Protonation and deprotonation of the M, N, and O intermediates during the bacteriorhodopsin photocycle.

Transient pH changes were measured with phenol red and chlorophenol red in the 30-microseconds-50-ms time range during the photocycle of bacteriorhodopsin (BR), the light-driven proton pump. At pH greater than or equal to 7, the results confirmed earlier data and suggestions that one proton is released during the L----M reaction, and taken up again during the decay of N. These are likely to be steps in the proton transport process. At pH less than 7, however, the time-resolved pH traces were complex and indicated additional protonation reactions. The data were explained by a model which assumed pH-dependent protonation states for M and N which varied from -1 to 0, and for O which varied from 0 to + 2, relative to BR. If the kinetics of the vectorial proton translocation process were taken as pH independent, this treatment of the data suggested that a residue with a pKa of 5.9 was made protonable in M and N and two residues with pKa's of 6.5 were made cooperatively protonable in O. The additional protons detected are not necessarily in the vectorial proton transfer pathway (i.e., they are probably "Bohr protons"), and while they must reflect conformational and/or neighboring ionization changes in the BR as it passes through the M, N, and O states, their role, if any, in the transport is uncertain.