Electron transport pump

The cytochrome bf complex is a proton pump and, during electron transport, pumps H+ ions from the stroma into the thylakoid space, creating an H+ gradient. H+ ions are also released into the thylakoid space when photosystem II oxidizes water to produce oxygen whilst the H+ ions used to reduce NADP+ to NADPH are taken up from the stroma. Both effects contribute to the H+ gradient. 

Oxidative phosphorylation occurs in the mitochondria of all animal and plant tissues, and is a coupled process between the oxidation of substrates and production of ATP. As the TCA cycle runs, hydrogen ions (or electrons) are carried by the two carrier molecules NAD or FAD to the electron transport pumps. Energy released by the electron transfer processes pumps the protons to the intermembrane region, where they accumulate in a high enough concentration to phosphorylate the ADP to ATP. The overall process is called oxidative phosphorylation. The cristae have the major coupling factors F, (a hydrophilic protein) and F0 (a hydrophobic lipoprotein complex). F, and F0 together comprise the ATPase (also called ATP synthase) complex activated by Mg2+. F0 forms a proton translocation pathway and Fj... 

Figure 13.11 The chemiosmotic hypothesis. Electron transport pumps protons (H ) out of the matrix. The formation of ATP accompanies the flow of protons through Fi-ATPase back into the matrix.

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... 

Photosynthetic electron transport, which pumps into the thylakoid lumen, can occur in two modes, both of which lead to the establishment of a transmembrane proton-motive force. Thus, both modes are coupled to ATP synthesis and are considered alternative mechanisms of photophosphorylation even though they are distinguished by differences in their electron transfer pathways. The two modes are cyclic and noncyclic photophosphorylation. 

The Stoichiometry of Proton Pumping by the Mitochondrial Electron-Transport Chain 129... 

Complex III 280 kDa 11 28 type hemes (b and bg) bound to same mitochondrially coded peptide 1 C heme (cytochrome c,) 1 Fe-S center Rieske factor Spans membrane, cytochrome b, and b in membrane, cytochrome c, and Fe-S center on outer face 0.25-0.53 Pumps protons out of matrix during electron transport/2e"... 

Complex IV 200 kDa (probably as a dimer) 13 2 A type hemes (a, aj) 2 or 3 Cu Spans inner membrane, cytochrome c site on outer face 0.16-1.00 Pumps protons out of matrix during electron transport/4e"... 

Figure 6. Pathways of protons and electrons during mitochondrial oxidations. The diagrams show the pathways of electrons which enter the electron chain at the level ofcomplexi (a)orcomplex II (b). Complexes I, III, and IV usethefreeenergy of electron transport to pump protons out of the matrix. This diagram also distinguishes formally between protons released by dehydrogenation and those which are pumped out of the matrix, although they all enter or leave the same pool.

Complexes III and IV have Fe-porphyrin prosthetic groups (hemes), complex IV also contains copper atoms which are involved in electron transport. Complexes I, III, and IV use the energy of electron transport to pump protons out of the matrix so as to maintain a pH gradient and an electrical potential difference across the inner membrane required for ATP synthesis (see below and Appendix 3). It is important to remember that all dehydrogenations of metabolic substrates remove two protons as well as two electrons and that a corresponding number of protons are consumed in the final reduction of dioxygen (Figures 5, 6). 

The AG of the redox span of segments of the electron-transport chain concerned with proton-pumping (Appendix 2) which is given by AG° = iFAE° in the standard state, or by... 

Although only two protons are pumped out of the matrix, two others from the matrix are consumed in the formation of H2O. There is therefore a net translocation of four positive charges out of the matrix which is equivalent to the extrusion of four protons. If four protons are required by the chemiosmotic mechanism to convert cytosolic ADP + Pj to ATP, then 0.5 mol ATP is made for the oxidation of one mol of ubiquinol and one mol ATP for the oxidation of 2 mols of reduced cytochrome c. These stoichiometries were obtained experimentally when ubiquinol was oxidized when complexes I, II, and IV were inhibited by rotenone, malonate, and cyanide, respectively, and when reduced cytochrome c was oxidized with complex III inhibited by antimycin (Hinkle et al., 1991). (In these experiments, of course, no protons were liberated in the matrix by substrate oxidation.) However, in the scheme illustrated in Figure 6, with the flow of two electrons through the complete electron transport chain from substrate to oxygen, it also appears valid to say that four protons are extmded by complex I, four by complex III, and two by complex 1. 

Boundary membranes play a key role in the cells of all contemporary organisms, and simple models of membrane function are therefore of considerable interest. The interface of two immiscible liquids has been widely used for this purpose. For example, the fundamental processes of photosynthesis, biocatalysis, membrane fusion and interactions of cells, ion pumping, and electron transport have all been investigated in such interfacial systems. 

P. Mitchell (Nobel Prize for Chemistry, 1978) explained these facts by his chemiosmotic theory. This theory is based on the ordering of successive oxidation processes into reaction sequences called loops. Each loop consists of two basic processes, one of which is oriented in the direction away from the matrix surface of the internal membrane into the intracristal space and connected with the transfer of electrons together with protons. The second process is oriented in the opposite direction and is connected with the transfer of electrons alone. Figure 6.27 depicts the first Mitchell loop, whose first step involves reduction of NAD+ (the oxidized form of nicotinamide adenosine dinucleotide) by the carbonaceous substrate, SH2. In this process, two electrons and two protons are transferred from the matrix space. The protons are accumulated in the intracristal space, while electrons are transferred in the opposite direction by the reduction of the oxidized form of the Fe-S protein. This reduces a further component of the electron transport chain on the matrix side of the membrane and the process is repeated. The final process is the reduction of molecular oxygen with the reduced form of cytochrome oxidase. It would appear that this reaction sequence includes not only loops but also a proton pump, i.e. an enzymatic system that can employ the energy of the redox step in the electron transfer chain for translocation of protons from the matrix space into the intracristal space. 

To explain how H+ transfer occurred across the membrane Mitchell suggested the protons were translocated by redox loops with different reducing equivalents in their two arms. The first loop would be associated with flavoprotein/non-heme iron interaction and the second, more controversially, with CoQ. Redox loops required an ordered arrangement of the components of the electron transport system across the inner mitochondrial membrane, which was substantiated from immunochemical studies with submitochondrial particles. Cytochrome c, for example, was located at the intermembranal face of the inner membrane and cytochrome oxidase was transmembranal. The alternative to redox loops, proton pumping, is now known to be a property of cytochrome oxidase. 

Along with electronic transport improvements must come attention to substrate transport in such porous structures. As discussed above, introduction of gas-phase diffusion or liquid-phase convection of reactants is a feasible approach to enabling high-current-density operation in electrodes of thicknesses exceeding 100 jxm. Such a solution is application specific, in the sense that neither gas-phase reactants nor convection can be introduced in a subclass of applications, such as devices implanted in human, animal, or plant tissue. In the context of physiologically implanted devices, the choice becomes either milliwatt to watt scale devices implanted in a blood vessel, where velocities of up to 10 cm/s can be present, or microwatt-scale devices implanted in tissue. Ex vivo applications are more flexible, partially because gas-phase oxygen from ambient air will almost always be utilized on the cathode side, but also because pumps can be used to provide convective flow of any substrate. However, power requirements for pump operation must be minimized to prevent substantial lowering of net power output. 

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. 

Proton transport via complexes I, III, and IV takes place vectorially from the matrix into the intermembrane space. When electrons are being transported through the respiratory chain, the concentration in this space increases—i. e., the pH value there is reduced by about one pH unit. For each H2O molecule formed, around 10 H ions are pumped into the intermembrane space. If the inner membrane is intact, then generally only ATP synthase (see p. 142) can allow protons to flow back into the matrix. This is the basis for the coupling of electron transport to ATP synthesis, which is important for regulation purposes (see p. 144). 

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