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The nuclear atpC1 gene encoding the ␥ subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis in Arabidopsis thaliana. In the seedling-lethal dpa1 (deficiency of plastid ATP synthase 1) mutant, the absence of detectable amounts of the ␥ subunit destabilizes the entire ATP synthase complex. The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. However, in vivo protein labeling analysis suggests that assembly of the ATP synthase ␣ and ␤ subunits into the thylakoid membrane still occurs in dpa1. As a consequence of the destabilized ATP synthase complex, photophosphorylation is abolished even under reducing conditions. Further effects of the mutation include an increased light sensitivity of the plant and an altered photosystem II activity. At low light intensity, chlorophyll fluorescence induction kinetics is close to those found in wild type, but non-photochemical quenching strongly increases with increasing actinic light intensity resulting in steady state fluorescence levels of about 60% of the minimal dark fluorescence. Most fluorescence quenching relaxed within 3 min after dark incubation. Spectroscopic and biochemical studies have shown that a high proton gradient is responsible for most quenching. Thylakoids of illuminated dpa1 plants were swollen due to an increased proton accumulation in the lumen. Expression profiling of 3292 nuclear genes encoding mainly chloroplast proteins demonstrates that most organelle functions are down-regulated. On the contrary, the mRNA expression of some photosynthesis genes is significantly up-regulated, probably to compensate for the defect in dpa1.

The H ϩ -translocating ATPase of chloroplasts (CF 0 CF 1 , 1 chloroplast ATP synthase) is a latent enzyme. Its physiological activation requires a transmembrane electrochemical proton potential difference (1-4). Hence, the proton gradient in addition to its role as the driving force of phosphorylation is a factor that controls CF 0 CF 1 activity. The obvious physiological meaning of this control mechanism is the suppression of unproductive ATP hydrolysis under conditions that would energetically allow this reaction, i.e. at low proton gradients (low light or dark) and high phosphate potentials.A superimposed regulatory device is the so-called thiol modulation of CF 0 CF 1 . The structural basis for thiol modulation is a sequence motif of nine amino acids comprising two cysteines in the ␥ subunit of CF 1 (5). This segment is present in higher plants (6) and green algae (7) but not in cyanobacteria (8 -10) or in diatoms (11) suggesting that thiol modulation is an acquisition of the chlorophyll a ϩ b plants only. In the demodulated (oxidized) state the two cysteines form a disulfide bond whereas the modulated state is obtained by reduction of this disulfide bridge. In vitro reduction can be achieved by dithiothreitol or other dithiols, but the natural reductant is a reduced thioredoxin. In chloroplasts at least two different thioredoxins occur, thioredoxin-m (Tr-m) and thioredoxin-f (Tr-f) (12). The former is thought to be involved in light/dark regulation of the chloroplast NADP-specific malate dehydrogenase, and the latter is responsible for the light/dark regulation of fructose bisphosphatase and other Calvin cycle enzymes (13,14). The thioredoxins are reduced via ferredoxin and ferredoxin-thioredoxin reductase (15) by electrons from the photosynthetic electron transport chain. In most of the experiments carried out so far, however, thiol modulation of CF 0 CF 1 was conducted with the artificial reductant dithiothreitol, and in a few studies Escherichia coli thioredoxin (Trx) was used (16, 17). Little information is known about the action of the naturally occurring chloroplast thioredoxins on CF 0 CF 1 (18 -20).Thiol modulation requires illumination of the chloroplasts to allow reduction of the disulfide bridge. Apparently, the regulatory segment of the ␥ subunit, which is hidden in the dark, becomes accessible as a consequence of ⌬H ϩ -induced CF 0 CF 1 activation (16,21). Decay of the proton gradient in the dark leads to deactivation of the ATP synthase. The most significant difference between the reduced and oxidized active states concerns the velocity of deactivation. While the oxidized form is immediately deactivated upon relaxation of the gradient, deactivation of thiolmodulated CF 0 CF 1 takes several minutes. For this reason only chloroplasts with thiol-modulated CF 0 CF 1 are capable of hydrolyzing added ATP after transition from light to dark (22,23).Deactivation of the modulated enzyme may proceed with or without reoxidation of the dithiol group (16). Most likely the natural actual oxidant is the oxid...

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In this paper the authors emphasise that the proton translocating ATP synthase from thiol-modulated chloroplasts and two cyanobaeterial strains has a coupling ratio of 4 protons per ATP synthesised or hydrolysed. This ratio is determined by several thermodynamic studies at equilibrium between phosphate potential (AGp) and proton gradient (zl/iu+), and is confirmed by measurement of proton flux during ATP hydrolysis. Ratios lower than 4 H+IATP that have been published in the past have predominantly been determined with the oxidised chloroplast enzyme. Errors in these measurements will be discussed.