The ATP synthase gamma subunit provides the primary site of activation of the chloroplast enzyme: experiments with a chloroplast-like Synechocystis 6803 mutant.

The activation characteristics of the F1Fo-ATP synthase (where F1 and Fo are the hydrophilic and membrane-bound parts respectively of the enzyme) from Synechocystis 6803 wild-type and a Synechocystis 6803 mutant with a chloroplast-like insertion in the gamma subunit have been studied. Activation of the ATP synthase in wild-type and mutant membrane vesicles was performed by acid-base transition-induced generation of a proton motive force (Delta mu H+). Since the mutant containing the regulatory segment of the chloroplast gamma subunit showed thiol-modulation (typical of the chloroplast enzyme), this segment is indeed involved in the regulation of enzyme activation. It is shown that the ATP synthase from Synechocystis 6803 wild type corresponds functionally to the reduced form of the chloroplast ATP synthase, in view of the low Delta mu H+ required for activation of the enzyme and the high stability of the active state. Both the cyanobacterial wild-type and mutant ATP synthases can be activated by methanol, which apparently does not require the presence of the gamma subunit regulatory segment.

Membrane vesicles fromSynechocystis 6803 showing proton and electron transport and high ATP synthase activities.

A simple procedure for the preparation of well-coupled and stable membrane vesicles from the transformable cyanobacteriumSynechocystis 6803 is described with the primary aim of producing vesicles suitable for the study of photosynthetic electron transport and phosphorylation. Spheroplasts were obtained from the cyanobacterium by lysozyme treatment and stored untill prior to measurement, thylakoid vesicles were obtained by osmotic shock. These vesicles showed very high and stable ATP synthesis rates either driven by light or by acid-base transition, and also performed light-induced ATP hydrolysis and linear electron transport. Formation of a proton gradient is studied by aminoacridines.

Modulation of the proton-translocation stoichiometry of H(+)-ATP synthases in two phototrophic prokaryotes by external pH.

The stoichiometry between proton translocation and ATP synthesis/hydrolysis was studied in two different photosynthetic prokaryotes, the thermophilic cyanobacterium Synechococcus 6716 and the purple bacterium Rhodospirillum rubrum. The H+/ATP ratio was determined by acid-base transitions as a function of the external pH. The H+/ATP ratio of the Synechococcus 6716 ATP synthase was found to increase with increasing pH. In contrast, in R. rubrum this ratio decreased with increasing pH. These results were qualitatively supported by experiments using the fluorescence probe 9-aminoacridine. The degree of coupling between the H+ flux and the ATP synthesis/hydrolysis reaction is apparently modulated by the conditions under which the proton pump has to work. Such modulation of the H+/ATP ratio may be of physiological significance for an organism, for example when ATP synthesis is necessary at low proton-electrochemical potential difference (delta mu H+ levels). The different pH dependencies of the H+/ATP ratios in these organisms are considered in relation to the differences in the charged amino acids that are present in the F0 subunits a and c.

On the activation mechanism of the H(+)-ATP synthase and unusual thermodynamic properties in the alkalophilic cyanobacterium Spirulina platensis.

The activation requirements and thermodynamic characteristics of ATP synthase from the alkalophilic cyanobacterium Spirulina platensis were studied in coupled membrane vesicles. Activation by methanol increased the Vmax, while the Km for MgATP was unaffected (0.7 mM). We propose that in Sp. platensis, as in chloroplasts, the activating effect of methanol is based on perturbation of the gamma-epsilon subunit interaction. Light-driven ATP synthesis by membrane vesicles of Sp. platensis was stimulated by dithiothreitol. The characteristics of the activation of the ATP synthase by the proton electrochemical potential difference (delta mu H+) were analyzed on the basis of the uncoupled rates of ATP hydrolysis as a function of a previously applied proton gradient. Two values of delta mu H+, at which 50% of the enzyme is active, were found; 13-14 kJ.mol-1 for untreated membrane vesicles, and 4-8 kJ.mol-1 for light-treated and dithiothreitol-treated membrane vesicles. These values are lower than the corresponding values for the oxidized and reduced forms, respectively, of the chloroplast enzyme. Although no bulk proton gradient could be observed, membrane vesicles of Sp. platensis were able to maintain an equilibrium phosphate potential (delta Gp) of 40-43.5 kJ.mol-1, comparable to values found for Synechococcus 6716 and Anabaena 7120 membrane vesicles. Acid/base-transition experiments showed that the thermodynamic threshold, delta mu H+, for ATP synthesis, catalyzed by light-treated and dithiothreitol-treated Spirulina membrane vesicles, was less than 5 kJ.mol-1. The activation characteristics and the low thermodynamic threshold allow ATP synthesis to occur at low delta mu H+ values. The findings are discussed, both with respect to differences and similarities with the enzymes from chloroplasts and other cyanobacteria, and with respect to the alkalophilic properties of Sp. platensis.