Replaceability of Schiff base proton donors in light-driven proton pump rhodopsins

Many H+-pump rhodopsins conserve "H+ donor" residues in cytoplasmic (CP) half channels to quickly transport H+ from the CP medium to Schiff bases at the center of these proteins. For conventional H+ pumps, the donors are conserved as Asp or Glu but are replaced by Lys in the minority, such as Exi-guobacterium sibiricum rhodopsin (ESR). In dark states, carboxyl donors are protonated, whereas the Lys donor is deprotonated. As a result, carboxyl donors first donate H+ to the Schiff bases and then capture the other H+ from the me-dium, whereas the Lys donor first captures H+ from the me-dium and then donates it to the Schiff base. Thus, carboxyl and Lys-type H+ pumps seem to have different mechanisms, which are probably optimized for their respective H+-transfer re-actions. Here, we examined these differences via replacement of donor residues. For Asp-type deltarhodopsin (DR), the embedded Lys residue distorted the protein conformation and did not act as the H+ donor. In contrast, for Glu-type proteo-rhodopsin (PR) and ESR, the embedded residues functioned well as H+ donors. These differences were further examined by focusing on the activation volumes during the H+-transfer re-actions. The results revealed essential differences between archaeal H+ pump (DR) and eubacterial H+ pumps PR and ESR. Archaeal DR requires significant hydration of the CP channel for the H+-transfer reactions; however, eubacterial PR and ESR require the swing-like motion of the donor residue rather than hydration. Given this common mechanism, donor residues might be replaceable between eubacterial PR and ESR.