Biology Facilitated by Heme Proteins as Seen in Cimex Nitrophorin and Ecdysone Inducible Protein 75

This dissertation is a study in how heme facilitates biology using two heme proteins as examples. I write about my mechanistic studies on Cimex nitrophorin and preliminary studies on Ecdysone inducible protein 75, respectively. Nitrophorins are salivary heme proteins used by bloodfeeding insects to deliver NO to the victim, leading to vasodilation and antihemostasis. The bedbug nitrophorin cNP, a thiolate heme protein accomplishes this via an unusual heme-assisted S-nitrosation reaction, requiring proximal ligand cleavage. This dissertation explores this mechanism through mutational, crystallographic and transient kinetic approaches. I present the detailed investigation of the two NO binding events, one at the heme and the other at the proximal cysteine. The heme nitrosyl shows marked pH dependence arising out of the apparent protonation of the proximal cysteine ligand, a feature crucial to cNP function. The structures and spectroscopy of cNP mutant proteins reveal the SNO modification to be regulatory in nature. Laser flash photolysis measurements and the structures of mutant proteins reveal the negative influence of steric hindrance on SNO stability.Studies of insect embryogenesis and metamorphosis reveal the regulatory role of the hormone ecdysone via its target, the ecdysone receptor. Ecdysone triggers expression of several nuclear receptors in a time and tissue dependant fashion, which in turn carry out gene regulation. Ecdysone inducible protein 75 (E75), a nuclear receptor and an early ecdysone responsive gene product, regulates a subset of the developmental activities attributed to ecdysone. We are investigating E75 from Aedes aegypti to uncover its role in ecdysone signaling in mosquitoes. I have expressed and partially purified the full length protein using the baculovirus driven expression in SF9 cells, and purified to homogeneity the heme binding domain resolubilized from inclusion bodies obtained by expression in E. coli. Preliminary characterization of the proteins using UV-visible spectroscopy indicates that E75 has a b type heme with a low spin six-coordinate ferric iron. In the E75 heme binding domain, the heme exhibits an unstable ferrous state and only binds NO and CO at high non-physiological levels. These data place into doubt the suggested roles for E75 as a gas regulated transcription regulator