Sonar S, Marti T, Rath P, Fischer W, Coleman M, Nilsson A, Khorana H G, Rothschild K J
Physics Department, Boston University, Massachusetts 02215.
J Biol Chem. 1994 Nov 18;269(46):28851-8.
Light-driven proton pumping in bacteriorhodopsin involves deprotonation of the retinylidene Schiff base during M formation and reprotonation during N formation as key steps. This study reports on the spectroscopic characterization of the bacteriorhodopsin mutant Tyr-57-->Asp (Y57D). The results reveal that although formation of the M intermediate and Schiff base deprotonation is blocked, the mutant still exhibits a significant level of light-driven proton translocation. The photocycle of Y57D involves formation of K and L intermediates accompanied by the normal chromophore isomerization and changes in the hydrogen bonding of Asp-96 and Asp-115. However, an additional Asp residue deprotonates during formation of the L intermediate along with a transmembrane alpha-helical structural change that normally occurs upon N formation. We postulate that proton transport in Y57D occurs through a redirected pathway that does not involve the deprotonation of the Schiff base. Chromophore isomerization, which normally results in the transfer of a proton from the Schiff base to Asp-85, instead causes the deprotonation of Asp-57 in Y57D, most likely through an interaction involving Asp-212. This deprotonation of Asp-57 causes the release of a proton into the extracellular medium. Reprotonation of Asp-57 occurs through the Schiff base reprotonation pathway, which consists of a hydrogen-bonded network of residues spanning from Asp-96 to Asp-212. The results also indicate that the transmembrane alpha-helical structural changes observed during N formation (Rothschild, K.J., Marti, T., Sonar, S., He, Y.W., Rath, P., Fischer, W., Bousche, O., and Khorana, H. G. (1993) J. Biol. Chem. 268, 27046-27052) do not require deprotonation of Asp-96 or of the Schiff base.
细菌视紫红质中光驱动的质子泵浦涉及在M态形成过程中视黄醛席夫碱的去质子化以及在N态形成过程中的再质子化，这是关键步骤。本研究报道了细菌视紫红质突变体Tyr-57→Asp（Y57D）的光谱特征。结果表明，虽然M中间体的形成和席夫碱的去质子化受阻，但该突变体仍表现出显著水平的光驱动质子转运。Y57D的光循环涉及K和L中间体的形成，伴随着正常的发色团异构化以及Asp-96和Asp-115氢键的变化。然而，在L中间体形成过程中，另一个Asp残基去质子化，同时发生了通常在N态形成时出现的跨膜α-螺旋结构变化。我们推测Y57D中的质子转运通过一条不涉及席夫碱去质子化的重定向途径发生。发色团异构化通常会导致质子从席夫碱转移到Asp-85，但在Y57D中，最有可能通过涉及Asp-212的相互作用，导致Asp-57去质子化。Asp-57的这种去质子化导致质子释放到细胞外介质中。Asp-57的再质子化通过席夫碱再质子化途径发生，该途径由从Asp-96到Asp-212的氢键残基网络组成。结果还表明，在N态形成过程中观察到的跨膜α-螺旋结构变化（Rothschild, K.J., Marti, T., Sonar, S., He, Y.W., Rath, P., Fischer, W., Bousche, O., and Khorana, H. G. (1993) J. Biol. Chem. 268, 27046 - 27052）不需要Asp-96或席夫碱去质子化。
