Reduction of NQO1 P187S by NADH

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.

NQO1ox P187S (7.5 μM) was mixed with NADH at concentrations ranging from 1:1 to 1:14 NQO1ox:NADH ratio. Reactions were studied in 20 mM HEPES-KOH, pH 7.4 with glucose/glucose oxidase (310 mM/10 units/mL), at 25 C.

Spectral evolution of NQO1 reductive half-reaction with NADH at different concentrations. Files are numbered from 1 to 7 representing the following NADD concentrations: 7.5, 10, 15, 20, 30, 50 and 100 uM of NADH.