Asp drives the protonation state of the glucose/H symporter.

The glucose/H symporter (GlcP) is a membrane transporter highly specific for glucose and a homolog of the human glucose transporters (GLUT, SLC2 family). Most GLUTs and their bacterial counterparts differ in the transport mechanism, adopting uniport and sugar/H symport, respectively. Unlike other bacterial GLUT homologs (for example, XylE), GlcP has a loose H/sugar coupling. Asp is part of the proton-binding site of GlcP and crucial for the glucose/H co-transport mechanism. To determine how pH variations affect the proton site and the transporter, we performed surface-enhanced IR absorption spectroscopy on the immobilized GlcP We found that Asp has a p of 8.5 ± 0.1, a value consistent with that determined previously for glucose transport, confirming the central role of this residue for the transport mechanism of GlcP A neutral replacement of the negatively charged Asp led to positive charge displacements over the entire pH range, suggesting that the polarity change of the WT reflects the protonation state of Asp We expected that the substitution of the residue Ile for a serine, located within hydrogen-bonding distance to Asp, would change the microenvironment, but the p of Asp corresponded to that of the WT. A167E mutation, selected in analogy to the XylE, introduced an additional protonatable site and perturbed the protonation state of Asp, with the latter now exhibiting a p of 6.4. These studies confirm that Asp is the proton-binding residue in GlcP and show that charged residues in its vicinity affect the p of glucose/H symport.