Redox potentials of the oriented film of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins

The redox potentials of the oriented films of the wild-type, the E194Q-, E204Q- and D96N-mutated bacteriorhodopsins (bR), prepared by adsorbing purple membrane (PM) sheets or its mutant on a Pt electrode, have been examined. The redox potentials (V) of the wild-type bR were −470 mV for the 13-cis configuration of the retinal Shiff base in bR and −757 mV for the all-trans configuration in H₂O, and −433 mV for the 13-cis configuration and −742 mV for the all-trans configuration in D₂O. The solvent isotope effect (ΔV=V(D₂O)−V(H₂O)), which shifts the redox potential to a higher value, originates from the cooperative rearrangements of the extensively hydrogen-bonded water molecules around the protonated CN part in the retinal Schiff base. The redox potential of bR was much higher for the 13-cis configuration than that for the all-trans configuration. The redox potentials for the E194Q mutant in the extracellular region were −507 mV for the 13-cis configuration and −788 mV for the all-trans configuration; and for the E204Q mutant they were −491 mV for the 13-cis configuration and −769 mV for the all-trans configuration. Replacement of the Glu¹⁹⁴ or Glu²⁰⁴ residues by Gln weakened the electron withdrawing interaction to the protonated CN bond in the retinal Schiff base. The E204 residue is less linked with the hydrogen-bonded network of the proton release pathway compared with E194. The redox potentials of the D96N mutant in the cytoplasmic region were −471 mV for the 13-cis configuration and −760 mV for the all-trans configuration which were virtually the same as those of the wild-type bR, indicating that the D to N point mutation of the 96 residue had no influence on the interaction between the D96 residue and the CN part in the Schiff base under the light-adapted condition. The results suggest that the redox potential of bR is closely correlated to the hydrogen-bonded network spanning from the retinal Schiff base to the extracellular surface of bR in the proton transfer pathway.