Pathways of proton release in the bacteriorhodopsin photocycle.
Pathways of proton release in the bacteriorhodopsin photocycle.
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DOI：
10.1021/bi00151a022
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发表时间：
1992-09
期刊：
影响因子：
2.9
通讯作者：
L. Zimányi;G. Varo;Man Chang;B. Ni;R. Needleman;J. Lanyi
中科院分区：
文献类型：
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作者：
L. Zimányi;G. Varo;Man Chang;B. Ni;R. Needleman;J. Lanyi
重组D96 N和D115 N/D96 N细菌视紫红质的光循环的速率常数的pH依赖性，确定从70 ns和420 ms之间的时间分辨的差异光谱光激发后。结果与先前针对含有D96 N取代的蛋白质提出的模型一致：BR hv-K-L-M1-M2-BR。只有M2----M1的反反应是pH依赖性的：其速率随着[H+]的增加而增加，pH 5和8之间。我们得出结论，从定量分析的pH值依赖性，其逆转，M1----M2反应，是链接到一个质子从一个组的pKa = 5.8的释放。这表明了野生型细菌视紫红质的一个模型，其中在pH大于5.8时，在100微秒的时间尺度上，在细胞外侧从这个未知的基团中释放出转运的质子，但在pH小于5.8时，质子从另一个残基中释放出来，随后在光循环中最有可能直接从D85在O-BR反应中释放出来。我们假设，另一方面，在细胞质侧的质子吸收将由D96和在N-O反应过程中，无论pH值。质子动力学与指示剂染料测定证实了这个模型的独特预测：在pH值大于6，质子释放质子吸收之前，但在pH值小于6，释放被延迟，直到后的摄取。结果进一步表明，整个M1-M2反应包括第二个动力学步骤，除了质子释放，这可能是早期假设的细胞外到细胞质的重定向开关的质子泵。
The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.