Paddock M L, McPherson P H, Feher G, Okamura M Y
Department of Physics, University of California, San Diego, La Jolla 92093.
Proc Natl Acad Sci U S A. 1990 Sep;87(17):6803-7. doi: 10.1073/pnas.87.17.6803.
The pathway of proton transfer in the reaction center (RC) from Rhodobacter sphaeroides was investigated by site-directed mutagenesis. Ser-L223, a putative proton donor that forms a hydrogen bond with the secondary quinone acceptor QB, was replaced with Ala and Thr. RCs with Ala-L223 displayed reduced electron transfer and proton uptake rates in the reaction QA-QB- + 2H+----QAQBH2. The rate constant for this reaction, k(2)AB, was found to be reduced approximately 350-fold to 4.0 +/- 0.2 s-1. Proton uptake measurements using a pH indicator dye showed a rapid uptake of 1 H+ per RC followed by a slower uptake of 1 H+ per RC at a rate of 4.1 +/- 0.1 s-1; native RCs showed a rapid uptake of 2H+ per RC. Evidence is provided that these changes were not due to gross structural changes in the binding site of QB. RCs with Thr-L223 showed little reduction in the rates of electron and proton transfer. These results indicate that proton transfer from the hydroxyl group of Ser-L223 or Thr-L223 is required for fast electron and proton transfer associated with the formation of the dihydroquinone QH2. In contrast, previous work showed that replacing Glu-L212, another putative proton donor to QB, with Gln slowed proton uptake from solution without significantly altering electron transfer. We propose a model that involves two distinct proton transfer steps. The first step occurs prior to transfer of the second electron to QB and involves proton transfer from Ser-L223. The second step occurs after this electron transfer through a pathway involving Glu-L212.
通过定点诱变研究了球形红杆菌反应中心（RC）中质子转移的途径。与次级醌受体QB形成氢键的假定质子供体Ser-L223被丙氨酸（Ala）和苏氨酸（Thr）取代。含有Ala-L223的反应中心在QA-QB- + 2H+----QAQBH2反应中显示出电子转移和质子摄取速率降低。发现该反应的速率常数k(2)AB降低了约350倍，降至4.0±0.2 s-1。使用pH指示剂染料进行的质子摄取测量表明，每个反应中心快速摄取1个H+，随后以4.1±0.1 s-1的速率每个反应中心缓慢摄取1个H+；天然反应中心每个反应中心快速摄取2个H+。有证据表明，这些变化不是由于QB结合位点的总体结构变化所致。含有Thr-L223的反应中心在电子和质子转移速率上几乎没有降低。这些结果表明，与二氢醌QH2形成相关的快速电子和质子转移需要Ser-L223或Thr-L223羟基的质子转移。相比之下，先前的研究表明，用谷氨酰胺（Gln）取代另一个假定的QB质子供体Glu-L212会减缓从溶液中的质子摄取，而不会显著改变电子转移。我们提出了一个涉及两个不同质子转移步骤的模型。第一步发生在第二个电子转移到QB之前，涉及从Ser-L223的质子转移。第二步发生在该电子转移之后，通过涉及Glu-L212的途径进行。
