Proton-translocating NADH

Yagi, T., and Matsuno-Yagi, A. 2003. The proton-translocating NADH-quinone oxidoreductase in the respiratory chain The secret unlocked, Biochemistry 42 2266-2274. 

Brandt, U. (1997) Proton-translocation by membrane-bound NADH ubiquinone-oxidoreductase (complex I) through redoxgated ligand conduction. Biochim Biophys. Acta 1318, 79-91. Advanced discussion of models for electron movement through Complex I. 

Fig. 5.3. The major components involved in mitochondrial NADH oxidation in facultative anaerobic mitochondria. In anaerobically functioning mitochondria, NADH is oxidized either by soluble enzymes (left) or by membrane-bound complexes of the electron-transport chain (middle). Under aerobic conditions, a classic respiratory chain is used to oxidize NADH (right). Proton translocation is indicated by H with arrows. Ovals represent the electron transporters RQ, UQ and cytochrome c (cyt. c), and electron transport is indicated by dashed arrows. The vertical bar represents a scale for the standard redox potentials in millivolts. Fum fumarate, NADH-DH NADH dehydrogenase, NADH-ECR soluble NADH enoyl-CoA reductase, RQH2 rhodoquinol, Succ succinate, UQH2 ubiquinol...

The maximum pmf generated in State 4 has been measured by various groups using several techniques (see Refs. 8,26,27). The results vary between about 160 and 240 mV. However, from the observed shifts of the redox equilibria in the respiratory chain imposed by the pmf (or by ATP via the pmf), it can be concluded that the functionally relevant pmf must be at least 200 mV in State 4 of well-coupled mitochondria [8]. (This need not be equivalent with the pmf between the bulk aqueous C and M phases). This limits the H /e ratio in State 4 to maximal values of 5.6 and 3.8 for the spans from NADH and ubiquinol to oxygen, respectively [8]. These are indeed upper limit stoicheiometries, attainable, of course, only at thermodynamic equilibrium between respiration and proton translocation. The true values must therefore be lower, particularly as the terminal step in the chain, i.e., oxidation of cytochrome c by Oj is irreversible (see Refs. 28, 29 and below). 

NADH-ubiquinone reductase (EC 1.6.5.3) or Complex I is structurally by far the most complicated member of the respiratory chain. It is also the least known in terms of structure, electron transfer pathway or mechanism of proton translocation. Even the nomenclature of the isolated enzyme entities and of the FeS centres is problematic because it differs between research groups. 

Oxidation of NADH by ubiquinone is linked to proton translocation. But also here the stoicheiometry is under debate the proposals range between 1 and 2H /e (see Table 3.1 and Section 2). 

Ragan and Hinkle [42) reported on proton translocation by Complex I reconstituted into liposomes. During oxidation of added NADH by ubiquinone-1 they observed an uptake of about 0.7 H /e in addition to the trivial proton uptake linked to reduction of quinone by NADH. Liposomes reconstituted with Complex I plus ATP synthase exhibited ATP synthesis linked to oxidation of NADH by Q-1 [311]. 

Great physiological significance can be attributed to the NADH oxidase of plasma membranes. These enzymes may indirectly contribute to oxygen activation by means of protein-bound iron reduction however, they are more important for transmembraneous electron transfer coupled to proton translocation and regulation of cell growth [22-24]. 

The coupling between electron transport from NADH (or FADH2) to O2 and proton transport across the inner mitochondrial membrane, which generates the proton-motive force, also can be demonstrated experimentally with Isolated mitochondria (Figure 8-14). As soon as O2 is added to a suspension of mitochondria, the medium outside the mitochondria becomes acidic. During electron transport from NADH to O2, protons translocate from the matrix to the Intermembrane space since the outer membrane Is freely permeable to protons, the pH of the outside medium Is lowered briefly. The measured change In pH Indicates that about 10 protons are transported out of the matrix for every electron pair transferred from NADH to O2. 

A FIGURE 8-17 Overview of multiprotein complexes, bound prosthetic groups, and associated mobile carriers in the respiratory chain. Blue arrows indicate electron flow red arrows indicate proton translocation. Left) Pathway from NADH. A total of 10 protons are translocated per pair of electrons that flow from NADH to 02.The protons released into the matrix space during oxidation of NADH by NADH-CoQ reductase are consumed in the formation of water from O2 by cytochrome c oxidase, resulting in no net proton translocation from these reactions. Right) Pathway... 

Current evidence suggests that a total of 10 protons are transported from the matrix space across the inner mitochondrial membrane for every electron pair that is transferred from NADH to O2 (see Figure 8-17). Since the succinate-CoQ reductase complex does not transport protons, only six protons are transported across the membrane for every electron pair that is transferred from succinate (or FADH2) to O2. Relatively little is known about the coupling of electron flow and proton translocation by the NADH-CoQ reductase complex. More is known about operation of the cytochrome c oxidase complex, which we discuss here. The coupled electron and proton movements mediated by the CoQH2-cytochrome c reductase complex, which involves a unique mechanism, are described separately. 

More than 20 redox centers are involved in the electron-transport chain. Figure 6.12 depicts a simplified view of the flow of electrons from NADH to O2 via this series of electron carriers. Electron flow through Complexes I, III, and IV is associated with the release of relatively large amounts of energy, which is coupled to proton translocation by these complexes (and therefore ATP production). The redox potentials of the electron carriers thus appear to play a role in determining the pathway of electron flow through the electron-transport chain. 

Overall, each NADH donates two electrons, equivalent to the reduction of V2 of an O2 molecule. A generally (but not universally) accepted estimate of the stoichiometry of ATP synthesis is that four protons are pumped at complex I, four protons at complex III, and two at complex IV. With four protons translocated for each ATP synthesized, an estimated 2.5 ATPs are formed for each NADH oxidized and 1.5 ATPs for each of the other FAD(2H)-containing flavoproteins that donate electrons to CoQ. (This calculation neglects proton requirements for the transport of phosphate and substrates from the cytosol, as well as the basal proton leak.) Thus, only approximately 30% of the energy available from NADH and FAD(2H) oxidation by O2 is used for ATP synthesis. Some of the remaining energy in the electrochemical potential is used for the transport of anions and Ca into the mitochondrion. The remainder of the energy is released as heat. Consequently, the electron transport chain is also our major source of heat. 

Because NADH enters the oxidative phosphorylation at Complex I, three steps of proton translocation result from electron transport, leading to three equivalents of ATP made by ATP synthase. Substrates... 

Analysis of data obtained used the relationships AG = -RTlnK, where K = [ADP] [Pi]/[ATP] = lO-, and AG = -nFEm, where Em is the membrane potential at Im = 0, one can calculate the quantity, n, the number of protons translocated across the membrane per mol of ATP hydrolyzed. This turns out to be about 3. For ATP synthesis. Lane et al. have recently reported that the entire mitochondrial electron transport chain (from NADH dehydrogenase to cytochrome oxidase) requires the translocation of at least 12 protons per oxygen atom. How these results are correlated remains to be seen . 

FIGURE 1. Respiratory chain the enzymes of the mitochondrial iimer membrane involved in oxidative phosphorylation. From complex I to V, they are NADH-dehydrogenase, succinate dehydrogenase, cytochrome bc complex, and cytochrome c oxidase. Protons are translocated across the membrane while electrons are transferred to Oj through the chain. The proton gradient is used by ATP synthase (complex V) to make ATP. (Reprinted with permission from Saraste, 1999, American Association for the Advancement of Science.)... 

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