The binding site of the strongly bound Eu3+ in Eu(3+)-regenerated bacteriorhodopsin

A Scatchard plot for the strongly bound Eu3+ to deionized bacteriorhodopsin (bR) was made using a method based on measuring the concentration of unbound Eu3+ from its fluorescence intensity. The results suggest that the first mole of Eu3+ added to a mole of bR is strongly bound by displacing 2-3 protons. In order to reconcile this result with the previous time-resolved fluorescence studies on Eu(3+)-regenerated bR, which showed the presence of 3 sites of comparable binding constants, one is forced to conclude that the emission from the strongly bound Eu3+ is completely quenched, e.g. by energy transfer to the retinal. For this to take place, the Eu3+ must be within a few A from the retinal, i.e. within the retinal pocket (the active site). The possible importance of this conclusion to the deprotonation mechanism of the protonated Schiff base, the switch of the proton pump in bR, is discussed.