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.
Highlights
Neuroglobin (Ngb) is a member of the globin protein family that is expressed in the brain ($1 mM) and the retina ($100 mM) of vertebrates (Burmester et al, 2000)
Observed in Mb*CO, (ii) a distance of 2.04 Abetween the heme iron and Npyrrol, which is in good agreement with that measured for Mb, (iii) an elongated distance between the heme and His(F8)96, yielding an Fe—N"(His) distance of 2.24 A, and (iv) a distance of 3.27 Abetween the heme iron and the C atom of the photolyzed CO, which is similar to that reported for the Fe—*CCO distance in Mb*CO, but somewhat different from the Fe— *CCO distance observed in Ngb*CO (2.7 A ) by X-ray diffraction (XRD) in this study
We have used different biophysical approaches to investigate the structure of Ngb hosting diatomic gaseous molecules, such as CO and O2, which can dock in the large cavity surrounding the heme
Summary
Neuroglobin (Ngb) is a member of the globin protein family that is expressed in the brain ($1 mM) and the retina ($100 mM) of vertebrates (Burmester et al, 2000). Ngb is a hexacoordinate protein in the ‘deoxy’ ferrous form, with the rate-limiting step for ligand binding being the spontaneous breakage of the distal His(E7)64–heme bond It has been shown by crystallography (Vallone, Nienhaus, Matthes et al, 2004) that a conformational transition involving a sliding of the heme has to take place in order to create the space to accommodate an external ligand, which is a unique mechanism for ligand-affinity modulation in the globin family. The three-dimensional structure of ferric Ngb (not competent for CO/O2 binding) under increasing O2 pressure was investigated in order to find out whether internal cavities in Ngb may act as storage niches for this small gaseous reactant as observed when using Xe or Kr as probes (Colloc’h et al, 2008; Duff et al, 2004; Lafumat et al, 2016)
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