Membrane intermembrane processes

From a theoretical perspective a number of processes could contribute to the intermembrane transport of lipids. These include (i) monomer solubility and diffusion, (ii) soluble carriers such as lipid transfer proteins, (iii) carrier vesicles, (iv) membrane apposition and lipid transfer, and (v) membrane fusion processes. Lipids such as fatty acids, lysophos-phatidic acid, and CDP-DG might have sufficient water solubility to allow for some monomeric transport, but most other lipids are likely to require one of the other potential mechanisms due to their extremely low solubility. 

NADH-coenzyme Q (CoQ) oxidoreductase, transfers electrons stepwise from NADH, through a flavoprotein (containing FMN as cofactor) to a series of iron-sulfur clusters (which will be discussed in Chapter 13) and ultimately to CoQ, a lipid-soluble quinone, which transfers its electrons to Complex III. A If, for the couple NADH/CoQ is 0.36 V, corresponding to a AG° of —69.5 kJ/mol and in the process of electron transfer, protons are exported into the intermembrane space (between the mitochondrial inner and outer membranes). 

Most of the mitochondrial proteins are nuclear encoded and thus must be targeted into mitochondria and sorted into some of their components after their synthesis at the cytosol. Because mitochondria have two membranes, there are four localization sites the matrix, the inner membrane, the intermembrane space, and the outer membrane (Fig. 6). Although there has been considerable progress in our understanding of these processes, some questions still remain. Moreover, the total picture is rather complicated and contains many exceptions. A simplified view is presented here based mainly on the view of Pfanner and Mihara (Mihara and Omura, 1996 Pfanner et al., 1997 Pfanner, 1998). There are also a number of other excellent reviews on this subject (Schatz, 1996 Stuart and Neupert, 1996 Neupert, 1997 Roise, 1997). 

The proton-motive Q-cycle model, put forward by Mitchell (references 80 and 81) and by Trumpower and co-workers, is invoked in the following manner (1) One electron is transferred from ubiquinol (ubiquinol oxidized to ubisemi-quinone see Figure 7.27) to the Rieske [2Fe-2S] center at the Qo site, the site nearest the intermembrane space or p side (2) this electron can leave the bci complex via an attached cytochrome c or be transferred to cytochrome Ci (3) the reactive ubisemiquinone reduces the low-potential heme bL located closer to the membrane s intermembrane (p) side (4) reduced heme bL quickly transfers an electron to high-potential heme bn near the membrane s matrix side and (5) ubiquinone or ubisemiquinone oxidizes the reduced bn at the Qi site nearest the matrix or n side. Proton translocation results from the deprotonation of ubiquinol at the Qo site and protonation of ubisemiquinone at the Qi site. Ubiquinol generated at the Qi site is reoxidized at the Qo site (see Figure 7.27). Additional protons are transported across the membrane from the matrix (see Figure 7.26 illustrating a similar process for cytochrome b(6)f). The overall reaction can be written... 

In the respiratory chain (see p. 140), electrons are transferred from NADH or ubiquinol (QH2) to O2. The energy obtained in this process is used to establish a proton gradient across the inner mitochondrial membrane. ATP synthesis is ultimately coupled to the return of protons from the intermembrane space into the matrix. 

Endocytosis involves the ceUular uptake of exogenous molecules or complexes inside plasma membrane-derived vesicles. This process can be divided into two major categories (1) adsorptive or phagocytic uptake of particles that have been bound to the membrane surface and (2) fluid or pinocytotic uptake, in which the particle enters the cell as part of the fluid phase. The solute within the vesicle is released intracellularly, possibly through lysosomal digestion of the vesicle membrane or by intermembrane fusion (Fig. 3.4). 

Proton pump Electron transport is coupled to the phosphorylation of ADP by the transport of protons (H+) across the inner mitochon drial membrane from the matrix to the intermembrane space. This process creates across the inner mitochondrial membrane an electrical gradient (with more positive charges on the outside of the membrane than on the inside) and a pH gradient (the outside of the... 

In eukaryotes, most of the reactions of aerobic energy metabolism occur in mitochondria. An inner membrane separates the mitochondrion into two spaces the internal matrix space and the intermembrane space. An electron-transport system in the inner membrane oxidizes NADH and succinate at the expense of 02, generating ATP in the process. The operation of the respiratory chain and its coupling to ATP synthesis can be summarized as follows ... 

To form the relatively undissociated water, 2 protons per electron pair, transported through complex IV, are removed from the mitochondrial matrix. An additional 2 protons per electron pair transported are extruded from the matrix by complex IV. The total number of protons lost by the mitochondrial matrix through the action of complexes I, III, and IV is thus 8-10 per electron pair, depending on the authority cited. The reason protons are extruded across the inner mitochondrial membrane is 2-fold complex IV apparently acts as a true proton pump with specific protein(s) of that complex acting as the transport particle(s). Complexes I and III, on the other hand, are associated with the so-called vectoral proton translocation process those enzymatic reactions that release protons (e.g., reoxidation of UQH2) take place at or near the intermembrane space surface on the inner mitochondrial membrane. This allows protons to be discharged into the intermembrane space rather than into the mitochondrial matrix. Overall, the pH differential between the cytosol and the mitochondrial matrix is about 1, or a 10-fold difference in [H+] (alkaline inside). 

In these mechanisms, electron transport through the various components of the electron-transport chain leads to structural changes in the proteins of the chain, such that changes in their pKa values (Chap. 3) of ionizable amino acid residues occurs. For example, an increase in the pKa of a residue adjacent to the matrix side of the membrane would lead to proton uptake from the matrix, while a decrease in the pKa of a residue adjacent to the intermembranous side of the membrane could lead to release of a proton. The net effect of these processes is the transfer of protons from the matrix to the intermembranous side of the membrane. However, proton-pump mechanisms do not make strong predictions of the H+/e stoichiometries. 

Import into the mitochondrial matrix and inner membrane may follow another route. Import is dependent on a membrane potential [86], probably goes via specific receptors on the outer membrane [76], and is usually accompanied by proteolytic processing of a precursor to the mature form. Fig. 12.5 summarizes the import pathways into the mitochondrial matrix, inner membrane and intermembrane space, exemplified by import of the various subunits of the cytochrome 6c, complex. 

Cytochromes, as components of electron transfer chains, must interact with the other components, accepting electrons from reduced donor molecules and transferring them to appropriate acceptors. In the respiratory chain of the mitochondria, the ubiquinolxytochrome c oxidoreductase, QCR or cytochrome bc complex, transfers electrons coming from Complexes 1 and 11 to cytochrome c. The bc complex oxidises a membrane-localised ubiquinol the redox process is coupled to the translocation of protons across the membrane, in the so-called proton-motive Q cycle, which is presented in a simplified form in Figure 13.14. This cycle was first proposed by Peter Mitchell 30 years ago and substantially confirmed experimentally since then. The Q cycle in fact consists of two turnovers of QH2 (Figure 13.14). In both turnovers, the lipid-soluble ubiquinol (QH2) is oxidized in a two-step reoxidation in which the semiquinone CoQ is a stable intermediate, at the intermembrane face of the mitochondrial inner membrane. It transfers one electron to the Rieske iron—sulfur protein (ISP), one electron to one of the two cytochrome b haems (bi), while two protons are transferred to the intermembrane space. In both of the Q cycles, the cytochrome bi reduces cytochrome bfj while the Reiske iron—sulfur cluster reduces cytochrome c/. The cytochrome ci in turn reduces the water-soluble cytochrome c, which transfers its electrons to the terminal oxidase, cytochrome c oxidase, described above. In one of the two Q cycles, reduced cytochrome bf reduces Q to the semiquinone, which is then reduced to QH2 by the second reduced cytochrome bn- The protons required for this step are derived from the matrix side of the membrane. The overall outcome of the two CoQ cycles (10) (/ — matrix o — intermembrane space) is... 

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