Evidence for the involvement of more than one metal cation in the Schiff base deprotonation process during the photocycle of bacteriorhodopsin

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The removal of metal cations inhibits the deprotonation process of the protonated Schiff base during the photocycle of bacteriorhodopsin. To understand the nature of the involvement of these cations, a spectroscopic and kinetic study was carried out on bacteriorhodopsin samples in which the native Ca2+ and Mg2+ were replaced by Eu3+, a luminescent cation. The decay of Eu3+ emission in bacteriorhodopsin can be fitted to a minimum of three decay components, which are assigned to Eu3+ emission from three different sites. This is supported by the response of the decay components to the presence of 2H2O and to the changes in the Eu3+/bR molar ratio. The number of water molecules coordinated to Eu3+ in each site is determined from the change in its emission lifetime when 2H2O replaces H2O. Most of the emission originates from two “wet” sites of low crystal-field symmetry—e.g., surface sites. Protonated Schiff base deprotonation has no discernable effect on the emission decay of protein-bound Eu3+, suggesting an indirect involvement of metal cations in the deprotonation process. Adding Eu3+ to deionized bacteriorhodopsin increases the emission intensity of each Eu3+ site linearly, but the extent of the deprotonation (and color) changes sigmoidally. This suggests that if only the emitting Eu3+ ions cause the deprotonation and bacteriorhodopsin color change, ions in more than one site must be involved—e.g., by inducing protein conformation changes. The latter could allow deprotonation by the interaction between the protonated Schiff base and a positive field of cations either on the surface or within the protein.

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