Ligand pathways in neuroglobin revealed by low-temperature photodissociation and docking experiments

Abstract

A combined biophysical approach was applied to map gas-docking sites within murine neuroglobin (Ngb), revealing snapshots of events that might govern activity and dynamics in this unique hexacoordinate globin, which is most likely to be involved in gas-sensing in the central nervous system and for which a precise mechanism of action remains to be elucidated. The application of UV–visible microspectroscopy in crystallo, solution X-ray absorption near-edge spectroscopy and X-ray diffraction experiments at 15–40 K provided the structural characterization of an Ngb photolytic intermediate by cryo-trapping and allowed direct observation of the relocation of carbon monoxide within the distal heme pocket after photodissociation. Moreover, X-ray diffraction at 100 K under a high pressure of dioxygen, a physiological ligand of Ngb, unravelled the existence of a storage site for O2 in Ngb which coincides with Xe-III, a previously described docking site for xenon or krypton. Notably, no other secondary sites were observed under our experimental conditions.

Keywords

• ultralow-temperature X-ray crystallography
• XANES
• heme protein
• neuroglobin
• oxygen binding
• cryo-trapping
• crystal microspectroscopy
• neuroprotection
• structural biology
• CO photolysis
• soak-and-freeze pressurization
• structure determination
• protein structure