Correlating Calmodulin Landscapes with Chemical Catalysis in Neuronal Nitric Oxide Synthase using Time-Resolved FRET and a 5-Deazaflavin Thermodynamic Trap.
Correlating Calmodulin Landscapes with Chemical Catalysis in Neuronal Nitric Oxide Synthase using Time-Resolved FRET and a 5-Deazaflavin Thermodynamic Trap.
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DOI：
10.1021/acscatal.6b01280
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发表时间：
2016-08-05
期刊：
影响因子：
12.9
通讯作者：
Scrutton NS
中科院分区：
文献类型：
--
作者：
Hedison TM;Leferink NG;Hay S;Scrutton NS
酶学的一个主要挑战是需要将酶的动态特性与它们的催化循环联系起来，并了解它们对催化循环的影响。对于像一氧化氮合酶（nos）这样的大型多中心酶来说尤其如此，其中动力学的重要性已经从各种结构、单分子和系综光谱方法中推断出来，但在实验中，运动并没有与反应周期中的机械步骤相关联。这里我们采取这样的方法。利用吸收光谱和Förster共振能量转移（FRET）的时间分辨光谱，利用黄素类似物（5-去氮黄素单核苷酸（5-dFMN））和同位素标记的烟酰胺辅酶的性质，我们将CaM与神经元一氧化氮合酶（nNOS）结合时结构变化的时间与nNOS催化循环联系起来。我们发现，CaM的重构发生在电子转移序列（FAD还原）的早期，而不是在反应周期的后期（例如，FMN还原）。构象变化与FAD还原动力学密切相关，反映了结合的CaM分子的短暂“打开”然后“关闭”。我们推断，结合NADPH的c端尾部位移和随后的FAD还原可能是构象变化的触发因素。通过结合使用辅助因子/辅酶类似物和时间分辨FRET/吸收分光光度法，我们展示了如何简化复杂酶的反应周期，从而能够详细研究蛋白质动力学和反应周期化学之间的关系-这种方法也可用于其他复杂的多中心酶。
A major challenge in enzymology is the need to correlate the dynamic properties of enzymes with, and understand the impact on, their catalytic cycles. This is especially the case with large, multicenter enzymes such as the nitric oxide synthases (NOSs), where the importance of dynamics has been inferred from a variety of structural, single-molecule, and ensemble spectroscopic approaches but where motions have not been correlated experimentally with mechanistic steps in the reaction cycle. Here we take such an approach. Using time-resolved spectroscopy employing absorbance and Förster resonance energy transfer (FRET) and exploiting the properties of a flavin analogue (5-deazaflavin mononucleotide (5-dFMN)) and isotopically labeled nicotinamide coenzymes, we correlate the timing of CaM structural changes when bound to neuronal nitric oxide synthase (nNOS) with the nNOS catalytic cycle. We show that remodeling of CaM occurs early in the electron transfer sequence (FAD reduction), not at later points in the reaction cycle (e.g., FMN reduction). Conformational changes are tightly correlated with FAD reduction kinetics and reflect a transient “opening” and then “closure” of the bound CaM molecule. We infer that displacement of the C-terminal tail on binding NADPH and subsequent FAD reduction are the likely triggers of conformational change. By combining the use of cofactor/coenzyme analogues and time-resolved FRET/absorbance spectrophotometry, we show how the reaction cycles of complex enzymes can be simplified, enabling a detailed study of the relationship between protein dynamics and reaction cycle chemistry—an approach that can also be used with other complex multicenter enzymes.