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Chemical Consequences of Heme Distortion and the Role of Heme Distortion in Signal Transduction of H-NOX Proteins

Abstract

Nitric oxide (NO) signaling in mammals controls important processes such as smooth muscle relaxation and neurotransmission by the activation of soluble guanylate cyclase (sGC). NO binding to the heme domain of sGC leads to dissociation of the iron-histidine (Fe-His) bond, which is required for enzyme activity. The heme domain of sGC belongs to a larger class of proteins called H-NOX (Heme-Nitric oxide/OXygen) binding domains. H-NOX proteins act as sensors for nitric oxide (NO) or oxygen (O2). The crystal structure of a H-NOX domain from Thermoanaerobacter tengcongensis (Tt H-NOX) contains one of the most distorted hemes reported to date. In this dissertation, I engineered Tt H-NOX to adopt a flatter heme by mutating a conserved residue in the H-NOX family. Decreasing heme distortion in Tt H-NOX increases affinity for O2 and decreases the reduction potential of the heme iron. Additionally, flattening the heme is associated with significant shifts at the N-terminus of the protein. These results show a clear link between the heme conformation and Tt H-NOX structure and demonstrate that heme distortion is an important determinant for maintaining biochemical properties in H-NOX proteins.

The reduction potential of Tt H-NOX was rationally modulated through mutations of the protein scaffold. The degree of heme distortion is directly correlated with the reduction potential of the heme iron. Inducing planarity in the heme causes overlap of the porphyrin orbitals with the d-orbitals of the iron, leading to increased electron density at the iron, which lowers the reduction potential. Rational design of Tt H-NOX to broaden the reduction potential range and change the function of the protein may potentially be used for further applications.

NMR solution structures of H-NOX proteins show a conformational change upon disconnection of the heme and proximal helix. The atomic details of these conformational changes are lacking in the NMR structures, however, especially at the heme pocket. I solved a high-resolution crystal structure of a H-NOX mutant mimicking a broken Fe-His bond. This mutant exhibits specific heme conformational changes and a major N-terminal displacement relative to the wild-type H-NOX protein. Fe-His ligation is ubiquitous in all H-NOX domains, thus the heme and protein conformational changes observed in this study are likely to occur throughout the H-NOX family when NO binding ruptures the Fe-His bond.

All mechanistic studies on H-NOX activation have been focused on the response to NO. However, little is known about the sub-class of H-NOX proteins that are predicted to sense O2. A key question is how these H-NOX proteins respond to O2 binding. I solved the crystal structure of an unligated mimic of Tt H-NOX at high resolution. When compared to wild-type, which is O2-bound, large conformation changes in the protein and heme are observed. Essentially, O2 acts as a link between the N- and C-terminal domains in Tt H-NOX and locks the protein and heme in a particular state. Tt H-NOX is part of a larger protein that contains a methyl-accepting chemotaxis domain that is predicted to sense O2 in T. tengcongensis. Follow-up experiments will focus on developing an assay in Tar4 that can test the response to O2 and the importance of heme distortion on activity.

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