Anion-specific interaction with human NQO1 inhibits flavin binding.

Ion binding to biomacromolecules can modulate their activity and stability in vivo. It is of particular interest to understand the structural and energetic basis of anion binding to functional sites of biomacromolecules. In this work, binding of anions to the FAD binding pocket of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with cancer due to a common polymorphism causing a P187S amino acid substitution, was investigated. It is known that NQO1 stability in vivo is strongly modulated by binding of its flavin cofactor. Herein, binding and protein stability analyses were carried out to show that anion binding to the apo-state of NQO1 P187S inhibits FAD binding with increasing strength following the chaotropic behavior of anions. These inhibitory effects were significant for some anions even at low millimolar concentrations. Additional pH dependent analyses suggested that protonation of histidine residues in the FAD binding pocket was not critical for anion or flavin binding. Overall, this detailed biophysical analysis helps to understanding how anions modulate NQO1 functionality in vitro, thus allowing hypothesize that NQO1 stability in vivo could be modulated by differential anion binding and subsequent inhibition of FAD binding.