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.

Defining NADH-Driven Allostery Regulating Apoptosis-Inducing Factor

“…Consistent with other reported H454 mutations (Churbanova and Sevrioukova, 2008; Villanueva et al, 2015), substitution of the active site histidine eliminates AIF’s ability to form a charge-transfer complex with NADH, and the protein rapidly converts excess NADH to NAD + through catalytic hydride removal (Figure S5A). Biochemical and SAXS analyses show that the H454A mutant exists as a dimer in solution in the absence of NADH and uphold H454 as the key active site residue that regulates AIF dimerization (Figure 5A–B, S2).…”

Apoptosis-Inducing Factor (AIF) in Physiology and Disease: The Tale of a Repented Natural Born Killer

“…The NADH-binding region possesses a sequence homology to bacterial NADH-dependent ferredoxin reductase and to the phylogenetically related yeast NADH:ubiquinone oxidoreductase Ndi1p ( Mate et al, 2002 ; Elguindy and Nakamaru-Ogiso, 2015 ). The folded AIF polypeptide undergoes dimerization upon NADH reduction, while the subsequent conformational change stabilizes the reduced form of the molecule, increasing AIF resistance to oxidation ( Sevrioukova, 2009 ; Churbanova and Sevrioukova, 2008 ; Villanueva et al, 2015 ). The C-terminal region contributes to the dimeric interface and determines the binding with other molecules, including the DNA.…”

WAH-1/AIF regulates mitochondrial oxidative phosphorylation in the nematode Caenorhabditis elegans

“…The mature form of AIF is mainly tethered to the mitochondrial inner membrane 16 , 20 , 21 . Structural and biochemical characterizations of in vitro purified AIF show that NADH incorporation determines the folding, the redox status, and the dimerization of the FAD-containing AIF protein 22 – 25 . In addition to its death-related role, AIF also contributes to cellular bioenergetics, as it assists in the assembly and/or stabilization of the electron transport chain (ETC) 26 .…”