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 enzyme supplies energy at 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 PP_i 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 Å 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), while binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significantly distance variations between two Acidic I motifs and two H622 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.

Huang, Y.T., Liu, T.H., Chen, Y.W., Lee, C.H., Chen, H.H., Huang, T.W., Hsu, S.H., Lin, S.M., Pan, Y.J., Lee, C.H., Hsu, Ian C., Tseng, F.G., Fu, C.C., and Pan, R.L. (2010) Distance varations between active sites of H⁺-pyrophosphatase determined by fluorescence resonance energy transfer. J Bio Chem (Accepted)