Human AIF-M2 is an unusual flavoprotein oxidoreductase that binds DNA, nicotinamide coenzyme, and the modified flavin 6-hydroxy-FAD. Using multiple solution methods to investigate the redox chemistry and binding interactions of AIF-M2, we demonstrate that binding of DNA and coenzyme to AIF-M2 is mutually exclusive. We also show that DNA binding does not perturb the redox chemistry of AIF-M2, but it has significant effects on the reduction kinetics of the 6-hydroxy-FAD cofactor by NAD(P)H. Based on quantitative analysis of ligand binding and redox chemistry, we propose a model for the function of AIF-M2. In this model, DNA binding suppresses the redox activity of AIF-M2 by preventing the binding of the reducing coenzyme NAD(P)H. This DNA-mediated suppression of AIF-M2 activity is expected to lower cellular levels of superoxide and peroxide, thereby lessening survival signaling by Ras, NF-κB, or AP-1, as suggested from knock-out studies of the related AIF in human colon cancer cell lines. We show marked differences between AIF-M2 and AIF. DNA and coenzyme binding activity is retained in the C-terminal deletion mutant AIF-M2-(Δ319-613), whereas DNA binds to the C-terminal D3 domain of AIF. Our work provides the first analysis of AIF-M2 ligand interactions and redox chemistry and identifies an important mechanistic connection between coenzyme and DNA binding, redox activity, and the apoptotic function of AIF-M2. Through its DNA binding activity, we suggest that AIF-M2 lessens survival cell signaling in the presence of foreign (e.g. bacterial and (retro)viral) cytosolic DNA, thus contributing to the onset of apoptosis. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.