Structures of recombinant human and mouse NAD(P)H:quinone oxidoreductases: species comparison and structural changes with substrate binding and release

Abstract

NAD(P)H/quinone acceptor oxidoreductase (QR1, NQO1, formerly DT-diaphorase; EC ) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. In this paper we report the apoenzyme structures of human (at 1.7-A resolution) and mouse (2.8 A) QR1 and the complex of the human enzyme with the substrate duroquinone (2.5 A) (2,3,5, 6-tetramethyl-p-benzoquinone). In addition to providing a description and rationale of the structural and catalytic differences among several species, these structures reveal the changes that accompany substrate or cofactor (NAD) binding and release. Tyrosine-128 and the loop spanning residues 232-236 close the binding site, partially occupying the space left vacant by the departing molecule (substrate or cofactor). These changes highlight the exquisite control of access to the catalytic site that is required by the ping-pong mechanism in which, after reducing the flavin, NAD(P)(+) leaves the catalytic site and allows substrate to bind at the vacated position. In the human QR1-duroquinone structure one ring carbon is significantly closer to the flavin N5, suggesting a direct hydride transfer to this atom.

Figure 1

Sequence alignment of hQR1 (H), mQR1 (M), and rQR1 (R). Secondary elements are named between the vertical lines that mark their beginning and end residues.

Figure 2

Portions of the electron density map (2F_o−F_c) of the three QR1 crystals. (A) apo hQR1 binding site showing the density around the FAD prosthetic group. (B) hQR1-DQ complex showing the density around the substrate. (C) apo mQR1 density in the active site.

Figure 3

Schematic representation of the QR1 dimer. The numbering of the secondary element is indicated. The bound FAD is shown in one of the two sites.

Figure 4

Hydrogen bonding and van der Waals interaction observed between FAD and protein in hQR1. Open radiated circles indicate hydrophobic interactions. Hydrogen bonds are represented by dashed green lines; water molecules are shown as blue filled circles.

Figure 5

Stereo view of the changes that take place upon binding of DQ to hQR1. The catalytic sites of apo hQR1 (blue), hQR1-DQ (black), and apo mQR1 (green) are shown for comparison. Only the FAD, DQ, water molecules, and nonglycine residues that participate in binding are shown.

Figure 6

Stereo view of the overlap of the two DQ complexes known to date. The ternary complex rQR1-CB/DQ is colored red and hQR1-DQ black. Only the FAD, DQ, water molecules, and nonglycine residues that participate in binding are shown.