Abstract
Human NQO1 [NAD(H):quinone oxidoreductase 1] is a multi-functional and stress-inducible dimeric protein involved in the antioxidant defense, the activation of cancer prodrugs and the stabilization of oncosuppressors. Despite its roles in human diseases, such as cancer and neurological disorders, a detailed characterization of its enzymatic cycle is still lacking. In this work, we provide a comprehensive analysis of the NQO1 catalytic cycle using rapid mixing techniques, including multiwavelength and spectral deconvolution studies, kinetic modeling and temperature-dependent kinetic isotope effects (KIEs). Our results systematically support the existence of two pathways for hydride transfer throughout the NQO1 catalytic cycle, likely reflecting that the two active sites in the dimer catalyze two-electron reduction with different rates, consistent with the cooperative binding of inhibitors such as dicoumarol. This negative cooperativity in NQO1 redox activity represents a sort of half-of-sites activity. Analysis of KIEs and their temperature dependence also show significantly different contributions from quantum tunneling, structural dynamics and reorganizations to catalysis at the two active sites. Our work will improve our understanding of the effects of cancer-associated single amino acid variants and post-translational modifications in this protein of high relevance in cancer progression and treatment.
Highlights
NAD(P)H quinone oxidoreductase 1 (NQO1; DT-diaphorase; EC 1.6.5.2) is a multi-functional and stress-inducible flavoprotein whose activity is associated with different pathologies, with cancer [1,2]
Such a lack of semiquinone intermediates indicates that reduction of the semiquinone to the hydroquinone state is thermodynamically more favorable and kinetically faster than the reduction of the oxidized to the semiquinone species [72]
This observation agrees with the absence of detectable semiquinone paramagnetic signals when evaluating the redox cycle of the enzyme and with mammalian quinone oxidoreductases, which are a notable exception in that they only function by a compulsory two-electron transfer [73]
Summary
NAD(P)H quinone oxidoreductase 1 (NQO1; DT-diaphorase; EC 1.6.5.2) is a multi-functional and stress-inducible flavoprotein whose activity is associated with different pathologies, with cancer [1,2]. NQO1 has a wide range of substrates and enzymatic functions associated with antioxidant defense and cancer development, including the NAD(P)H-dependent two-electron reduction of quinones to form hydroquinones, avoiding the formation of highly reactive and cytotoxic semiquinones [3,4], the maintenance of vitamin K3 and ubiquinone in their reduced state [5,6,7,8] and scavenging of superoxide anions [9]. The activation of the antioxidant response induces the expression of NQO1, as well as that of enzymes involved in NADPH generation [21,22,23,24,25]. The expression of NQO1 can be induced by some antioxidants (such as resveratrol) through the antioxidant response pathway [26,27]
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