Experimental evidence for secondary protein-chromophore interactions at the Schiff base linkage in bacteriorhodopsin:...

Resonance Raman spectroscopy of the retinylidene chromophore in various isotopically labeled membrane environments together with spectra of isotopically labeled model compounds demonstrates that a secondary protein interaction is present at the protonated Schiff base linkage in bacteriorhodopsin. The data indicate that although the interaction is present in all protonated bacteriorhodopsin species it is absent in unprotonated intermediates. Furthermore, kinetic resonance Raman spectroscopy has been used to monitor the dynamics of Schiff base deprotonation as a function of pH. All our results are consistent with lysine as the interacting group. A structure for the interaction is proposed in which the interacting protein group in an unprotonated configuration is complexed through the Schiff base proton to the Schiff base nitrogen. These data suggest a molecular mechanism for proton pumping and ion gate molecular regulation. In this mechanism, light causes electron redistribution in the retinylidene chromophore, which results in the deprotonation of an amino acid side chain with pK >10.2 +/- 0.3 (e.g., arginine). This induces subsequent retinal and protein conformational transitions which eventually lower the pK of the Schiff base complex from >12 before light absorption to 10.2 +/- 0.3 in microseconds after photon absorption. Finally, in this low pK state the complex can reprotonate the proton-deficient high pK group generated by light, and the complex is then reprotonated from the opposite side of the membrane.