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Redox-linked conformational dynamics in apoptosis-inducing factor.

ABSTRACT: Apoptosis-inducing factor (AIF) is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis, whose exact role in normal mitochondria remains unknown. Upon reduction with NADH, AIF undergoes dimerization and forms tight, long-lived FADH(2)-NAD charge-transfer complexes (CTC) that are proposed to be functionally important. To obtain a deeper insight into structure/function relations and redox mechanism of this vitally important protein, we determined the X-ray structures of oxidized and NADH-reduced forms of naturally folded recombinant murine AIF. Our structures reveal that CTC with the pyridine nucleotide is stabilized by (i) pi-stacking interactions between coplanar nicotinamide, isoalloxazine, and Phe309 rings; (ii) rearrangement of multiple aromatic residues in the C-terminal domain, likely serving as an electron delocalization site; and (iii) an extensive hydrogen-bonding network involving His453, a key residue that undergoes a conformational switch to directly interact with and optimally orient the nicotinamide for charge transfer. Via the His453-containing peptide, redox changes in the active site are transmitted to the surface, promoting AIF dimerization and restricting access to a primary nuclear localization signal through which the apoptogenic form is transported to the nucleus. Structural findings agree with biochemical data and support the hypothesis that both normal and apoptogenic functions of AIF are controlled by NADH.

SUBMITTER: Sevrioukova IF

PROVIDER: S-EPMC2726748 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Redox-linked conformational dynamics in apoptosis-inducing factor.

Sevrioukova Irina F IF

Journal of molecular biology 20090515 5

Apoptosis-inducing factor (AIF) is a bifunctional mitochondrial flavoprotein critical for energy metabolism and induction of caspase-independent apoptosis, whose exact role in normal mitochondria remains unknown. Upon reduction with NADH, AIF undergoes dimerization and forms tight, long-lived FADH(2)-NAD charge-transfer complexes (CTC) that are proposed to be functionally important. To obtain a deeper insight into structure/function relations and redox mechanism of this vitally important protein ...[more]

PMID: 19447115

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Project description:This Account summarizes recent findings centered on the role that redox partner binding, allostery, and conformational dynamics plays in cytochrome P450 proton coupled electron transfer. P450s are one of Nature's largest enzyme families and it is not uncommon to find a P450 wherever substrate oxidation is required in the formation of essential molecules critical to the life of the organism or in xenobiotic detoxification. P450s can operate on a remarkably large range of substrates from the very small to the very large, yet the overall P450 three-dimensional structure is conserved. Given this conservation of structure, it is generally assumed that the basic catalytic mechanism is conserved. In nearly all P450s, the O2 O-O bond must be cleaved heterolytically enabling one oxygen atom, the distal oxygen, to depart as water and leave behind a heme iron-linked O atom as the powerful oxidant that is used to activate the nearby substrate. For this process to proceed efficiently, externally supplied electrons and protons are required. Two protons must be added to the departing O atom while an electron is transferred from a redox partner that typically contains either a Fe2S2 or FMN redox center. The paradigm P450 used to unravel the details of these mechanisms has been the bacterial CYP101A1 or P450cam. P450cam is specific for its own Fe2S2 redox partner, putidaredoxin or Pdx, and it has long been postulated that Pdx plays an effector/allosteric role by possibly switching P450cam to an active conformation. Crystal structures, spectroscopic data, and direct binding experiments of the P450cam-Pdx complex provide some answers. Pdx shifts the conformation of P450cam to a more open state, a transition that is postulated to trigger the proton relay network required for O2 activation. An essential part of this proton relay network is a highly conserved Asp (sometimes Glu) that is known to be critical for activity in a number of P450s. How this Asp and proton delivery networks are connected to redox partner binding is quite simple. In the closed state, this Asp is tied down by salt bridges, but these salt bridges are ruptured when Pdx binds, leaving the Asp free to serve its role in proton transfer. An alternative hypothesis suggests that a specific proton relay network is not really necessary. In this scenario, the Asp plays a structural role in the open/close transition and merely opening the active site access channel is sufficient to enable solvent protons in for O2 protonation. Experiments designed to test these various hypotheses have revealed some surprises in both P450cam and other bacterial P450s. Molecular dynamics and crystallography show that P450cam can undergo rather significant conformational gymnastics that result in a large restructuring of the active site requiring multiple cis/trans proline isomerizations. It also has been found that X-ray driven substrate hydroxylation is a useful tool for better understanding the role that the essential Asp and surrounding residues play in catalysis. Here we summarize these recent results which provide a much more dynamic picture of P450 catalysis.

Dataset Information

Redox-linked conformational dynamics in apoptosis-inducing factor.

Publications

Redox-linked conformational dynamics in apoptosis-inducing factor.

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