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].

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