The structure of carbonmonoxy neuroglobin reveals a heme-sliding mechanism for control of ligand affinity

Abstract

Neuroglobin (Ngb), a globular heme protein expressed in the brain of vertebrates, binds oxygen reversibly, with an affinity comparable to myoglobin (Mb). Despite low sequence identity, the overall 3D fold of Ngb and Mb is very similar. Unlike in Mb, in Ngb the sixth coordination position of the heme iron is occupied by the distal histidine, in the absence of an exogenous ligand. Endogenous ligation has been proposed as a unique mechanism for affinity regulation and ligand discrimination in heme proteins. This peculiarity might be related to the still-unknown physiological function of Ngb. Here, we present the x-ray structure of CO-bound ferrous murine Ngb at 1.7 A and a comparison with the 1.5-A structure of ferric bis-histidine Ngb. We have also used Fourier transform IR spectroscopy of WT and mutant CO-ligated Ngb to examine structural heterogeneity in the active site. Upon CO binding, the distal histidine retains (by and large) its position, whereas the heme group slides deeper into a preformed crevice, thereby reshaping the large cavity ( approximately 290 A(3)) connecting the distal and proximal heme sides with the bulk. The heme relocation is accompanied by a significant decrease of structural disorder, especially of the EF loop, which may be the signal whereby Ngb communicates hypoxic conditions. This unexpected structural change unveils a heme-sliding mechanism of affinity control that may be of significance to understanding Ngb's role in the pathophysiology of the brain.

Fig. 1.

Stereo diagrams of the structures of NgbCO (red) and unligated metNgb (magenta) in ribbon representation. (A) Overall structures, the distal histidine (His-64), the proximal histidine (His-96), Phe-106, the heme group, and loops EF and FG are highlighted. Green regions indicate the positions of the two point mutations, Cys-55–Ser and Cys-120–Ser. (B) Close-up view of the active site, also including Tyr-44 and Phe-106.

Fig. 2.

Plots of rmsds versus primary sequence position. (A) rmsds between C atoms of pairs of structures after optimal alignment of swMbCO and deoxy swMb (gray dashed line), NgbCO and metNgb (gray solid line), NgbCO and swMbCO (black dashed line), and metNgb and met swMb (black solid line). (B) rmsds representing disorder within a single structure estimated from isotropic B factor refinement of the structures of NgbCO (solid line) and metNgb (dashed line). Bars and capital letters indicate the helical segments in murine Ngb.

Fig. 3.

Cavities in NgbCO (blue) and unligated metNgb (yellow) as determined by using surfnet (46). The position of Ser-55 is highlighted in green.

Fig. 4.

FTIR absorbance difference spectra of the stretching bands of hemebound CO in WT Ngb at pH 7.3 (solid line) and pH 5.3 (dashed line) (A), mutant Lys-67–Leu at pH 7.3 (solid line) and pH 5.5 (dashed line) (B), and mutants His-64–Leu (pH 7.4) and His-64–Leu–Lys-67–Leu (pH 7.3) (C). Difference spectra were calculated from absorbance spectra taken before and after illumination with 532-nm light at 3 K.