ATP hydrolysis and synthesis by the F 0 F 1 -ATP synthase are coupled to proton translocation across the membrane in the presence of magnesium. Calcium is known, however, to disrupt this coupling in the photosynthetic enzyme in a unique way: it does not support ATP synthesis, and CaATP hydrolysis is decoupled from any proton translocation, but the membrane does not become leaky to protons. Understanding the molecular basis of these calcium-dependent effects can shed light on the as yet unclear mechanism of coupling between proton transport and rotational catalysis. We show here, using an actin filament ␥-rotation assay, that CaATP is capable of sustaining rotational motion in a highly active hybrid photosynthetic F 1 -ATPase consisting of ␣ and  subunits from Rhodospirillum rubrum and ␥ subunit from spinach chloroplasts (␣ Recent experiments have provided strong evidence for the rotational catalysis mechanism proposed for the ubiquitous enzyme F 0 F 1 -ATPase/ATP synthase (1, 2). Particularly striking is the effort led by several laboratories to measure the rotational motion of the F 1 part of the enzyme on the singlemolecule level. F 1 is constructed of five subunits, with a stoichiometry of ␣ 3  3 ␥␦⑀. Fluorescent actin filaments attached to the ␥ subunit of engineered surface-immobilized ␣ 3  3 ␥ subcomplexes of the enzyme, either from a thermophyilic bacterium, TF 1 1 (3), or from Escherichia coli, EF 1 (4, 5), were shown to rotate unidirectionally while hydrolyzing MgATP. This rotation was not only dependent on ATP concentration but was shown to have almost 100% efficiency of chemical to mechanical energy conversion. At very low ATP concentration the stepwise motion of the enzyme was exposed (6). Small beads attached to ␥ in place of the actin filament allowed Yasuda et al.(7) to follow the motion at the limit of small load and watch rotation at a rate as high as 130 revolutions per second. Most spectacularly, forced clockwise rotation was shown very recently to lead to ATP synthesis, thus confirming the essential role of ␥ subunit rotation in both directions of the reaction (8). Some single-molecule work also addressed the operation of the F 0 part of the F 0 F 1 -ATP synthase. It was shown that the F 0 -c subunit oligomer, which consists of 10 -14 identical c subunits, is capable of rotating together with ␥ (9, 10).While much new information has been collected regarding rotational catalysis in the ATP synthase, there are still many open questions, mainly in relation to the coupling between enzymatic activity and rotational motion. The photosynthetic version of the ATP synthase is an attractive system to probe some of these questions, since it presents several distinct functional properties. These include the tight regulation of ATP hydrolysis, which is especially important in photosynthetic cells, where it prevents the depletion of essential ATP pools in the dark (11-13). Indeed, plant chloroplasts have a unique regulatory system, termed thiol modulation, which leads to reduction of a disulfide bond fo...
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