Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine.

The vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNF) was genetically incorporated individually at three sites (5, 36, and 78) in the heme protein H-NOX to probe local hydration environments. The UAA pCNF was incorporated site-specifically using an engineered, orthogonal tRNA synthetase in . The ability of all of the pCNF-containing H-NOX proteins to form the ferrous CO, NO, or O ligated and unligated states was confirmed with UV-Vis spectroscopy.