Bacteriorhodopsin mutants D85N, D85T and D85,96N as proton pumps.

Proton translocation in the BR mutants D85N, D85T and D85,96N was studied by attachment of purple membranes to planar lipid bilayers. Pump currents in these mutants were measured via capacitive coupling and by use of the appropriate ionophores. All mutants have a reduced pK of their Schiff bases around 8-8.5 in common. At physiological pH, a mixture of chromophores absorbing at 410 nm (deprotonated form) and around 600 nm (protonated form) coexists. Excitation with continuous blue light induces in all three mutants an outwardly directed stationary pump current. These currents are enhanced upon addition of azide in D85N and D85,96N by a factor of 50, but no azide enhancement is observed in D85T. Yellow light alone induces transient inwardly directed currents in the mutants but additional blue light leads to a stationary current with the same direction. All the observed currents are carried by protons, so that the consecutive absorption of a yellow and a blue photon leads to inverted stationary photocurrents by the mutants, as observed with halorhodopsin (HR). A mechanistic model describing the inversion of proton pumping is discussed by the cis-trans, trans-cis isomerization of the retinal and the different proton accessibility of the Schiff base from the extracellular or the cytoplasmic side of the membrane.