Structural characterization of spectroscopic substates in carbonmonoxy neuroglobin

Relating structure and spectroscopy is fundamental in characterizing the conformational dynamics and elucidating function at an atomistic level in condensed-phase environments. In particular, the combination of infrared spectroscopy and atomistic simulations has provided fundamental insight into structural assignments of spectroscopic bands. Infrared spectroscopy and Molecular Dynamics (MD) simulations on carbonmonoxy myoglobin (MbCO) were able to partially identify three major CO infrared bands (A0, A1 and A3) which are related to different conformational substates in the active site of the protein. Recently, two similar CO bands were identified from experiments in human carbonmonoxy neuroglobin (NgbCO), named N0 and N3. Time-dependent frequency changes found in the N0 band and a large variation of relaxation times for these bands made the characterization of these substates considerably more difficult compared to MbCO. In this work we discuss the structure-spectroscopy relationship for three different His64 protonation states in human and murine NgbCO using MD simulations and density functional theory (DFT) calculations. The present work assigns the N3 band to the His(epsilon)64 tautomer having its side chain in hydrogen bonding contact to CO. Frequencies of the corresponding His64H+ and His(delta)64 tautomers show characteristic contributions to the N0 band.