Vectorial transport by pumps requires a switch in the accessibility (or affinity) of the ion binding site from the extracelullar to the cytoplamic side, or vice versa. In the proton-pump bacteriorhodopsin (bR) the nature of this switch mechanism is still controversial, although it is expected to occur during the transition between two M substates. Here, we characterized this transition by time-resolved Fourier transform infrared (FT-IR) spectroscopy under functional conditions, using a novel approach for the analysis of kinetic data: the regularized inversion of an eigenvalue problem. The use of I R spectroscopy allowed the simultaneous evaluation of the involvement of the protein backbone, retinal, amino acid side chains, and internal water molecules in the switch mechanism. We provide solid evidence that the switch is not associated with protein backbone conformational changes. On the other hand, changes in the retinal conformation (or in the orientation of the Schiff Base (SB) during the switch) are reasonably although not completely discarded. We found that the proton release group (PRG), a delocalized proton characterized by a broad continuum band in the infrared, deprotonates in the transition between two M substates. Vectorial proton transport is most likely guaranteed by the coupled proton affinity changes resulting from the PRG deprotonation, favoring an affinity-based over an accessibility-based switch mechanism.