Suppression of Electron Transfer to Dioxygen by Charge Transfer and Electron Transfer Complexes in the FAD-dependent Reductase Component of Toluene Dioxygenase*
Suppression of Electron Transfer to Dioxygen by Charge Transfer and Electron Transfer Complexes in the FAD-dependent Reductase Component of Toluene Dioxygenase*
复制标题
DOI：
10.1074/jbc.m112.374918
复制
发表时间：
2012-09
期刊：
影响因子：
--
通讯作者：
Tzong-Yuan Lin;T. Werther;J. Jeoung;H. Dobbek
中科院分区：
文献类型：
--
作者：
Tzong-Yuan Lin;T. Werther;J. Jeoung;H. Dobbek
背景：甲苯双加氧酶还原酶在好氧条件下转化还原还原还原当量的NADH。结果：还原型还原酶与NAD+形成稳定的复合物，显示出较慢的电子转移到分子氧。结论：电荷转移复合物的形成调节了还原酶的反应活性。意义：黄素和烟酰胺之间的电荷转移复合物是丰富的，可能有不同的机制功能。三组分甲苯双加氧酶系统由含FAD的还原酶、Rieske型[2Fe-2S]铁氧还蛋白和Rieske型双加氧酶组成。含FAD的还原酶的任务是将电子从NADH穿梭到铁氧还蛋白，这是在分子氧存在下酶必须催化的反应。我们研究了还原酶在还原和氧化半反应中的动力学，并在还原半反应结束时检测到还原还原酶和NAD+之间的稳定电荷转移复合物，其对分子氧的反应性比在不存在NAD+的情况下还原还原酶的反应性低得多。NAD+和还原还原酶之间的复合物的晶体结构揭示了对分子氧的低反应性的一个合理原因，这表明烟酰胺环和蛋白质基质屏蔽了异咯嗪环的反应性C4 a位置，并迫使三环形成非典型的平面构象，这两个因素都不利于还原黄素与分子氧的反应。从电荷转移复合物到电子受体的快速电子转移进一步降低了不需要的副反应的风险，并且还原酶与其同源铁氧还蛋白之间的复合物的晶体结构显示出电子供体和电子受体辅因子之间的短距离。两种蛋白质之间的吸引力可能是由复杂界面的一大块上的相反电荷介导的。所观察到的电荷转移和电子转移复合物的稳定性、特异性和反应性被认为防止了reductaseTOL与分子氧的反应，从而为相互冲突的要求提供了解决方案。
Background: Toluene dioxygenase reductase converts the redox equivalents of NADH under oxic conditions. Results: Reduced reductase forms a stable complex with NAD+ that shows slower electron transfer to dioxygen. Conclusion: Charge transfer complex formation regulates the reactivity of the reductase. Significance: Charge transfer complexes between flavins and nicotinamides are abundant and likely to have diverse mechanistic functions. The three-component toluene dioxygenase system consists of an FAD-containing reductase, a Rieske-type [2Fe-2S] ferredoxin, and a Rieske-type dioxygenase. The task of the FAD-containing reductase is to shuttle electrons from NADH to the ferredoxin, a reaction the enzyme has to catalyze in the presence of dioxygen. We investigated the kinetics of the reductase in the reductive and oxidative half-reaction and detected a stable charge transfer complex between the reduced reductase and NAD+ at the end of the reductive half-reaction, which is substantially less reactive toward dioxygen than the reduced reductase in the absence of NAD+. A plausible reason for the low reactivity toward dioxygen is revealed by the crystal structure of the complex between NAD+ and reduced reductase, which shows that the nicotinamide ring and the protein matrix shield the reactive C4a position of the isoalloxazine ring and force the tricycle into an atypical planar conformation, both factors disfavoring the reaction of the reduced flavin with dioxygen. A rapid electron transfer from the charge transfer complex to electron acceptors further reduces the risk of unwanted side reactions, and the crystal structure of a complex between the reductase and its cognate ferredoxin shows a short distance between the electron-donating and -accepting cofactors. Attraction between the two proteins is likely mediated by opposite charges at one large patch of the complex interface. The stability, specificity, and reactivity of the observed charge transfer and electron transfer complexes are thought to prevent the reaction of reductaseTOL with dioxygen and thus present a solution toward conflicting requirements.