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J. Biol. Chem., Vol. 282, Issue 41, 30331-30340, October 12, 2007

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DNA Binding Suppresses Human AIF-M2 Activity and Provides a Connection between Redox Chemistry, Reactive Oxygen Species, and Apoptosis^*

Min Gong, Sam Hay, Ker R. Marshall, Andrew W. Munro, and Nigel S. Scrutton¹

From the Manchester Interdisciplinary Biocentre and Faculty of Life Sciences, University of Manchester, M1 7DN Manchester and the Department of Biochemistry, University of Leicester, Leicester, United Kingdom

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-B, 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-(319-613), 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.

Received for publication, May 4, 2007 , and in revised form, July 23, 2007.

^* This work was funded in part by the UK Biotechnology and Biological Sciences Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S6, Table S1, and additional references.

¹ Biotechnology and Biological Sciences Research Council Professorial Research Fellow. To whom correspondence should be addressed: Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess St., M1 7DN Manchester, UK. Tel.: 44-161-306-51-52; E-mail: nigel.scrutton{at}manchester.ac.uk.

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