On the molecular mechanisms of the Schiff base deprotonation during the bacteriorhodopsin photocycle.
Using optical flash photolysis and time-resolved Raman methods, we examined intermediates formed during the photocycle of bacteriorhodopsin (bR), as well as the bR color change, as a function of pH (in the 7.0-1.5 region) and as a function of the number of bound Ca(2+) ions. It is found that at a pH just below 3 or with less than two bound Ca(2+) per bR, the deprotonation (the L(550) --> M(412)) step ceases, yet the K(610) and L(550) analogues are still formed as in native bR. The lack of deprotonation in the photocycle of both acid blue and deionized blue bR and the similarity of their Raman spectra as well as of their K(610) and L(550) analogues strongly suggest that both blue samples have nearly the same retinal active site. It is suggested that in both blue species, bound cations are removed via a proton-cation exchange equilibrium, either on the cation exchange column for the deionized sample or in solution for the acid blue sample. The proton-cation exchange equilibrium is found to quantitatively account for the pH dependence of the purple-to-blue color change. The different mechanisms responsible for the large reduction ( approximately 11 units) of the pK(a) value of the protonated Schiff base (PSB) during the photocycle are discussed. The absence of the L(550) --> M(412) deprotonation process in both blue species is discussed in terms of the previously proposed cation model for the deprotonation of the PSB during the photocycle of native bR. The extent of the deprotonation and the blue-to-purple color change are found to follow the same dependence on either the pH or the amount of cations added to deionized blue bR. This observed correlation is briefly discussed.
Department of Chemistry and Biochemistry, University of California, Los Angeles, 405 Hilgard Ave., Los Angeles, CA 90024.
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Chronister
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Corcoran
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Song
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El-Sayed