Paddock M L, Rongey S H, McPherson P H, Juth A, Feher G, Okamura M Y
Department of Physics, University of California, San Diego, La Jolla 92093-0319.
Biochemistry. 1994 Jan 25;33(3):734-45. doi: 10.1021/bi00169a015.
The role of Asp-L213 in proton transfer to reduced quinone QB in the reaction center (RC) from Rhodobacter sphaeroides was studied by site-directed replacement of Asp with residues having different proton donor properties. Reaction centers (RCs) with Asn, Leu, Thr, and Ser at L213 had greatly reduced (approximately 6000-fold) proton-coupled electron transfer [kAB(2)] and proton uptake rates associated with the second electron reduction of QB (QA- QB- + 2H(+)-->QAQBH2) compared to native RCs. RCs containing Glu at L213 showed faster (approximately 90-fold) electron and proton transfer rates than the other mutant RCs but were still reduced (approximately 70-fold) compared with native RCs. These results show that kAB(2) is larger when a carboxylic acid occupies the L213 site, consistent with the proposal that Asp-L213 is a component of a proton transfer chain. The reduced kAB(2) observed with Glu versus Asp at L213 suggests that Asp at L213 is important for proton transfer for some other reason in addition to its proton transfer capabilities. Glu-L213 is estimated to have a higher apparent pKa (pKa > or = 7) than Asp-L213 (pKa < or = 4), as indicated by the slower rate of charge recombination (D+QAQB(-)-->DQAQB) in the mutant RCs. The importance of the pKa and charge of the residue at L213 for proton transfer are discussed. Based on these studies, a model for proton transfer is proposed in which Asp-L213 contributes to proton transfer in native RCs in two ways: (1) it is a component of a proton transfer chain connecting the buried QB molecule with the solvent and/or (2) it provides a negative charge that stabilizes a proton on or near QB.
通过定点替换天冬氨酸（Asp）为具有不同质子供体特性的氨基酸残基，研究了天冬氨酸 - L213在球形红细菌反应中心（RC）中向还原醌QB转移质子的作用。与天然反应中心相比，L213位点为天冬酰胺（Asn）、亮氨酸（Leu）、苏氨酸（Thr）和丝氨酸（Ser）的反应中心，其质子耦合电子转移[kAB(2)]以及与QB第二次电子还原（QA - QB- + 2H(+) --> QAQBH2）相关的质子摄取速率大幅降低（约6000倍）。L213位点为谷氨酸（Glu）的反应中心，其电子和质子转移速率比其他突变反应中心更快（约90倍），但与天然反应中心相比仍降低了（约70倍）。这些结果表明，当羧酸占据L213位点时，kAB(2)更大，这与天冬氨酸 - L213是质子转移链的一个组成部分的提议一致。在L213位点观察到谷氨酸与天冬氨酸相比kAB(2)降低，这表明L213位点的天冬氨酸除了其质子转移能力外，由于其他某些原因对质子转移也很重要。如突变反应中心中电荷复合（D+QAQB(-) --> DQAQB）速率较慢所示，估计谷氨酸 - L213的表观pKa（pKa ≥ 7）高于天冬氨酸 - L213（pKa ≤ 4）。讨论了L213位点残基的pKa和电荷对质子转移的重要性。基于这些研究，提出了一个质子转移模型，其中天冬氨酸 - L213以两种方式促进天然反应中心中的质子转移：（1）它是将埋藏的QB分子与溶剂连接的质子转移链的一个组成部分；和/或（2）它提供一个负电荷，使QB上或其附近的质子稳定。
