Environmental effects on formation and photoreaction of the M412 photoproduct of bacteriorhodopsin: implications for the mechanism of proton pumping.

Photochemical studies of the effects of temperature, pH, and dehydration on the formation and back photoreaction of the M412 intermediate in the photocycle of light-adapted bacteriorhodopsin (bR570) are carried out. Continuous illumination experiments in the range between -40 and -90 degrees C indicate that at low temperatures branching occurs at the stage of the L550 intermediate in which a back reaction to the parent pigment competes with the formation of M412. At low temperatures the yield of M412 is markedly increased at high pH. The effect is attributed to the catalytic action of a protein group of pK congruent to 10 on the rate of the L550 leads to M412 process. Our results, taken together with previous evidence for deprotonation of a tyrosine during the L550 leads to M412 transition, suggest that the formation of a tyrosinate ion is a prerequisite for deprotonation of the Schiff base. A model is proposed in which both the Schiff base and the tyrosine translocate their protons to two acceptor groups, A1 and A2, accessible to the outside of the cell through a segment of a proton wire. The model accounts for the observation that up to two photons may be pumped per cycle. The proton-pump mechanism is analyzed in terms of a generalized kinetic scheme for pumping. In contrast to current models for proton pumping which are based on a (primary) light-induced accessibility change of the chromophore (class I models), we introduce a new class (II) of models based exclusively on pK changes. We suggest that in bR570 the Schiff base and the tyrosine are accessible to protons on the outside surface of the membrane. An analysis of the back photoreaction from M412 tends to favor class II models over previous class I models.