Reduction of NQO1 wildtype by NADH

Raw data published in: Anoz-Carbonell E, Timson DJ, Pey AL, Medina M. The Catalytic Cycle of the Antioxidant and Cancer-Associated Human NQO1 Enzyme: Hydride Transfer, Conformational Dynamics and Functional Cooperativity. Antioxidants (Basel). 2020 Aug 20;9(9):772. doi: 10.3390/antiox9090772. PMID: 32825392; PMCID: PMC7554937.

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 (7.5 μM) was mixed with NADH/D 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 NADH concentrations: 7.5, 10, 15, 20, 30, 50 and 100 uM of NADH.