Proton pumping across membranes

When light-driven proton pumping across the thylakoid membrane occurs, a concomitant efflux of Mg ions from vesicles into the stroma is observed. This efflux of Mg somewhat counteracts the charge accumulation due to H ... 

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. 

In plants, both the water-splitting reaction and electron flow through the cytochrome baf complex are accompanied by proton pumping across the thylakoid membrane. The proton-motive force thus created drives ATP synthesis by a CF0CF complex similar to the mitochondrial F0Fi complex. 

Photoprotonic signals can be generated by light induced proton release, this might happen for the quaternary N-R+ derivatives of 117, for instance [8.233]. The photoproduction of proton gradients across membranes could serve as a light-powered proton pump for inducing vectorial processes such as the transport of protons [8.233, 8.234] or H+-ATPase model reactions [8.274]. 

The crystal structure of bovine cytochrome c oxidase (Complex IV) homodimer has been determined to a resolution of 1.8 A (Shinzawa-Itoh et al., 2007). This integral membrane protein complex composed of thirteen different subunits per monomer is responsible for the reduction of molecular oxygen to water during aerobic respiration, with concomitant proton pumping across the mitochondrial inner membrane. A combination of high resolution X-ray structure analysis of the integral lipids in bovine Complex IV with mass spectroscopy analysis of their chain lengths and the positions of the unsaturated bonds of the... 

Electron transport leads to proton pumping across the inner mitochondrial membrane... 

Reflect and Apply What are some of the difficulties in determining the exact number of protons pumped across the inner mitochondrial membrane by the respiratory complexes ... 

Reflect and Apply It has taken considerable amounts of research to establish the number of protons pumped across the mitochondrial membrane at the various stages of electron transport. Would you expect to encounter difficulties in determining the number of protons pumped in electron transport across the thylakoid membrane Why or why not ... 

Exact values for P/O ratios are difficult to determine because of the complexity of the systems that pump protons and phosphorylate ADP. The number of ADP molecules phosphorylated is direcdy related to the number of protons pumped across the membrane. This figure has been a matter of some controversy. It has been difficult for chemists and biochemists to accept uncertain stoichiometry. 

It would take much work to establish the number of protons pumped across the thylakoid membrane. This is partly the result of experience with mitochondria and partly a prediction based on the greater complexity of structure in the chloroplast. 

Explain the roles of cytochrome Ci and the b cytochromes (bi and bu) in the oxidation of ubiquinol to ubiquinone. Are protons pumped across the inner mitochondrial membrane during these reactions ... 

Two light-activated cyclic electron transfer systems have been reincorporated into lipid vesicles in such a way that proton pumping across the membranes may be observed under appropriate conditions. The first of these has been constructed from mammalian cytochrome bc] complex and reaction centres isolated from Rhodopseudomonas sphaeroides (RCbc vesicles), a combination used previously by Packham et al. (1980) for single turnover studies in solution. In order to maintain adequate multiple turnover electron flux under our conditions, it was necessary to add both cytochrome c and ubiquinone-2. In the presence of valinomycin, light activation caused the translocation of four protons outwards across the vesicles for each pair of electrons completing a cycle, although this ratio appeared to fall to two after a significant ApH had built up. 

ITowever, membrane proteins can also be distributed in nonrandom ways across the surface of a membrane. This can occur for several reasons. Some proteins must interact intimately with certain other proteins, forming multisubunit complexes that perform specific functions in the membrane. A few integral membrane proteins are known to self-associate in the membrane, forming large multimeric clusters. Bacteriorhodopsin, a light-driven proton pump protein, forms such clusters, known as purple patches, in the membranes of Halobacterium halobium (Eigure 9.9). The bacteriorhodopsin protein in these purple patches forms highly ordered, two-dimensional crystals. 

Each of the respiratory chain complexes I, III, and IV (Figures 12-7 and 12-8) acts as a proton pump. The inner membrane is impermeable to ions in general but particularly to protons, which accumulate outside the membrane, creating an electrochemical potential difference across the membrane (A iH )-This consists of a chemical potential (difference in pH) and an electrical potential. 

Complex III (CoQ cytochrome c oxidoreductase) transfers electrons from CoQ to cytochrome c, through a sequence of cytochrome and iron-sulfur cofactors. Here, Alf for the couple CoQ/cytochrome c is 0.19 V, corresponding to a AG° of —36.7 kJ/mol, again enough to power the synthesis of an ATP molecule and to ensure that protons are pumped across the inner mitochondrial membrane. 

We now turn our attention to how the gradient of protons pumped by Complexes I, III and IV across the inner mitochondrial membrane into the intermembrane space, together with the associated membrane potential, is used to turn the molecular rotor that ensures... 

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. 

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