Conformational Changes of H+ ATP Synthase Upon ATP Hydrolysis Detected by Fluorescence Correlation Spectroscopy

Abstract

H+ -ATPases catalyze the synthesis of ATP from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. The enzyme consists of two parts: the hydrophobic, membrane integrated F0-part is involved in proton transport and contains the subunits a, b and c with a likely stoichiometry ab2c12 for the Escherichia coli enzyme. The nucleotide binding sites are located in the hydrophilic F1-part with subunit composition α3β3γδε [1]. During ATP hydrolysis intersubunit rotation of the γ-subunit in the F1-part (which was dissociated from the holoenzyme) was observed with single fluorescence labeled enzymes in realtime using enhanced videomicroscopy [2 - 4]. ATP synthesis and hydrolysis occur at the catalytic binding sites of the β-subunits. In the crystal structure of the F1-ATPase three different conformations of the three β-subunits were detected [5]. From these structural data a detailed mechanistic model of the F1-ATPase as a ‘stepped rotatory motor protein’ was developed [6]. According to this model, large conformational changes in the lower part of the β-subunits are expected during catalysis. We investigated the diffusion of the F1-part before and after binding of ATP with fluorescence correlation spectroscopy (FCS). The β-subunit was specifically labeled with a newly synthesized sulfonyl fluoride derivative of Sulforhodamine G. The labeled amino acid is not known yet. Preliminary FCS measurements with EF1 are shown (Fig.1.). Upon ATP binding the translational diffusion time is decreased by about 15 percent due to changes in size and shape of the enzymes during the catalytic cycle. Similar results were obtained for EF1-ATPase with a fluorescence label on the γ-subunit [7]. These FCS measurements are supported by new electron microscopy data, which show a significant shrinking up of F1-part of the enzyme upon binding of the non-hydrolysable ATP derivative AMPPNP. The diameter of the F1-part decreased mainly in the upper half of the F1-part upon binding of AMPPNP [8].