LOCAL-ACCESS MODEL FOR PROTON-TRANSFER IN BACTERIORHODOPSIN

The accessibility of the retinal Schiff base in bacteriorhodopsin was studied in the D85N/D96N mutant where the proton acceptor and donor ar e absent. Protonation and deprotonation of the Schiff base after pH ju mp without illumination and in the photocycle of the unprotonated Schi ff base were measured in the visible and the infrared. Whether access is extracellular (EC) or cytoplasmic (CP) was decided from the effect of millimolar concentrations of azide on the rates of proton transfers . The results, together with earlier work on the wild-type protein, su ggest a new hypothesis for the proton-transfer switch: (i) In the meta stable 13-cis,15-anti and all-trans,15-syn photoproducts, but not in t he stable isomeric states, access flickers between the EC and CP direc tions. (ii) The direction of proton transfer is decided both by this l ocal access and by the presence of a suitable donor or acceptor group (in the wild type), or the proton conductivity in the EC and CP half-c hannels (in D85N/D96N). (iii) Thermal reisomerization of the retinal c an occur only when the Schiff base is protonated, as is well-known. In the wild-type transport cycle, the concurrent local EC and CP access during the lifetime of the metastable 13-cis,15-anti state enables the changing pK(a)'s of the proton acceptor and donor to determine the di rection of proton transfer. Proton transfer from the Schiff base to As p-85 in the EC direction is followed by reprotonation by Asp-96 from t he CP direction because proton release to the EC surface raises the pK (a) of Asp-85 and a large-scale protein conformation change lowers the pK(a) of Asp-96. The unexpected finding we report here for D85N/D96N, that when the retinal is in the stable all-trans,15-anti and 13-cis,1 5-syn isomeric forms access of the Schiff base is locked (in the EC an d CP directions, respectively), suggests that in this protein reisomer ization, rather than changes in the proton conductivities of the EC an d CP half-channels, provides the switch function. With this mechanism, the various modes of transport reported for Asp-85 mutants (CP to EC direction with blue Light, and EC to CP direction with blue plus green light) are understood also in terms of rules i-iii.