VIBRATIONAL SPECTROSCOPY OF BACTERIORHODOPSIN MUTANTS .1. TYROSINE-185 PROTONATES AND DEPROTONATES DURING THE PHOTOCYCLE
VIBRATIONAL SPECTROSCOPY OF BACTERIORHODOPSIN MUTANTS .1. TYROSINE-185 PROTONATES AND DEPROTONATES DURING THE PHOTOCYCLE
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DOI：
10.1002/prot.340030403
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发表时间：
1988-01-01
影响因子：
2.9
通讯作者：
ROTHSCHILD, KJ
中科院分区：
文献类型：
--
作者：
BRAIMAN, MS;MOGI, T;ROTHSCHILD, KJ
结合FTIR差分光谱技术和定点诱变技术，研究了酪氨酸侧链在细菌视紫红质（bR）质子泵送机制中的作用。对于含有单个Tyr.fwdarw的11个可能的bR突变体中的每一个。通过苯置换，得到了bR.fwdarw的差谱。K和bR。光反应。只有Tyr-185。Phe突变导致一组条带的消失，这些条带先前被证明是由于bR .fwdarw期间酪氨酸的质子化。K光反应[Rothschild et al.: Proceedings of National Academy of Sciences of United States of America .][1986]。tyr185 .fwdarw。Phe突变还消除了bR .fwdarw中的一组条带。与Tyr去质子化有关的M差谱这些波段中的大多数（例如，正1272厘米-1峰）完全不受其他十个波段的影响。板式换热器突变。因此，酪氨酸-185在核聚变过程中获得一个质子。K反应并在M形成时再次失去它。我们的FTIR光谱也提供了证据，表明Tyr-185与视网膜发色团的质子化希夫碱键相互作用，因为负的C = NH+拉伸带从野生型的1640 cm-1移动到Tyr-185 .fwdarw的1636 cm-1。板式换热器突变。一个与这些结果一致的模型是，tyr185通常是电离的，并作为质子化的希夫碱的反离子。发色团的初级光异构化使希夫碱基从tyr1 -185转移，从而提高后者的pKa并导致其质子化。
The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr.fwdarw. Phe substituion, difference spectra have been obtained for the bR.fwdarw. K and bR .fwdarw. M photoreactions. Only the Tyr-185 .fwdarw. Phe mutation results in the disappearance of a set of bands that were previously shown to be due to the protonation of a tyrosinate during the bR .fwdarw. K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United States of America 83:347, (1986)]. The Tyr-185 .fwdarw. Phe mutation also eliminates a set of bands in the bR .fwdarw. M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr .fwdarw. Phe mutations. Thus, tyrosinate-185 gains a proton during the bR .fwdarw. K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C = NH+ stretch band shift sfrom 1640 cm-1 in the wild tyupe to 1636 cm-1 in the Tyr-185 .fwdarw. Phe mutant. A model that is consistent with these results in that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.