Chloroplast membrane carriers

Photosynthesis occurs in the plant cell organelle called the chloroplast. During the process of photosynthesis, electrons are transferred from H20 to NADP+ via an electron carrier system. The energy released by electron transport is converted into the form of a proton gradient and coupled to ADP phosphorylation. In this experiment a method is introduced to demonstrate the formation of the proton gradient across the chloroplast membranes. 

Electron transport through oxidases in the plasma membrane contributes to, or controls, part of the proton release from the cell. The details of oxidase function and the mechanism of control remain to be elucidated. The NADPH oxidase of neutrophils is a special case in which proton transport is coupled to the cytochrome >557 electron carrier. This type of proton transport has its precedents in the well-characterized proton pumping through electron carriers in mitochondrial and chloroplast membranes and prokaryotic plasma membranes. 

Chloroplast respiration is a novel form of respiration that has only recently been discovered. Chloroplast membranes exhibit a nonphosphorylation ETS that consumes oxygen (48). Ferredoxin and reduced nicotinamide adenine dinucleotide phosphate (NADPH) serve as electron carriers and glyceraldehyde-3-phosphate serves as the electron donor. This system probably originated in the respiratory ETS of the chloroplast s free-living ancestors, the cyanobacteria (49). 

Fig. 11. Various hypotheses proposed by which higher plants may attain high levels of unsaturated fatty acids in their chloroplast membrane galactolipids. (a) Phosphatidylcholine acts as a carrier molecule involved in the desaturation, (b) Desaturation of fatty acids occurs after formation of the galactolipid molecule, (c) Desaturation occurs before formation of the galactolipid molecule. In the first hypothesis, all the desaturases involved are confined in the chloroplast in the second hypothesis, the conversion of 18 1 to 18 2 is maximal in microsomes," whereas desaturation of 18 2 to 18 3 is highest in chloroplast membranes, (d) Deacylation-reacylation mechanism in which X can be a CoA-thioester, a polar lipid, etc. D, Desaturases T, acyl-ACP thioesterase e.r., endoplasmic reticulum.

In plants, the photosynthesis reaction takes place in specialized organelles termed chloroplasts. The chloroplasts are bounded in a two-membrane envelope with an additional third internal membrane called thylakoid membrane. This thylakoid membrane is a highly folded structure, which encloses a distinct compartment called thylakoid lumen. The chlorophyll found in chloroplasts is bound to the protein in the thylakoid membrane. The major photosensitive molecules in plants are the chlorophylls chlorophyll a and chlorophyll b. They are coupled through electron transfer chains to other molecules that act as electron carriers. Structures of chlorophyll a, chlorophyll b, and pheophytin a are shown in Figure 7.9. 

Let us start our examination with the prototypical blue protein plastocyanin, found in the thylacoid membrane of chloroplasts, where it acts as an electron carrier in photosynthesis (see Figure 1). As Figure 30 illustrates, the active site of plastocyanin is formed of a Cu(II) ion (pseudo)tetrahedrally coordinated to two histidine nitrogen atoms and... 

In addition to NAD and flavoproteins, three other types of electron-carrying molecules function in the respiratory chain a hydrophobic quinone (ubiquinone) and two different types of iron-containing proteins (cytochromes and iron-sulfur proteins). Ubiquinone (also called coenzyme Q, or simply Q) is a lipid-soluble ben-zoquinone with a long isoprenoid side chain (Fig. 19-2). The closely related compounds plastoquinone (of plant chloroplasts) and menaquinone (of bacteria) play roles analogous to that of ubiquinone, carrying electrons in membrane-associated electron-transfer chains. Ubiquinone can accept one electron to become the semi-quinone radical ( QH) or two electrons to form ubiquinol (QH2) (Fig. 19-2) and, like flavoprotein carriers, it can act at the junction between a two-electron donor and a one-electron acceptor. Because ubiquinone is both small and hydrophobic, it is freely diffusible within the lipid bilayer of the inner mitochondrial membrane and can shuttle reducing equivalents between other, less mobile electron carriers in the membrane. And because it carries both electrons and protons, it plays a central role in coupling electron flow to proton movement. 

Photophosphoiylation in the chloroplasts of green plants and in cyanobacteria involves electron flow through a series of membrane-bound carriers. 

Like Complex III of mitochondria, cytochrome b6f conveys electrons from a reduced quinone—a mobile, lipid-soluble carrier of two electrons (Q in mitochondria, PQb in chloroplasts)—to a water-soluble protein that carries one electron (cytochrome c in mitochondria, plastocyanin in chloroplasts). As in mitochondria, the function of this complex involves a Q cycle (Fig. 19-12) in which electrons pass, one at a time, from PQBH2 to cytochrome bs. This cycle results in the pumping of protons across the membrane in chloroplasts, the direction of proton movement is from the stromal compartment to the thylakoid lumen, up to four protons moving for each pair of electrons. The result is production of a proton gradient across the thylakoid membrane as electrons pass from PSII to PSI. Because the volume of the flattened thylakoid lumen is small, the influx of a small number of protons has a relatively large effect on lumenal pH. The measured difference in pH between the stroma (pH 8) and the thylakoid lumen (pH 5) represents a 1,000-fold difference in proton concentration—a powerful driving force for ATP synthesis. 

A crucially important finding is that submitochon-drial particles or vesicles from broken chloroplasts will synthesize ATP from ADP and P , when an artificial pH gradient is imposed.172186 Isolated purified FjF0 ATPase from a thermophilic Bacillus has been coreconstituted into liposomes with the light-driven proton pump bacteiiorhodopsin (Chapter 23). Illumination induced ATP synthesis.187 These observations support Mitchell s proposal that the ATP synthase is both spatially separate from the electron carriers in the membrane and utilizes the protonmotive force to make ATP. Thus, the passage of protons from the outside of the mitochondria back in through the ATP synthase induces the formation of ATP. What is the stoichiometry of this process ... 

It is very difficult to measure the flux of protons across the membrane either out of the mitochondria into the cytoplasm or from the cytoplasm through the ATP synthase into the mitochondria. Therefore, estimates of the stoichiometry have often been indirect. One argument is based on thermodynamics. If Ap attains values no more negative than -160 mV and Rp within mitochondria reaches at least 104 M 1, we must couple AGh of -15.4 kj/ mol to AG of formation of ATP of +57.3 kj/ mol. To do this four H+ must be translocated per ATP formed. Recent experimental measurements with chloroplast ATP synthase188 also favor four H+. It is often proposed that one of these protons is used to pump ADP into the mitochondria via the ATP-ADP exchange carrier (Section D). Furthermore, if Rp reaches 106 M 1 in the cytoplasm, it must exceed 104 M 1 in the mitochondrial matrix. 

ATP synthesis in chloroplasts. The flow of electrons between PSII and PSI (Fig. 23-18) is of great importance for ATP formation. As previously mentioned, plastocyanin is usually the immediate donor to P700 and serves as a mobile carrier to bring electrons to this reaction center. In this function it is analogous to cytochrome c of mitochondrial membranes. The essentiality of plastocyanin was shown by study of copper-deficient Scenedesmus (Fig. 1-11). The photoreduction of C02 by H2 is impaired in these cells, but the Hill reaction occurs at a normal rate. 

The photosynthetic process in green plants occurs in subcellular organelles called chloroplasts. These organelles resemble mitochondria they have two outer membranes and a folded inner membrane called the thy-lakoid. The apparatus for photosynthesis, including the chlorophyll reaction centers and electron carriers, is in the thylakoid membrane. The chemical reactions of the Calvin cycle take place in the stroma, the region around the thylakoid membrane. 

The plastocyanins are found in plant chloroplasts and other photosynthetic organisms, and act as membrane-bound electron carriers between photosystems II and I in the photosynthetic pathway of higher plants, green algae and some blue-green algae. 

Figure 4. Scheme for proton transfer by plastoquinone as a mobile carrier in membrane lipid. Electrons are transferred one by one to a bound plastoquinone A (PQA) which in turn reduces external plastoquinone. When reduced, the anionic plastoquinone takes up protons to become a hydroquinone which is oxidized by the cytochrome bb f complex on the inside of the membrane to release protons. A second quinone, vitamin K, (KQ) is also involved in chloroplast electron transport, but its role in proton movement is not known. 

Chloroplasts are enclosed by two membranes. The outer membrane is freely permeable to small molecules (up to about 10 kDa) due to the presence of a porin and the inner membrane is the osmotic barrier and the site where specific transport occurs. The specificity of envelope permeability is strikingly highlighted by the contrast between Pi and PP the former being among the most rapidly translocated molecules and the latter among those to which the envelope is relatively impermeable. Carrier-mediated anion transport can be classified as ... 

Most of the components involved in electron transport in mitochondria are contained in four supramolecular protein complexes that traverse the inner mitochondrial membrane. Complex I, which contains FMN and various iron-sulfur clusters as active sites, transfers electrons from NADH to ubiquinone (Fig. 6-8). Complex II, which contains FAD, various iron-sulfur clusters, and a Cyt >, transfers electrons from succinate also to a ubiquinone. Ubiquinone functions as a pool of two-electron carriers, analogous to the function of plastoquinone A in the lamellar membranes of chloroplasts, which accepts electrons from Complexes I and II and delivers them to the... 

The photosynthetic apparatus of green plants and cyanobacteria oxidizes water and transfers electrons to NADP, with a net gain in electrochemical potential of 1.13 eV (at pH 7), utilizing the energy of two light quanta per electron. The complete system is contained in the chloroplasts, and is localized within the thylakoid membranes, with the exception of the electron carrier ferredoxin, which is in solution in the stroma, and serves to transfer electrons from the reducing end of photosystem I (PS I) to a membrane-bound flavoprotein which then reduces NADP, and of the copper protein plastocyanin (PC, the electron donor to PS I), which is in solution in the internal phase of thylakoids. 

If the model proposed by Andersson and Anderson [109] of total separation of PS I and PS II in the granal chloroplasts were to be accepted, electron transport from the PS II acceptors to P-700 would require a mobile electron carrier(s) which should diffuse laterally in the membrane fast enough to account for the observed electron transport rate. Plastoquinone [112] and plastocyanin are the candidates of choice for this role. The former has been shown to be present at approximately the same activity in the partitions and in the stroma-exposed membranes [43], while PC is known to be located in the intrathylakoid space [113],... 

The chloroplast is encircled by a double membrane called the envelope. Of the two membranes, the inner is practically impermeable to hydrophylic compounds, such as Pj, phosphate esters, dicarboxylates, glucose and sucrose. Transport of certain of these metabolites is accomplished by carrier proteins, specific for groups of compounds. Individual carriers have been shown to facilitate the transport of Pj and phosphate esters, dicarboxylates, ATP and ADP, and glucose. >... 

The carrier protein facilitating Pj and phosphate ester transport is of particular interest in leaves in connection with carbon processing - i.e., the synthesis, transport and degradation of carbohydrate, all of which occur in the cytosol [51]. This metabolite carrier, called the phosphate translocator, is a polypeptide with a molecular mass of 29 kDa and is a major component of the inner envelope membrane [52,53]. The phosphate translocator mediates the counter-transport of 3-PGA, DHAP and Pj. The rate of Pj transport alone is three orders of magnitude lower than with simultaneous DHAP or 3-PGA counter-transport [54]. Consequently operation of the phosphate translocator keeps the total amount of esterified phosphate and Pj constant inside the chloroplast. Significantly, the carrier is specific for the divalent anion of phosphate. 

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