Figure 4.

Proton transfer processes in ReaChR. Shown is the C=O stretching region of the steady state of the wild type at pH 7.4 in H₂O (gray filled curves) and D₂O (black) and the mutants E130Q (red), E163T (blue), D293N (green), and D196N (purple) at 80 (A) and 293 K (B), respectively. Additionally, the region around the 1753 cm⁻¹ band is shown for measurements at 260 K. Spectra at 80 K represent the D → K transition. At 293 K, the steady states represent a mixture of the succeeding intermediates with a large contribution of O. Band assignment is indicated by colored bands in the upper line: mutational analysis at 80 K indicates an early hydrogen bond change of Glu¹³⁰. Spectra at 293 K show that Glu¹³⁰ deprotonates, the counter-ion complex protonates, and Asp¹⁹⁶ also deprotonates during the photocycle, the latter being supported by spectra at 260 K.