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 Exiguobacterium 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 medium, whereas the Lys donor first captures H⁺ from the medium 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 reactions. 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 proteorhodopsin (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 reactions. 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.