Biophysical Characterisation of Neuroglobin of the Icefish, a Natural Knockout for Hemoglobin and Myoglobin. Comparison with Human Neuroglobin

Abstract

The Antarctic icefish Chaenocephalus aceratus lacks the globins common to most vertebrates, hemoglobin and myoglobin, but has retained neuroglobin in the brain. This conserved globin has been cloned, over-expressed and purified. To highlight similarities and differences, the structural features of the neuroglobin of this colourless-blooded fish were compared with those of the well characterised human neuroglobin as well as with the neuroglobin from the retina of the red blooded, hemoglobin and myoglobin-containing, closely related Antarctic notothenioid Dissostichus mawsoni. A detailed structural and functional analysis of the two Antarctic fish neuroglobins was carried out by UV-visible and Resonance Raman spectroscopies, molecular dynamics simulations and laser-flash photolysis. Similar to the human protein, Antarctic fish neuroglobins can reversibly bind oxygen and CO in the Fe2+ form, and show six-coordination by distal His in the absence of exogenous ligands. A very large and structured internal cavity, with discrete docking sites, was identified in the modelled three-dimensional structures of the Antarctic neuroglobins. Estimate of the free-energy barriers from laser-flash photolysis and Implicit Ligand Sampling showed that the cavities are accessible from the solvent in both proteins.Comparison of structural and functional properties suggests that the two Antarctic fish neuroglobins most likely preserved and possibly improved the function recently proposed for human neuroglobin in ligand multichemistry. Despite subtle differences, the adaptation of Antarctic fish neuroglobins does not seem to parallel the dramatic adaptation of the oxygen carrying globins, hemoglobin and myoglobin, in the same organisms.

A distal ligand mutes the interaction of hydrogen sulfide with human neuroglobin

“…2A). The spectrum of sulfide-treated neuroglobin under aerobic conditions appears to be a mixture of species and resembles that of O 2 -bound ferrous neuroglobin, which displays absorbance maxima at 413, 543, and 571 nm (34,35). However, accumulation of O 2 -Fe II -Ngb is excluded because similar spectral changes were induced by sulfide under anaerobic conditions with the exception that a slight increase in the intensity at 558 nm was observed, indicating the presence of a small amount of Fe II -Ngb (not shown).…”

“…We assign this band to the S-S vibration of an iron-bound hydropolysulfide product. This assignment is based on its similarity to the 498 cm Ϫ1 band in a Cys-SS-ligated [2Fe-2S] 2ϩ cluster in the FNR transcriptional factor, which was assigned using 34 S isotope labeling to the S-S stretching mode of the persulfide (57). In the resonance Raman spectrum of Na 2 S 4 , a 482 cm Ϫ1 band was assigned to the S-S vibration (58).…”

Engineered chimeras reveal the structural basis of hexacoordination in globins: A case study of neuroglobin and myoglobin

“…To turn this qualitative observation into a quantitative estimate of rate constants for the microscopic processes, we have analyzed the CO rebinding kinetics with the previously proposed kinetic schemes [22,27,28] or suitably modified in order to account for the observed kinetic heterogeneity, paying attention to the differences in bimolecular rebinding, distal His binding and dissociation kinetics (additional information on geminate recombination can be found in the supporting information). Analysis of CO rebinding kinetics to Mb requires the use of a kinetic model detailed in Scheme 2, where the non-exponential geminate recombination is attributed to migration to an internal cavity and is followed by a bimolecular rebinding step [29].…”

“…Interestingly, recent theoretical work has shown that for Ngb a relevant escape pathway is located in proximity of the CD region [22] and thus the observed change in k −2 may reflect the modulation introduced by replacing the Mb CD region for the Ngb one. From the microscopic rates it is possible to estimate k on,CO , which for Mb is 0.7 × 10 6 M − 1 s − 1 , a value perfectly in line with the literature [14][15][16].…”

“…In the absence of exogenous bound ligands, Ngb is found as a bis-histidyl, 6c species, with the distal HisE7 occupying the sixth coordination site, as revealed by X-ray crystallography and spectroscopy [17,18]. The competition between exogenous ligands and the endogenous HisE7 results in complex patterns of ligand binding kinetics [19][20][21][22]. Once oxygen is bound, formation of a hydrogen bond (HB) with HisE7 results in a small dissociation rate (k off = 0.5-1.5 s − 1 ) [19,21] and an oxygen affinity similar to that of Mb [23].…”