Thiol modulation of the chloroplast protonmotive ATPase and its effect on photophosphorylation |
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Authors: | John D Mills Peter Mitchell |
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Institution: | Glynn Research Institute, Glynn House, Bodmin, Cornwall, PL30 4AU U.K. |
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Abstract: | Thiol modulation of the chloroplast protonmotive ATPase (CF0-CF1) by preillumination of broken chloroplasts in the presence of dithiothreitol (or preillumination of intact chloroplasts in the absence of added thiols) had the following effects on photophosphorylation. (1) When assayed at pH 8 and saturating light, the initial rate of photophosphorylation was increased by 10–40%. There was an accompanying increase in the rate of coupled electron transport with no significant change in the overall ratio. (2) On lowering the pH of the assay medium to pH 7, the stimulatory effect of thiol modulation on photophosphorylation and coupled electron flow was enhanced. At pH 7, there was also a small increase in ratio. (3) Addition of a non-saturating amount of uncoupler to the assay medium enhanced the stimulatory effect of thiol modulation on photophosphorylation. In the presence of 1 mM NH4Cl, there was only a small increase in coupled electron flow and a correspondingly larger increase in ratio. (4) Lowering the light intensity, or inhibiting electron transport, diminished the stimulatory effect of thiol modulation on photophosphorylation, coupled electron transport and ratio. (5) Under all the above conditions, the ΔpH maintained across the thylakoid membrane was lower after thiol modulation, even when photophosphorylation markedly increased in rate. (6) Thiol modulation of CF0-CF1 increased the observed Michaelis constant for ADP (Km(ADP)) and the apparent maximum rate (Vapp of photophosphorylation by the same factor, so that ratio was not altered. was also unaffected by changing the medium pH, but was significantly decreased upon addition of uncouplers to the medium. These results indicate that the observed rate of ATP synthesis catalysed by thiol demodulated chloroplasts is limited kinetically by the fraction (α) of enzyme molecules that are active during photophosphorylation. A model based on a dual pH optimum requirement for activation of CF0-CF1 is presented to explain the dependence of α on ΔpH. Thiol modulation of CF0-CF1 is proposed to stimulate photophosphorylation by causing the enzyme to become active over a lower range of ΔpH, thereby reducing the kinetic limitation on ATP synthesis imposed by the activation process. |
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Keywords: | Chloroplast ATPase Photophosphorylation Thiol modulation Electron transport ATPase Chl chlorophyll reversible protonmotive ATPase pH values of the external medium intrathylakoid space difference in the electrochemical potential of protons ΔpH between the intrathylakoid and aqueous medium free energy change of ATP synthesis or hydrolysis Tricine Mes 4-morpholineethanesulphonic acid Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid DCMU 3-(3 4-dichlorophenyl)-1 1-dimethylurea |
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