Elucidation of the mechanism of ion transport by bacteriorhodopsin and halorhodopsin by electrical measuements

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In the first year, I constructed an experimental system which enabled to measure the fast photoelectric response of the membrane fractions containing bacteriorhodopsin (bR) or halorhodopsin (hR) by adsorbing them onto a thin polymer film, In the second year, I examined the photocurrent of bR and its mutant, two kinds of hRs from different halophytic bacteria as a function of pH or Cl-concentrations. The result suggested that the pH dependency of the photoelectric current by bR reflected the affinities of the entrance and exit of the proton pathway of bR for protons. similar results were obtained for hRs and it was suggested that the way of ion translocation in the pump proteins were similar to th at of a multiion channel. By using a pulsed laser flash, I could measure the charge movement within the pump protein in a single turnover. The charge movement in bR occurred in the musec and msec time region whereas the charge movement in two hRs occurred in the msec and sub-msec time region. By comparing the data obtained in the electrical measurements with the data obtained in the photochemical cycle, it was suggested that the charge movement occurs during the formation and decay of the N intermediate.On the other hand, I carried out a theoretical model study based on the properties of proton translocation by bR The model assumes three ion binding sites and the affinity change induce the uni-directional ion translocation. Furthermore, the distribution of the high affinity state and low affinity state must deviate from equilibrium distribution in order to induce active transport. I propose this is the critical difference between ion pumps and ion channels. The experimental results obtained in the second year fairly matches this idea. Thus it seems reasonable to assume that an ion pump is a multiion channel in which the affinity change for transported ion induces active transport.