pH-dependent Ca2+ binding to the F0 c-subunit affects proton translocation of the ATP synthase from Synechocystis 6803.

It was shown before (Wooten, D. C., and Dilley, R.

Introduction of a carboxyl group in the loop of the F0 c-subunit affects the H+/ATP coupling ratio of the ATP synthase from Synechocystis 6803.

The proton translocation stoichiometry (H+/ATP ratio) was investigated in membrane vesicles from a Synechocystis 6803 mutant in which the serine at position 37 in the hydrophilic loop of the c-subunit from the wild type was replaced by a negatively charged glutamic acid residue (strain plc37). At this position the c-subunit of chloroplasts and the cyanobacterium Synechococcus 6716 already contains glutamic acid. H+/ATP ratios were determined with active ATP synthase in thermodynamic equilibrium between phosphate potential (deltaGp) and the proton gradient (deltamuH+) induced by acid-base transition.

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.

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.

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.

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).