Biophys J, September 1998, p. 1455-1465, Vol. 75, No. 3

Connectivity of the Retinal Schiff Base to Asp⁸⁵ and Asp⁹⁶ during the Bacteriorhodopsin Photocycle: The Local-Access Model

 ^*Department of Physiology and Biophysics, University of California, Irvine, California 92697, and  ^#Department of Biochemistry, Wayne State University, Detroit, Michigan 48201 USA

In the recently proposed local-access model for proton transfers in the bacteriorhodopsin transport cycle (Brown et al. 1998. Biochemistry. 37:3982-3993), connection between the retinal Schiff base and Asp⁸⁵ (in the extracellular direction) and Asp⁹⁶ (in the cytoplasmic direction) is maintained as long as the retinal is in its photoisomerized state. The directionality of the proton translocation is determined by influences in the protein that make Asp⁸⁵ a proton acceptor and, subsequently, Asp⁹⁶ a proton donor. The idea of concurrent local access of the Schiff base in the two directions is now put to a test in the photocycle of the D115N/D96N mutant. The kinetics had suggested that there is a single sequence of intermediates, L  M₁  M₂  N, and the M₂  M₁ reaction depends on whether a proton is released to the extracellular surface. This is now confirmed. We find that at pH 5, where proton release does not occur, but not at higher pH, the photostationary state created by illumination with yellow light contains not only the M₁ and M₂ states, but also the L and the N intermediates. Because the L and M₁ states decay rapidly, they can be present only if they are in equilibrium with later intermediates of the photocycle. Perturbation of this mixture with a blue flash caused depletion of the M intermediate, followed by its partial recovery at the expense of the L state. The changes in the amplitude of the C==O stretch band at 1759 cm¹ demonstrated protonation of Asp⁸⁵ in this process. Thus, during the reequilibration the Schiff base lost its proton to Asp⁸⁵. Because the N state, also present in the mixture, arises by protonation of the Schiff base from the cytoplasmic surface, these results fulfill the expectation that under the conditions tested the extracellular access of the Schiff base would not be lost at the time when there is access in the cytoplasmic direction. Instead, the connectivity of the Schiff base flickers rapidly (with the time constant of the M₁  M₂ equilibration) between the two directions during the entire L-to-N segment of the photocycle.

Biophys J, September 1998, p. 1455-1465, Vol. 75, No. 3
© 1998 by the Biophysical Society   0006-3495/98/09/1455/11  $2.00

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