title: Key Residues Regulating the Reductase Activity of the Human Mitochondrial Apoptosis Inducing Factor.
abstract: The human Apoptosis Inducing Factor (hAIF) is a bifunctional NAD(P)H-dependent flavoreductase involved in both mitochondrial energy metabolism and caspase-independent cell death. Even though several studies indicate that both functions are redox controlled by NADH binding, the exact role of hAIF as a reductase in healthy mitochondria remains unknown. Upon reduction by NADH, hAIF dimerizes and produces very stable flavin/nicotinamide charge transfer complexes (CTC), by stacking of the oxidized nicotinamide moiety of the NAD(+) coenzyme against the re-face of the reduced flavin ring of its FAD cofactor. Such complexes are critical to restrict the hAIF efficiency as a reductase. The molecular basis of the hAIF reductase activity is here investigated by analyzing the role played by residues contributing to the interaction of the FAD isoalloxazine ring and of the nicotinamide moiety of NADH at the active site. Mutations at K177 and E314 produced drastic effects on the hAIF ability to retain the FAD cofactor, indicating that these residues are important to set up the holo-enzyme active site conformation. Characterization of P173G hAIF indicates that the stacking of P173 against the isoalloxazine ring is relevant to determine the flavin environment and to modulate the enzyme affinity for NADH. Finally, the properties of the F310G and H454S hAIF mutants indicate that these two positions contribute to form a compact active site essential for NADH binding, CTC stabilization, and NAD(+) affinity for the reduced state of hAIF. These features are key determinants of the particular behavior of hAIF as a NADH-dependent oxidoreductase.
authors.forename: Raquel
authors.initials: R
authors.lastname: Villanueva
authors.forename: Patricia
authors.initials: P
authors.lastname: Ferreira
authors.forename: Carlos
authors.initials: C
authors.lastname: Marcuello
authors.forename: Alejandro
authors.initials: A
authors.lastname: Usón
authors.forename: M Dolores
authors.initials: MD
authors.lastname: Miramar
authors.forename: M Luisa
authors.initials: ML
authors.lastname: Peleato
authors.affiliation: Fundación ARAID , 50018 Zaragoza, Spain.
authors.forename: Anabel
authors.initials: A
authors.lastname: Lostao
authors.affiliation: INSERM U1138, Cell Death and Drug Resistance in Lymphoproliferative Disorders Team, Centre de Recherche des Cordeliers , F-75006, Paris, France.|Université Pierre et Marie Curie-Sorbonne Universités , F-75006, Paris, France.|Université Paris Descartes-Sorbonne Paris Cité , F-75006, Paris, France.
authors.forename: Santos A
authors.initials: SA
authors.lastname: Susin
authors.forename: Milagros
authors.initials: M
authors.lastname: Medina
mesh_terms: D000595:Amino Acid Sequence; D051033:Apoptosis Inducing Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D017124:Conserved Sequence; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009243:NAD / Q000378:metabolism; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: C494829:AIFM1 protein, human; D051033:Apoptosis Inducing Factor; D009243:NAD