170380 20260420103354.0 doi 10.1111/febs.70511 sideral 148927 ART-2026-148927 eng Rivero, Maribel Tyrosine residues at the substrate binding site in human NQO1 homodimer: Protein conformational dynamics and optimization of substrate binding geometry 2026 Human NQO1 is a homodimeric flavoenzyme essential for the redox metabolism of many substances and implicated in major global health challenges such as cancer and Alzheimer's disease. X-ray crystallographic studies have identified several residues within its substrate binding site (including Tyr126 and Tyr128) that may regulate catalytic competent binding of substrates, cofactor redox properties, half-site reactivity, and/or functional inter-active site negative cooperativity. To elucidate the functional role of Tyr126 and Tyr128, we generated point mutants at these positions and assessed their dynamics and kinetic properties. Hydrogen-deuterium exchange coupled to mass spectrometry revealed that non-conservative mutations, particularly at Tyr126, notably disrupted dynamics not only within the substrate binding site but also in structural elements connecting the two active sites of the NQO1 homodimer. Rapid-mixing pre-steady-state kinetics experiments of the reduction of NQO1 by NAD(P)H showed that mutations to Phe caused a mild decrease in hydride transfer (HT) efficiency from the coenzyme to the FAD cofactor. In contrast, mutations to Ala resulted in a significantly greater impact and mutations to Glu nearly abolished HT. Despite these effects, some mutations moderately affected the non-synchronous catalysis between the two alternating active sites, but hardly produced an impact on the selectivity for NADPH versus NADH as hydride donor coenzymes. However, all variants exhibited markedly impaired enzyme turnover, highlighting alterations in the enzyme's substrate specificity toward quinones. The data presented here demonstrate that Tyr126 and Tyr128 optimize both substrate binding geometry as well as overall enzyme conformational dynamics during the asymmetric catalytic cycle of the NQO1 homodimer. Access copy available to the general public Unrestricted info:eu-repo/grantAgreement/ES/DGA-FEDER/E35-23R info:eu-repo/grantAgreement/ES/MICINN/PID2022-136369NB-I00 info:eu-repo/semantics/openAccess by-nc-nd https://creativecommons.org/licenses/by-nc-nd/4.0/deed.es info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Pacheco-Garcia, Juan Luis Vankova, Pavla Loginov, Dmitry Quereda-Moraleda, Isabel Martin-Garcia, Jose Manuel Man, Petr Pey, Angel Luis Medina, Milagros Universidad de Zaragoza (orcid)0000-0001-8743-0182 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. (2026), [27 pp.] FEBS J. FEBS Journal 1742-464X 4399366 http://zaguan.unizar.es/record/170380/files/texto_completo.pdf Versión publicada 2537655 http://zaguan.unizar.es/record/170380/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:170380 articulos driver 2026-04-18-10:48:25 ARTICLE