Cavity effect on NQO1

Raw data published in: Pacheco-Garcia JL, Loginov DS, Anoz-Carbonell E, Vankova P, Palomino-Morales R, Salido E, Man P, Medina M, Naganathan AN, Pey AL. Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations. Antioxidants (Basel). 2022 Jun 2;11(6):1110. doi: 10.3390/antiox11061110. PMID: 35740007; PMCID: PMC9219786.

Data measured using a stopped-flow spectrophotometer from Applied Photophysics (SX.18MV, Applied Photophysics Ltd., Leatherhead, UK) interfaced with a photodiode array detector and under anaerobic conditions, following previously established protocols. Multiple wavelength absorption data in the flavin absorption region (400-900 nm) were collected and processed using the ProData-SX software (Applied Photophysics Ltd.). Time-dependent spectral deconvolution was performed by global analysis and numerical integration methods using Pro-Kineticist (Applied Photophysics Ltd.). Collected data were fitted to either single- or multi-step (A→B→n….→Z) models allowing for estimation of the corresponding observed conversion rate constants at each NADH concentration, as well of the spectra of intermediate and final species.

Experimental section:

Fast hydride- and deuteride-transfer reactions (HT and DT, respectively) were carried out under anaerobic conditions using a stopped-flow spectrophotometer as described [15]. Briefly, the reductive half reaction was measured by mixing NQO1holo variants (7.5 µM) with NADH ranging from 7.5 to 100 µM (these refer to final concentrations). Reactions were performed in 20 mM HEPES-KOH, pH 7.4. Data were collected using either NADH or NADD, but using only one of these reducing species in a given experiment. Multiple-wavelength absorption data in the flavin absorption region were collected and processed as described [15]. Time-dependent spectral deconvolution was performed by global fitting analysis and numerical integration using previously described procedures [15]. This deconvolution procedure was carried out considering sequential and irreversible steps in the context of a two-step mechanism (A⟶B⟶C) and was used to determine observed rate constants (kobs) for these steps as well as the spectroscopic properties of these species (A, B and C). 

To determine primary kinetic isotopic effects (KIEs) in the HT process [20], the kobs for HT and DT was determined by mixing NADH/D with NQO1holo using equimolar concentrations of NQO1holo and NADH or [4R-2H]-NADD (7.5 µM of each component again experiments at higher NADH concentrations where limited but reactions becoming too fast for detection upon increasing temperature). These apparent KIEs were determined as the ratio of kobs values using NADH and NADD. Experiments were carried out at temperatures ranging 6–20 °C. Activation parameters (frequency factor, A, and the activation energy, Ea) were determined using the Arrhenius equation as described [15].

Password: nqo1

本研究原始数据发表于：Pacheco-Garcia JL、Loginov DS、Anoz-Carbonell E、Vankova P、Palomino-Morales R、Salido E、Man P、Medina M、Naganathan AN、Pey AL。《通过空腔构建突变体研究多功能氧化还原NQO1蛋白的别构通信》，*Antioxidants (Basel)*，2022年6月2日，11(6):1110。DOI: 10.3390/antiox11061110；PMID: 35740007；PMCID: PMC9219786。

本数据集采用英国莱瑟黑德Applied Photophysics有限公司生产的SX.18MV型停流分光光度计（stopped-flow spectrophotometer）采集，该设备搭配光电二极管阵列检测器，并在厌氧条件下依照已发表的标准实验方案完成测量。采集了黄素蛋白吸收区域（400~900 nm）的多波长吸收光谱数据，并通过ProData-SX软件（Applied Photophysics有限公司）进行预处理。采用Pro-Kineticist软件（Applied Photophysics有限公司）通过全局分析与数值积分方法完成时间分辨光谱解卷积。将采集到的数据拟合至单步或多步（A→B→n…→Z）模型，以此估算各烟酰胺腺嘌呤二核苷酸（NADH）浓度下对应的表观转化速率常数，以及中间产物与终产物的光谱特征。

实验部分：
快速氢化物转移与氘化物转移反应（分别记为HT与DT）依照文献[15]所述方法，在厌氧条件下通过停流分光光度计完成。简言之，通过将全载辅基NQO1变体（终浓度7.5 μM）与终浓度为7.5~100 μM的NADH混合，完成还原半反应的测量。反应体系为20 mM HEPES-KOH缓冲液，pH 7.4。实验可采用NADH或NADD作为还原剂，但单次实验仅使用其中一种还原试剂。依照文献[15]所述流程采集并处理黄素蛋白吸收区域的多波长吸收光谱数据，采用全局拟合分析与数值积分方法完成时间分辨光谱解卷积，该解卷积流程基于两步级联不可逆反应机制（A⟶B⟶C）开展，用于确定上述反应步骤的表观速率常数（kobs）以及各物种（A、B、C）的光谱特性。

为测定HT过程中的一级动力学同位素效应（KIEs）[20]，通过将NADH/NADD与全载辅基NQO1混合，测定HT与DT的kobs值，实验中全载辅基NQO1与NADH或[4R-2H]-NADD的浓度均为等摩尔（每组初始浓度为7.5 μM；当NADH浓度较高时，反应速率过快难以检测，需适当提高反应温度以调控反应速率）。表观KIEs通过NADH与NADD对应的kobs值之比计算得到。实验温度设置为6~20 ℃。依照文献[15]所述的阿伦尼乌斯方程，计算得到活化参数（频率因子A与活化能Ea）。

数据集访问密码：nqo1