BACTERIORHODOPSIN MUTANTS D85N, D85T AND D85,96N AS PROTON PUMPS

Proton translocation in the BR mutants D85N, D85T and D85,96N was stud ied by attachment of purple membranes to planar lipid bilayers. Pump c urrents in these mutants were measured via capacitive coupling and by use of the appropriate ionophores. All mutants have a reduced pK of th eir Schiff bases around 8-8.5 in common. At physiological pH, a mixtur e of chromophores absorbing at 410 nm (deprotonated form) and around 6 00 nm (protonated form) coexists. Excitation with continuous blue ligh t induces in all three mutants an outwardly directed stationary pump c urrent. 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 D8 5T. Yellow light alone induces transient inwardly directed currents in the mutants but additional blue light leads to a stationary current w ith the same direction. All the observed currents are carried by proto ns, so that the consecutive absorption of a yellow and a blue photon l eads 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 isomerizati on of the retinal and the different proton accessibility of the Schiff base from the extracellular or the cytoplasmic side of the membrane.