Previous mutagenesis studies with bacteriorhodopsin have shown that reprotonation of the Schiff's base is the rate-limiting step in the photocycle of the D96N mutant, whereas retinal re-isomerization and return of the protein to the initial state constitute the rate-limiting events in the photocycle of the L93A mutant. Thus, in the D96N mutant, decay of the M intermediate is slowed down by more than 100-fold at pH 7. In the L93A mutant, decay of the O intermediate is slowed down by 250-fold. We report here that in the L93A, D96N double mutant, decay of the M intermediate, as well as the formation and decay of the O intermediate, are slowed down dramatically. The photocycle is completed by the decay of a long-lived O intermediate, as in the L93A mutant. The decay of the M and O intermediates in the double mutant parallels the behavior seen in the single mutants over a wide temperature and pH range, arguing that the observed independence is an intrinsic property of the mutant. The slow decay of the M and O intermediates can be selectively and independently reversed under conditions identical to those used for the corresponding intermediates in the D96N and L93A single mutants. Because the effects of the two individual mutations are preserved in the double mutant and can be independently reversed, we conclude that residues Asp 96 and Leu 93 act independently and at different stages of the bacteriorhodopsin photocycle. These results also show that formation of the O intermediate only requires protonation of the Schiff's base and is independent of the protonation of Asp 96 from the aqueous medium.

译文

先前使用细菌视紫红质的诱变研究表明,席夫氏碱的质子化是D96N突变体光循环中的限速步骤,而视网膜重新异构化和蛋白质返回至初始状态构成了光循环中的限速事件L93A突变体的序列。因此,在D96N突变体中,pH值为7时,M中间体的衰减被减慢了100倍以上。在L93A突变体中,O中间体的衰减被减慢了250倍。我们在此报告,在L93A,D96N双重突变体中,M中间产物的衰减以及O中间产物的形成和衰减都大大减慢了。像L93A突变体一样,光循环是通过长寿命的O中间体的衰变完成的。双重突变体中M和O中间体的衰变与单个突变体在较宽的温度和pH范围内观察到的行为相似,认为观察到的独立性是突变体的固有特性。 M和O中间体的缓慢衰变可以在与D96N和L93A单突变体中相应中间体相同的条件下有选择地独立逆转。因为两个单独的突变的影响保留在双突变体中,并且可以独立逆转,所以我们得出结论,残基Asp 96和Leu 93分别在细菌视紫红质光循环的不同阶段起作用。这些结果还表明,O中间体的形成仅需要席夫碱的质子化,而与来自水性介质的Asp 96的质子化无关。

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