The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv→ K ↔ L ↔ M1 ↔ M2 BR. Only the M2→M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1 → M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH >5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-µs time scale, but at pH <5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O → BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N → O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH >6, proton release preceded proton uptake, but at pH <6, the release was delayed until after the uptake. The results indicated further that the overall M1 → M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.
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