Reduction of NQO1 wildtype by NADD

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.

NQO1_ox (7.5 μM) was mixed with NADD at concentrations ranging from 1:1 to 1:14 NQO1_ox: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 NADD 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 NADD.