S82D mutation affects NQO1 activity

Raw data published in: Pacheco-Garcia JL, Anoz-Carbonell E, Vankova P, Kannan A, Palomino-Morales R, Mesa-Torres N, Salido E, Man P, Medina M, Naganathan AN, Pey AL. Structural basis of the pleiotropic and specific phenotypic consequences of missense mutations in the multifunctional NAD(P)H:quinone oxidoreductase 1 and their pharmacological rescue. Redox Biol. 2021 Oct;46:102112. doi: 10.1016/j.redox.2021.102112. Epub 2021 Aug 18. PMID: 34537677; PMCID: PMC8455868.

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-(HT) or deuteride-(DT) transfer reactions were carried out under anaerobic conditions using a stopped-flow spectrophotometer (SX.18 MV, Applied Photophysics Ltd.) interfaced with a photodiode array detector, essentially as described [24]. Briefly, the reductive half-reaction was measured by mixing NQO1holo variants (7.5 μM) with either NADH ranging from 7.5 to 100 μM or with NADPH or 4R-2H-NADH (NADD) at stoichiometric concentrations (7.5 μM). To study the oxidative half-reaction, 7.5 μM NQO1hq (formed by reaction of NQO1holo with stoichiometric amounts of NADH) was mixed with 7.5 μM of 2,6-Dichlorophenolindophenol (DCPIP). Reactions were performed in 20 mM HEPES-KOH, pH 7.4. Multiple wavelength absorption data in the flavin absorption region were collected and processed as described [24]. Time-dependent spectral deconvolution was performed by global analysis and numerical integration methods using previously described procedures [24]. Basically, this deconvolution procedure was carried out considering sequential and irreversible steps in the context of two (A→B→C) or three (A→B→C→D) step mechanisms, where A-D are spectral species (not necessarily a given state), and allowed to determine observed rate constants (kobs) for these steps as well as spectroscopic properties of A and D are initial and final, but not intermediate states A to D species. According to a recent study, catalytically relevant processes involved steps A→B→C [24].

For estimation of primary kinetic isotopic effects (KIEs) in the HT process [34], HT or DT kobs from NADH/D to NQO1holo were evaluated at different temperatures in samples containing equimolecular mixtures of protein and coenzyme (7.5 μM of each component) using NADH and [4R-2H]-NADD. KIEs were thus determined as the ratio of the kobs values using NADH and NADD at a given temperature. Activation parameters (frequency factor, A, and the activation energy, Ea) were determined using the Arrhenius equation as described [24].

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