Cornelius Taabazuing

Abstract:

Hypoxia Inducible Factor (HIF) is a transcription activator that acts as the primary regulator of O₂ homeostasis in mammals. HIF controls over 100 genes in response to variations in O₂ tension, inducing the expression of genes central to processes such as angiogenesis, erythropoiesis, and metabolism. As hypoxia is implicated in the progression of cancer, hypertension, and myocardial ischemia, understanding how O₂ sensing leads to induction of genes that are involved in the pathogenesis of these diseases is crucial for therapeutic development. Although HIF regulates O₂ homeostasis, it does not directly sense O₂, rather, HIF relies on two hydroxylases, Prolyl Hydroxylase Domain 2 (PHD2) and Factor Inhibiting HIF (FIH) to sense O₂ and control HIF transcriptional activity via posttranslational modifications.

FIH is an Fe(II)/αKG-dependent dioxygenase that inactivates HIF transcriptional activity through hydroxylation of an asparagine residue (Asn⁸⁰³) in the C-terminal transactivation domain (CTAD) of HIF. The Fe(II)/αKG-dependent enzymes are a large and functionally diverse superfamily of mononuclear non-heme oxygenases that couple the oxidative decarboxylation of the co-substrate, αKG, to various chemical reactions, including hydroxylation, desaturation, and ring expansions. Early steps in catalysis, specifically O₂ binding and activation, are not well understood for the αKG-oxygenases yet these are key to understanding their chemistry. As FIH is one of the primary O₂ sensors, elucidating these steps are important for understanding its function.

We report here our use of NO as an O₂ mimic to probe for the gas binding pocket of FIH, and to identify structural factors that induce O₂ binding and activation through a combination of {FeNO}⁷ EPR spectroscopy, and UV-Visible spectroscopy. We demonstrate that substrate binding triggers O₂ binding as the affinity for NO increased. Importantly, the target residue (Asn⁸⁰³) on the HIF-1α substrate is vital for O₂ binding and activation as a distinct electronic environment corresponding to the O₂ activated state is formed upon HIF-1α binding. Mutation of Asn⁸⁰³ to Gln/Ala results in loss of the proper electronic environment which promotes chemistry. The active site aspartate is also important for O₂ activation as the electronic structure suggests a constitutively activated enzyme complex upon mutation of this residue. Structural and computational analysis indicate rotation about the Fe-NO bond induces O₂ activation and this mechanism may be conserved for the broader class of enzymes.