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.

CO bonding in hexa‐ and pentacoordinate carboxy‐neuroglobin: A quantum mechanics/molecular mechanics and local vibrational mode study

“…Initial CO positions in the active site of the wild type protein and the protein mutations were created manually in accordance with previous experimental and theoretical studies of NgbCO (see Figure 1). 30,34,109,114 As suggested in the literature, the dissociated CO preferentially adapts two different orientations, where either CO is located perpendicular to the plane defined by the distal histidine (form A) or parallel to that plane (form B). Furthermore, in each of the two docking forms A and B CO can be placed initially in two opposite orientations before the geometry optimization, one where the C atom is closer to the heme center (denoted in our study as 1) and one where the O atom is closer to the heme center (denoted as 2) which led to 36 protein systems denoted in this work as: F106A δ 1, F106A δ 2, F106A ϵ 1, F106A ϵ 2, F28L δ 1, F28L δ 2, F28L ϵ 1, F28L ϵ 2, F28W δ 1, F28W δ 2, F28W ϵ 1, F28W ϵ 2, H64A1, H64A2, H64Q1, H64Q2, H64V1, H64V2, H64 δ 1, H64 δ 2, H64 ϵ 1, H64 ϵ 2, H64 δϵ 1, H64 δϵ 2, K67A δ 1, K67A δ 2, K67A ϵ 1, K67A ϵ 2, K67R δ 1, K67R δ 2, K67R ϵ 1, K67R ϵ 2, K67T δ 1, K67T δ 2, K67T ϵ 1, and K67T ϵ2 , where 1 and 2 denote complexes obtained from different starting orientations.…”

“…This triggers a larger conformational reorganization of the protein, as confirmed for the crystal structure of carbon monoxide bound murine Ngb. 34 Far-IR and THz domain spectroscopy, 35 resonance Raman, 26,36,37 and time-resolved resonance Raman, 38 complemented with computational investigations 39,40 have suggested that the biological function of Ngb and other globins is related in particular to the strength of the Fe-L bond, however these effects were not quantified.…”

Lack of orientation selectivity of the heme insertion in murine neuroglobin revealed by resonance Raman spectroscopy

“…A unique structural feature of murine Ngb is the heme sliding within the heme crevice upon ligand binding, resulting in an iron displacement of 2 A [18][19][20][21]. Recently, a spectroscopic and structural characterization of the CO complexes of the wild-type (WT) murine Ngb, the CD loop mutant 'Gly-loop [44][45][46][47] ' endowed with enhanced flexibility, the F106A mutant, and the double Gly-loop [44][45][46][47] /F106A mutant (Fig.…”

Probing the Role of Murine Neuroglobin CDloop–D-Helix Unit in CO Ligand Binding and Structural Dynamics

“…The possible involvement of nearby temporary docking sites is suggested by the presence of a process of small amplitude endowed with a lifetime of ∼7 μs that can be identified only in the CDless mutant ( Supporting Information Table S5 ) and that may arise from the migration of photodissociated CO molecules to kinetic traps, as observed for murine and icefish Ngbs at low temperature. 27 , 31 , 38 We used a simple model ( eq 1 ) to describe the geminate rebinding on the nanosecond time scale when His64 recombination is negligible, where we assume that the photodissociated ligand may either exit the protein matrix or rebind from the distal pocket …”