Extended Data Fig. 2: Spectroscopic analysis of sodium binding mode.

a, b, Time-resolved IR absorption changes from KR2 microcrystals at pH 8 (a) and at pH 4 (b) recorded with tunable quantum cascade lasers as described⁵⁴. The maximum concentration of the O intermediate is reached at around 1–20 ms after pulsed excitation at pH 8 and is characterized by specific marker bands that are absent at pH 4, in particular the C=C stretching vibrational band of retinal at 1,516 cm⁻¹ of the O state. The band at 1,688 cm⁻¹ has previously been suggested to originate from the C=O stretching mode as a result of sodium binding to an asparagine residue, presumably N112⁶¹. The band at 1,554 cm⁻¹ is tentatively assigned to the asymmetric stretching vibration of a carboxylate that rises upon binding of a sodium ion in bidendate or pseudo-bridging fashion where one of the carboxylate oxygens is interacting with another partner^62,63. For a detailed analysis of the ligation, the corresponding symmetrical mode needs to be assigned, as the frequency difference between the COO⁻ asymmetric and symmetric stretch is dependent on the mode of sodium ligation^64,65. c, O(like)-KR2 (ground state) difference spectra recorded under different conditions. Spectra have been scaled to the ground-state bleaching band measured between 1,530 and 1,540 cm⁻¹. It is well-established that KR2 operates exclusively as a sodium pump at neutral pH and in the presence of sodium ions. KR2 acts as proton pump in the presence of KCl but has no (known) function at pH 4^1,28. It is evident that the band at 1,688 cm⁻¹ is most pronounced if sodium is pumped, which supports the assignment to the C=O stretching vibration of N112 upon binding of sodium. d, Time traces of the ethylenic stretch of the O state vibrating at 1,518 cm^–1 and the two candidate vibrational bands at 1,420 cm^–1 and 1,392 cm⁻¹ of the symmetric carboxylate stretching vibration. The band at 1,420 cm^–1 exhibits similar kinetic behaviour to the one at 1,518 cm^–1. Hence, the former vibrational band is tentatively assigned as the symmetrical mode that relates to the asymmetrical vibration at 1,554 cm⁻¹ of deprotonated D251 upon ligation of a sodium ion. The difference in frequency between the symmetric and asymmetric modes is with 135 cm⁻¹ at the edge of binding in bidentate to pseudobridging mode of model compounds^62,64,65. This indicates that the two oxygens of D251 are not equidistant from the sodium ion. Such asymmetric ligation is expected in the heterogenous environment of the protein interior as documented by our X-ray structures in the millisecond regime.