Structural characterization of spectroscopic substates in carbonmonoxy neuroglobin

Abstract

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_ε64 tautomer having its side chain in hydrogen bonding contact to CO. Frequencies of the corresponding His64H⁺ and His_δ64 tautomers show characteristic contributions to the N₀ band.