REPLACEMENT OF ASPARTIC RESIDUE-85, RESIDUE-96, RESIDUE-115, OR RESIDUE-212 AFFECTS THE QUANTUM YIELD AND KINETICS OF PROTON RELEASE AND UPTAKE BY BACTERIORHODOPSIN
REPLACEMENT OF ASPARTIC RESIDUE-85, RESIDUE-96, RESIDUE-115, OR RESIDUE-212 AFFECTS THE QUANTUM YIELD AND KINETICS OF PROTON RELEASE AND UPTAKE BY BACTERIORHODOPSIN
复制标题
DOI：
10.1073/pnas.86.2.529
复制
发表时间：
1989-01-01
影响因子：
11.1
通讯作者：
KHORANA, HG
中科院分区：
文献类型：
--
作者：
MARINETTI, T;SUBRAMANIAM, S;KHORANA, HG
最近，一些细菌视紫红质的天冬氨酸突变体被证明在稳态质子运输中存在缺陷。在这里，我们报告了这些突变体的光诱导质子释放和摄取的时间分辨测量。通过测量细菌视紫红质胶束溶液的体积电导率变化，直接监测蛋白质和水相之间的质子转移。对于Asp-90.fwdarw。在突变体中，质子摄取减慢了1个数量级，但对释放步长没有明显影响。asp - 85. fwdarw。Asn、H+的吸收动力学正常，但产率明显低于asp -96 - fwdarw。突变型或野生型，尤其在pH为6时。谷氨酸取代Asp-85或Asp-96对质子运动的动力学和量子产率有较小但可检测的影响。在115位和212位替换天冬氨酸和谷氨酸降低了质子量子产率。其中，只有Asp-115.fwdarw。Asn突变体对质子释放步骤有影响，只有asp212 . fww有影响。谷氨酸突变降低了质子摄取速率。这些实验表明，在细菌视紫红质质子泵的正常运行中，Asp-96在H+摄取中起着必不可少的作用。结果还表明，氨基酸取代只会影响H+释放或H+摄取的动力学，但不会同时影响两者。这意味着这两个步骤在光循环开始后相互独立地发生。
Recently, a number of aspartic acid mutants of bacteriorhodopsin have been shown to be defective in steady-state proton transport. Here we report time-resolved measurements of light-induced proton release and uptake for these mutants. Proton transfers between the protein and the aqueous phase were directly monitored by measuring changes in the bulk conductivity of a micellar solution of bacteriorhodopsin. For the Asp-90.fwdarw. Asn mutant, proton uptake was slowed by >1 order of magnitude with no observable effect on the release step. For Asp-85.fwdarw. Asn, H+ uptake occurred with normal kinetics, but the yield was significantly lower compared with either the Asp-96.fwdarw. Asn mutant or wild type, especially at pH 6. Substitution of glutamate for Asp-85 or Asp-96 had smaller but detectable effects on the kinetics and quantum yield of proton movements. Both asparagine and glutamate substitutions of aspartates at positions 115 and 212 lowered the proton quantum yields. Of these, only the Asp-115.fwdarw. Asn mutant showed an effect on the proton release step, and only the Asp212.fwdarw. Glu mutation decreased the proton uptake rate. These experiments imply an obligatory role for Asp-96 in H+ uptake in the normal operation of the bacteriorhodopsin proton pump. The results also indicate that the amino acid substitutions affect the kinetics of either H+ release or H+ uptake, but not both. This implies that the two steps occur independently of each other after initiation of the photocycle.