Observation of Calcium-dependent Unidirectional Rotational Motion in Recombinant Photosynthetic F1-ATPase Molecules

Ward C Tucker,Alon Schwarz,Tiferet Levine,Ziyun Du,Zippora Gromet-Elhanan,Mark L Richter,Gilad Haran

doi:10.1074/jbc.c400269200

Abstract

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 gamma-rotation assay, that CaATP is capable of sustaining rotational motion in a highly active hybrid photosynthetic F(1)-ATPase consisting of alpha and beta subunits from Rhodospirillum rubrum and gamma subunit from spinach chloroplasts (alpha(R)(3)beta(R)(3)gamma(C)). The rotation was found to be similar to that induced by MgATP in Escherichia coli F(1)-ATPase molecules. Our results suggest a possible long range pathway that enables the bound CaATP to induce full rotational motion of gamma but might block transmission of this rotational motion into proton translocation by the F(0) part of the ATP synthase.

Highlights

From the ‡Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045 and the Departments of ʈChemical Physics and §Biological Chemistry, Weizmann Institute of Science, Rehovot 71600, Israel
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
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 [11,12,13]

Summary

Accelerated Publication

A second unique feature of ATP synthases of both chloroplasts [16] and chromatophores [17] is their high sensitivity to inhibition by excess free Mg2ϩ ions, which results in a drastic reduction of their MgATPase activities at a Mg/ATP ratio exceeding 0.5 Another very interesting phenomenon, found in both chloroplasts [18] and chromatophores [17], is the decoupling of Ca2ϩ-induced ATP hydrolysis from proton translocation, their membranes do not become leaky to protons. Calcium-dependent Rotation in Photosynthetic F1-ATPase in our laboratory [19], shows rotational motion in the presence of CaATP This result indicates that the decoupling effect of calcium, which does not interrupt the connection between ATP hydrolysis and ␥C rotation, might be mediated by a long range effect on the membrane bound F0 subunits

EXPERIMENTAL PROCEDURES

TABLE I

RESULTS AND DISCUSSION