Probing Local Environments of an Oxygen-Binding Heme-Protein using a Spectroscopically Active Unnatural Amino Acid

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 vital functions such as chemotaxis and signal transduction. The heme-binding pocket of the H-NOX protein from Thermoanaerobacter tencongensis (Tt) has been shown to bind diatomic molecules such as O2, NO, and CO and the protein has been proposed to be able to sense O2 in its native environment. Crystal structures of Tt H-NOX have provided static structural images of the protein that suggest the heme pocket is inaccessible to solvent. Our current study focuses on probing the nature of the local hydration state of the heme pocket in addition to other sites in the protein by genetically incorporating the spectroscopic reporter unnatural amino acid (UAA) p-cyano-L-phenylalanine (pCNF) in a site-specific manner. pCNF is an effective vibrational reporter of local protein environments due to the sensitivity of the nitrile symmetric stretching frequency of this UAA to local environment. The hydration status of the heme pocket and other sites in the protein will be presented as determined utilizing the vibrational reporter UAA pCNF.