Dynamics of pH-induced spectral changes in bacteriorhodopsin

The kinetics of the spectral shift induced in bacteriorhodopsin by low pH are investigated by using the rapid-mixing, stopped-flow technique. The generation of the acid form of the chromophore (A605) occurs in two distinct steps: a fast process (t1/2I = 21 +/- 4 ms) is followed by a much slower reaction (t1/2II = 6 +/- 2 s). The observations are interpreted in terms of neutralization of an acid group in the neighborhood of the retinyl chromophore, the double-staged kinetics being attributed to cooperative effects between chromophores. The method may serve as a tool for studying the kinetics of proton migration across the purple membrane.