The regulation of oxygen homeostasis is crucial to our existence as aerobes. At the cellular level, oxygen sensing is implicated in a large number of biomedically relevant processes, including the immune response, pre-natal lung development, erythropoiesis, and angiogenesis. When oxygen levels become too low (hypoxia), the hypoxia inducible factor (HIF) induces the expression of over 70 genes, controlling cellular O2 regulation. The key players in the [O2] response are two enzymes known as HIF-hydroxylases, which regulate the activity level of HIF. The long-range goal of this proposal is to understand the oxygen dependent reactivity of the HIF-hydroxylases.

The HIF-hydroxylases belong to the Fe(II), alpha-ketoglutarate dependent oxygenase superfamily. The two known varieties of HIF-hydroxylases are HIF-prolyl hydroxylase (PHD) and HIF-asparaginyl hydroxylase (FIH), which hydroxylate discrete domains of the alpha subunit of HIF (HIF ). PHD and FIH are the primary O2 sensors in humans, inactivating HIF  under conditions of normal or elevated [O2]. Consequently, understanding the mechanism of these enzymes may provide avenues to control cellular responses to O2 levels. 


  • Regulating angiogenesis - hypoxia sensing by non-heme oxygenases

While much is known about  KG oxygenases in general, the molecular details of O2 activation remain largely speculative, and the structural link between substrate and O2 binding is unclear. We are developing a combined biophysical, mechanistic, and spectroscopic approach to understand the oxygen-dependent activity of HIF hydroxylases and related enzymes. Kinetic isotope effects, mutagenesis, and single turnover kinetics are being used to delineate the chemical mechanism of O2 activation. The chemical and structural relationship between substrate and O2 binding is being tested by amide exchange methods. Non-productive reactions with O2 are also being explored, as they may represent a physiological inactivation pathway for this class of enzyme. This research will identify structures unique to the HIF hydroxylases which regulate their oxygen-dependent reactivity, as well as better understanding of O2-activation in the broad class of  KG oxygenases.
  • Controlling charge transfer chemistry in nanoparticle/metalloenzyme hybrids

Increasing the efficiency of dye-sensitized solar cells is crucial to harvesting the sun's energy for our use.  Solar cells of the future will ideally combine the efficient electron/hole separation of semiconductors within a self-assembled architecture. We are collaborating with other groups to explore hybrid nanoparticle/protein systems as an approach to developing self-assembled structures with protein-based charge-transport.  We are using surface-functionalized nanoparticles to recognize redox proteins to explore hybrid systems which support charge transfer, with the ultimate goal being the creation of "green" systems for solar cell applications.
We have focused our attention on developing nanoparticle hybrids with cytochrome c (Cyt c) and cytochrome c peroxidase (CcP) due to their facile spectroscopy, the simple chemistry of reducing H2O2, and their well-characterized binding interfaces.  We have shown that polymeric and gold nanoparticles with simple functionalities interrupt the Cc:CcP electron transfer by selectively binding to the protein surface, and showed that gold nanoparticles functionalized with single amino-acids bind to Cyt c on the same surfaces as natural redox partners.  We recently mapped the surface of Cyt c which binds to different nanoparticles through the use of amide exchange methods (amide HDX).  This work showed that the functionality of the nanoparticle led to different binding modes on Cyt c, and implicates a combination of coulombics and hydrophobicity in determing facial specificity for binding.  We are extending this work to correlate redox reactivity with binding mode in both Cyt c and CcP.