Grossman Iris, Yuval Aviram Haim, Armony Gad, Horovitz Amnon, Hofmann Hagen, Haran Gilad, Fass Deborah
Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel.
Department of Chemical Physics, Weizmann Institute of Science, Rehovot 7610001, Israel.
Nat Commun. 2015 Oct 15;6:8624. doi: 10.1038/ncomms9624.
The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a multidomain catalyst of disulfide-bond formation that relays electrons from substrate cysteines through two redox-active sites to molecular oxygen. The chemical steps in electron transfer have been delineated, but the conformational changes accompanying these steps are poorly characterized. Here we use single-molecule Förster resonance energy transfer (smFRET) to probe QSOX conformation in resting and cycling enzyme populations. We report the discovery of unanticipated roles for conformational changes in QSOX beyond mediating electron transfer between redox-active sites. In particular, a state of the enzyme not previously postulated or experimentally detected is shown to gate, via a conformational transition, the entrance into a sub-cycle within an expanded QSOX kinetic scheme. By tightly constraining mechanistic models, smFRET data can reveal the coupling between conformational and chemical transitions in complex enzymatic cycles.
对酶分子进行逐个查询的能力正在改变我们对催化机制的看法。巯基氧化酶（QSOX）是一种形成二硫键的多结构域催化剂，它将电子从底物半胱氨酸通过两个氧化还原活性位点传递给分子氧。电子转移的化学步骤已被阐明，但伴随这些步骤的构象变化却鲜有描述。在这里，我们使用单分子荧光共振能量转移（smFRET）来探测处于静止和循环状态的酶群体中的QSOX构象。我们报告了QSOX构象变化在介导氧化还原活性位点之间的电子转移之外的意外作用。特别是，一种以前未被假设或实验检测到的酶状态被证明通过构象转变控制着进入扩展的QSOX动力学方案中的一个子循环。通过严格限制机制模型，smFRET数据可以揭示复杂酶循环中构象和化学转变之间的耦合。
