATP synthase, while the pumped protons have only limited access to the bulk phase inside and/or outside the mitochondrion [42]. This more refined model can again be tested by deriving from it flux-force relations according to the MNET approach. A discussion of the refined model can be found in Ref. 43. 

During electron transport, protons are pumped out of the mitochondrion at each of the major sites except for ... 

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