A single evolutionarily divergent mutation determines the different FAD-binding affinities of human and rat NQO1 due to site-specific phosphorylation Pacheco García, Juan Luis Mesa Torres, Noel Pey Rodríguez, Ángel Luis Epistasis Flavoprotein Molecular evolution Protein phosphorylation ALP thanks Professors Jose Manuel Sanchez-Ruiz and Beatriz Ibarra-Molero (both from the University of Granada) for providing access and advice on their home-built software for electrostatic calculations. BR acknowledges kind hospitality and use of computational resources in the European Magnetic Resonance Center (CERM), Sesto Fiorentino (Florence), Italy. This work was supported by Spanish Ministry of Economy and Competitiveness and European ERDF Funds (MCIU/AEI/FEDER, EU) [RTI2018-097991-BI00 to JLN and RTI2018-096246-B-I00 to ALP]; FEDER/Junta de Andalucia-Consejeria de Transformacion Economica, Industria, Conocimiento y Universidades [Grant P18-RT-2413 to ALP]. Financial support from EU Horizon 2020 project EU FT-ICR MS (731077) as well as institutional (CZ.1.05/1.1.00/02.0109) and MS facility support (LM2018127 CIISB) are gratefully acknowledged. Funding for open charge: Universidad de Granada/CBUA. The phosphomimetic mutation S82D in the cancer-associated, FADdependent human NADP(H):quinone oxidoreductase 1 (hNQO1) causes a decrease in flavin-adenine dinucleotide-binding affinity and intracellular stability. We test in this work whether the evolutionarily recent neutral mutation R80H in the vicinity of S82 may alter the strong functional effects of S82 phosphorylation through electrostatic interactions. We show using biophysical and bioinformatic analyses that the reverse mutation H80R prevents the effects of S82D phosphorylation on hNQO1 by modulating the local stability. Consistently, in rat NQO1 (rNQO1) which contains R80, the effects of phosphorylation were milder, resembling the behaviour found in hNQO1 when this residue was humanized in rNQO1 (by the R80H mutation). Thus, apparently neutral and evolutionarily divergent mutations may determine the functional response of mammalian orthologues towards phosphorylation. 2021-12-20T11:46:15Z 2021-12-20T11:46:15Z 2021-11-24 journal article Pacheco-Garcia, J.L... [et al.] (2021), A single evolutionarily divergent mutation determines the different FAD-binding affinities of human and rat NQO1 due to site-specific phosphorylation. FEBS Lett. [https://doi.org/10.1002/1873-3468.14238] http://hdl.handle.net/10481/72134 10.1002/1873-3468.14238 eng info:eu-repo/grantAgreement/EC/H2020/731077 http://creativecommons.org/licenses/by/3.0/es/ open access Atribución 3.0 España John Wiley & Sons