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 (PP_i), 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 Å, respectively. Furthermore, putative PP_i binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), 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.