Low-temperature FTIR spectroscopy provides evidence for protein-bound water molecules in eubacterial light-driven ion pumps.

Light-driven H+, Na+ and Cl- pumps have been found in eubacteria, which convert light energy into a transmembrane electrochemical potential. A recent mutation study revealed asymmetric functional conversion between the two pumps, where successful functional conversions are achieved exclusively when mutagenesis reverses the evolutionary amino acid sequence changes. Although this fact suggests that the essential structural mechanism of an ancestral function is retained even after gaining a new function, questions regarding the essential structural mechanism remain unanswered. Light-induced difference FTIR spectroscopy was used to monitor the presence of strongly hydrogen-bonded water molecules for all eubacterial H+, Na+ and Cl- pumps, including a functionally converted mutant. This fact suggests that the strongly hydrogen-bonded water molecules are maintained for these new functions during evolution, which could be the reason for successful functional conversion from Na+ to H+, and from Cl- to H+ pumps. This also explains the successful conversion of the Cl- to the H+ pump only for eubacteria, but not for archaea. It is concluded that water-containing hydrogen-bonding networks constitute one of the essential structural mechanisms in eubacterial light-driven ion pumps.

A natural light-driven inward proton pump.

Light-driven outward H+ pumps are widely distributed in nature, converting sunlight energy into proton motive force. Here we report the characterization of an oppositely directed H+ pump with a similar architecture to outward pumps. A deep-ocean marine bacterium, Parvularcula oceani, contains three rhodopsins, one of which functions as a light-driven inward H+ pump when expressed in Escherichia coli and mouse neural cells. Detailed mechanistic analyses of the purified proteins reveal that small differences in the interactions established at the active centre determine the direction of primary H+ transfer. Outward H+ pumps establish strong electrostatic interactions between the primary H+ donor and the extracellular acceptor. In the inward H+ pump these electrostatic interactions are weaker, inducing a more relaxed chromophore structure that leads to the long-distance transfer of H+ to the cytoplasmic side. These results demonstrate an elaborate molecular design to control the direction of H+ transfers in proteins.

Asymmetric Functional Conversion of Eubacterial Light-driven Ion Pumps.

In addition to the well-known light-driven outward proton pumps, novel ion-pumping rhodopsins functioning as outward Na(+) and inward Cl(-) pumps have been recently found in eubacteria. They convert light energy into transmembrane electrochemical potential difference, similar to the prototypical archaeal H(+) pump bacteriorhodopsin (BR) and Cl(-) pump halorhodopsin (HR). The H(+), Na(+), and Cl(-) pumps possess the conserved respective DTE, NDQ, and NTQ motifs in the helix C, which likely serve as their functional determinants. To verify this hypothesis, we attempted functional interconversion between selected pumps from each category by mutagenesis. Introduction of the proton-pumping motif resulted in successful Na(+) → H(+) functional conversion. Introduction of the respective characteristic motifs with several additional mutations leads to successful Na(+) → Cl(-) and Cl(-) → H(+) functional conversions, whereas remaining conversions (H(+) → Na(+), H(+) → Cl(-), Cl(-) → Na(+)) were unsuccessful when mutagenesis of 4-6 residues was used. Phylogenetic analysis suggests that a H(+) pump is the common ancestor of all of these rhodopsins, from which Cl(-) pumps emerged followed by Na(+) pumps. We propose that successful functional conversions of these ion pumps are achieved exclusively when mutagenesis reverses the evolutionary amino acid sequence changes. Dependence of the observed functional conversions on the direction of evolution strongly suggests that the essential structural mechanism of an ancestral function is retained even after the gain of a new function during natural evolution, which can be evoked by a few mutations. By contrast, the gain of a new function needs accumulation of multiple mutations, which may not be easily reproduced by limited mutagenesis in vitro.