Probing Local Solvation Environments in H-NOX Proteins using 4-Cyano-L-Phenylalanine

Abstract

Heme Nitric Oxide and/or Oxygen (H-NOX) binding domains are gas-sensing domains found in both eukaryotic and prokaryotic cells and are involved in a variety of functions in the cell. The heme pocket in Caldanaerobacter subterraneus H-NOX binds diatomic molecules such as O2, NO, and CO. Crystal structures of Cs H-NOX have provided static structural images of the protein that suggest solvent is inaccessible to the heme pocket. The present study focuses on site-specifically genetically incorporating the unnatural amino acid (UAA) 4-cyano-L-phenylalanine (pCNF) in various distinct sites within the protein. Specifically, pCNF was individually incorporated at sites 5, 36 and 78. Site 36 is a surface site while sites 5 and 78 are in the heme pocket. UV/Vis spectroscopy was utilized to confirm successful heme incorporation, and heme oxidation and ligation state of each construct. FTIR spectral analysis of the pCNF containing protein constructs were recorded for the unligated protein, NO bound, CO bound and O2 bound proteins revealing a distinct blue shift at each ligation state for the 36 site in comparison to the 5 and 78 sites indicative of differing solvation states. This data will be presented illustrating the ability of 4-cyano-L-phenylalanine to be an effective probe of local solvation environments in a biologically relevant protein.