Formation of a Proton-Conducting Hydrogen-Bond Network during the L/M Transition of <i>Ns</i> XeR Uncovered by Light-Induced FTIR Spectroscopy

XeR) exhibits particularly high activity, yet the determinants of efficient inward pumping remain unclear. We used temperature-dependent light-induced FTIR difference spectroscopy to probe L and M formation beyond the K intermediate at 77 K. In L, the Schiff-base N-D stretching modes shift markedly to lower frequency, indicating a strongly hydrogen-bonded Schiff base, while an intense water O-D band emerges, consistent with assembly of a multiwater network. The carboxylic C═O stretch of Asp220 shows intermediate-specific changes that distinguish L from M and implicate Asp220 in handling protons on the cytoplasmic side. Together, the spectra support a model in which a water-mediated hydrogen-bond network organized during L-to-M formation promotes rapid proton transfer in the cytoplasmic side, consistent with a Grotthuss-type mechanism.