Studies of embryogenesis and metamorphosis in insects reveal the regulatory role of the steroid hormone ecdysone. Ecdysone binds specifically to its target, the ecdysone receptor. Response to ecdysone involves a complex hierarchy of nuclear receptor proteins expressed in a time and tissue dependant fashion, carrying out a variety of gene regulatory activities.

Ecdysone inducible protein 75 (E75), a nuclear receptor (NR) and an early ecdysone responsive gene product, regulates a subset of the developmental activities attributed to ecdysone signaling. D. melanogaster E75 has been recently identified as a hemeprotein and can potentially act as a heme based sensor and transcription factor. The suggested function for E75 is to repress the transcriptional activation by another NR, HR3. The proposed ligand for E75 is nitric oxide [Reinking et. al., Cell, 122, 2005, 195-207].

Investigating the role of E75 as a gas responsive nuclear receptor participating in the ecdysone response is at the heart of our effort. We have cloned the full length protein (AaE75) and the heme binding domain (AaE75HBD) from the yellow fever mosquito Aedes aegypti. We have refolded and purified to homogeneity AaE75HBD from inclusion bodies obtained by recombinant expression in E. coli. The characterization of the HBD using UV-Visible and EPR spectroscopies is underway and will be described. AaE75HBD appears to function as a ferrous histidine ligated heme protein. NO binding, but not CO binding, leads to the apparent heme proximal ligand release, which could be linked to a conformational change of importance for signaling.