Mechanism of NAD(P)H:Quinone reductase: Ab initio studies of reduced flavin

Abstract

NAD(P)H:quinone oxidoreductase type 1 (QR1, NQO1, formerly DT-diaphorase; EC 1.6.99.2) is an FAD-containing enzyme that catalyzes the nicotinamide nucleotide-dependent reduction of quinones, quinoneimines, azo dyes, and nitro groups. Animal cells are protected by QR1 from the toxic and neoplastic effects of quinones and other electrophiles. Alternatively, in tumor cells QR can activate a number of cancer chemotherapeutic agents such as mitomycins and aziridylbenzoquinones. Thus, the same enzyme that protects the organism from the deleterious effects of quinones can activate cytotoxic chemotherapeutic prodrugs and cause cancer cell death. The catalytic mechanism of QR includes an important initial step in which FAD is reduced by NAD(P)H. The unfavorable charge separation that results must be stabilized by the protein. The details of this charge stabilization step are inaccessible to easy experimental verification but can be studied by quantum chemistry methods. Here we report ab initio quantum mechanical calculations in and around the active site of the enzyme that provide information about the fine details of the contribution of the protein to the stabilization of the reduced flavin. The results show that (1) protein interactions provide approximately 2 kcal/mol to stabilize the planar conformation of the reduced flavin isoalloxazine ring observed in the X-ray structure; (2) the charge separation present in the reduced planar form of the flavin is stabilized by interactions with groups of the protein; (3) even after stabilization, the reduction potential of the cofactor remains more negative than that of the free flavin, making it a better reductant for a larger variety of quinones; and (4) the more negative reduction potential may also result in faster kinetics for the quinone reduction step.

Curcumin ameliorates cognitive deficits heavy ion irradiation-induced learning and memory deficits through enhancing of Nrf2 antioxidant signaling pathways

“…We show in the present study that curcumin can upregulate the antioxidant enzyme NQO1 by activating the Nrf2 pathway. In addition to its potency to catalyze reduction of quinone to hydroquinone (Joseph et al, 2000;Cavelier and Amzel, 2001), NQO1 is known to maintain both α-tocopherol and coenzyme Q10 in their reduced antioxidant state (Beyer et al, 1997;Siegel et al, 1997). It seems that the cytoprotective effect of curcumin related to Nrf2 activation plays a major role in mouse cerebral injury by heavy ion IR, at least in our experimental conditions.…”

Indolequinone Inhibitors of NRH:Quinone Oxidoreductase 2. Characterization of the Mechanism of Inhibition in both Cell-Free and Cellular Systems

“…6D) in the active site of NQO2 were very similar. As observed in the co-crystallized structure (27) the indolequinone ring was aligned parallel to the isoalloxazine ring of FAD and the C7 carbonyl of the quinone moiety was positioned directly above the N-5 of the FAD, enabling efficient hydride transfer from the FAD during reduction (8, 39). The hydrophobic interaction between the indole 2-methyl and residues Phe178 and Phe106 also contributes favorably to the binding energy.…”

“…Molecular docking simulations were performed for indolequinones 1–9 in the active site of NQO2 with the anionic form of the reduced FAD cofactor which represents the physiological situation where quinone encounters reduced FAD (8, 39). High-ranking binding conformations of the indolequinones in the active site of NQO2 were identified and shown in Figure 6 ( 1–3, 5 and 7) and Figure S1 ( 4, 6, 8, 9 ).…”

Human NAD(P)H:Quinone Oxidoreductase Type I (hNQO1) Activation of Quinone Propionic Acid Trigger Groups

“…During NAD + displacement, it is thought that the O(2) of the flavin is protonated to form FADH 2 . 34, 40, 91, 92 Subsequent reduction of the quinone occurs via a mechanism that is effectively the reverse of that for formation of FADH 2 . These unproven mechanistic processes are outlined in Scheme 2.…”