Thylakoid spaces

FIGURE 22.27 Light-induced pH changes in chloroplast compartments. Illumination of chloroplasts leads to proton pumping and pH changes in the chloroplast, such that the pH within the thylakoid space falls and the pH of the stroma rises. These pH changes modulate the activity of key Calvin cycle enzymes. 

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. 

The simplest ATP-driven reaction, ATP-driven proton uptake, has already been discussed. After activation the membrane-bound ATP synthase pumps protons into the inner thylakoid space, coupled to ATP hydrolysis. Both 4pH and Aip are produced with magnitudes similar to those produced by light-driven proton transport [51.77]. 

In 1966, Andre Jagendorf showed that chloroplasts synthesize ATP in the dark when an artificial pH gradient is imposed across the thylakoid membrane. To create this transient pH gradient, he soaked chloroplasts in a pH 4 buffer for several hours and then rapidly mixed them with a pH 8 buffer containing ADP and Pj. The pH of the stroma suddenly increased to 8, whereas the pH of the thylakoid space remained at 4. A burst of ATP synthesis then accompanied the disappearance of the pH gradient across the thylakoid membrane (Figure 19.24). This incisive experiment was one of the first to unequivocally support the hypothesis put forth by Peter Mitchell that ATP synthesis is driven by proton-motive force. 

A more direct confirmation of the inside-out character of the B3 vesicles was provided by freeze-fracture electron microscopy. Representative replicas ofthe T2 and B3 vesicles are shown in Fig. 19 (D). As expected for the B3 inside-out vesicles, large distinct particles appear in the EF faces next to the intra-thylakoid space [Fig. 19 (D), left], while closely packed small particles appear in the PF faces next to the stroma [Fig. 19 (D), right]. A large number of vesicular faces were examined and the results revealed that on average the B3 fraction contained 74% inside-out and 26% rightside-out vesicles while the T2 fraction contained 89% rightside-out and only 11% inside-out vesicles. 

In chloroplasts, the magnitude of Apn+ is due to the pH gradient. The reason for this is that the thyla-koid membrane is quite permeable to other ions such as CF and [see Fig. 8 (A)]. Consequently, light-induced transfer of H into the thylakoid space is readily accompanied by the transfer of CFions in the same direction and Mg ions in the opposite direction. As a result, electrical neutrality is very nearly maintained, virtually no membrane potential is generated, and the contribution by the AT term is negligible. 

The synthesis of each ATP molecule is believed to require pumping approximately 3 protons across the membrane into the thylakoid space. The ATP synthase is found in thylakoid membrane that is directly in contact with the stroma. 

As stated earlier, the rate-limiting step in the Calvin cycle is the carboxy-lation of ribulose 1,5-bisphosphate to form two molecules of 3-phospho-glycerate. The activity of rubisco increases markedly on illumination. The addition of CO2 to lysine 201 of rubisco to form the carbamate is essential for Mg coordination and, hence, catalytic activity (Section 20.1.1). Carbamate formation is favored by alkaline pH and high concentrations of Mg ion in the stroma, both of which are consequences of the light-driven pumping of protons from the stroma into the thylakoid space. Magnesium ion concentration rises because Mg ions from the thylakoid space are released into the stroma to compensate for the influx of protons. 

Describe the structure of the chloroplast. Locate the outer, inner, and thylakoid membranes the thylakoid space the granum and the stroma. Associate these structures with the functions they perform. 

When isolated thylakoid membranes were suspended at pH 7 6 and illuminated in presence of an e"-acceptor, protons were pumped across the thylakoid membrane and the H -concentration in the inner-thylakoid space, [H j ] increased. When [H j ] exceeded a critical threshold, the yield of variable fluorescence, 5p decreased (Fig.la)... 

Willey et al. (1) have monitored carboxypeptidase digestion of cytochrome f (Cyt f) in intact thylakoid membranes. They have presented a model of the orientation of Cyt f in the thylakoid membrane where residues 251-270 form a transmembrane alpha-helix, residues 271-285 are in the stroma and the remainder of the molecule resides in the intra-thylakoid space. It has been postulated that the solubility of purified Cyt f, from members of the Cruciferae, is due to the loss of the transmembrane alpha-helix during purification. Turnip Cyt f is soluble and, upon electrophoresis in the presence of SDS (SDS-PAGE), is resolved into diffuse double bands with Mp of 28 and 32 Kd. The predicted Mp of sequenced cytochromes f is 31 Kd and a loss of 4 Kd from tne C-terminus would include all of the presumed membrane spanning alpha-helix. The remaining Cyt f would be more negative by 2 units. 

In the leaf cells of C3 plants there are at least five main compartments separated by membranes through which protons cannot freely pass. These are the thylakoid space and the stroma of the chloroplasts, the stroma of mitochondria and peroxisomes and the cytosol. As a result, achieving the correct pH in the separate compartments must be exerted metabolically, by the diffusion of gases such as carbon dioxide or by special pumps transporting species such as the bicarbonate ion. In the steady state, it is necessary that biochemical reactions producing alkalinity or acidity be compensated by some means, if pH is not to change. 

PSIIq to a low-efficient (low-fluorescent) quenching state, PSIIg (which operates as a non-photochemical trap), depending on the energetic balance of the leaf, and that this conversion is mediated by acidification of the inner thylakoid space PSIIq + H <—>... 

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