Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer

Homodimeric H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H+-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H+ translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PPi binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H+-PPase are 49.3 +/- 4.0 and 67.2 +/- 5.7 angstrom, respectively. Furthermore, putative PPi binding motifs on individual subunits are found to be relatively far away from each other (70.8 +/- 4.8 angstrom), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H+-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H+-PPase upon substrate binding.