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 Å and a comparison with the 1.5-Å 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 (Ϸ290 Å 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.protein crystallography ͉ hypoxia signaling ͉ conformational changes ͉ binding N euroglobin (Ngb) is a recently discovered vertebrate heme protein expressed in the brain (1). The 3D structure of ferric Ngb (metNgb) from human and mouse has recently been published (2, 3). The protein displays all key determinants of the canonical 3-over-3 ␣-helical globin fold (4) despite the very low sequence identify with canonical hemoglobins and myoglobins (Mbs). However, in contrast to Mbs, the heme iron in metNgb is hexacoordinated by the distal and proximal histidines (His-64 and His-96) in the absence of an exogenous ligand (2, 3); spectroscopic data show hexacoordination also for ferrous deoxy Ngb (1,5,6). Endogenous ligation at the sixth coordination position has already been reported for nonsymbiotic plant Hbs and bacterial Hbs (7,8). Interestingly, whereas sperm whale Mb (swMb) displays only small matrix cavities (9), metNgb contains a huge internal tunnel (Ϸ290 Å 3 ) that connects the distal and proximal sides of the heme to the bulk (2, 3). Such a large cavity implies a substantial free energy cost estimated at several kcal͞mol (10).Hexacoordination has been proposed as a novel mechanism to regulate ligand affinity in heme proteins (5-8, 11, 12) because the covalent bond between the heme iron and the distal His has to be broken for an exogenous ligand to bind. In Ngb, the latter reaction has been observed in flash photolysis experiments, in which recombination of CO was found to occur in two steps. The first process (on microsecond time scales at [CO] ϭ ...

Neuroglobin, a recently discovered globin predominantly expressed in neuronal tissue of vertebrates, binds small, gaseous ligands at the sixth coordination position of the heme iron. In the absence of an exogenous ligand, the distal histidine (His64) binds to the heme iron in the ferrous and ferric states. The crystal structure of murine ferric (met) neuroglobin at 1.5 A reveals interesting features relevant to the ligand binding mechanism. Only weak selectivity is observed for the two possible heme orientations, the occupancy ratio being 70:30. Two small internal cavities are present on the heme distal side, which enable the His64(E7) side chain to move out of the way upon exogenous ligand binding. Moreover, a third, huge cavity (volume approximately 290 A3) connecting both sides of the heme, is open towards the exterior and provides a potential passageway for ligands. The CD and EF corners exhibit substantial flexibility, which may assist ligands in entering the protein and accessing the active site. Based on this high-resolution structure, further structure-function studies can be planned to elucidate the role of neuroglobin in physiological responses to hypoxia.

Neuroglobin (Ngb) is a globin expressed in the nervous system of humans and other organisms that is involved in the protection of the brain from ischemic damage. Despite considerable interest, however, the in vivo function of Ngb is still a conundrum. In this paper we report a number of kinetic experiments with O 2 and NO that we have interpreted on the basis of the 3D structure of Ngb, now available for human and murine metNgb and murine NgbCO. kinetics ͉ neuron protection ͉ nitric oxide reduction ͉ physiological role ͉ structural changes N euroglobin (Ngb) is a heme protein belonging to the extended globin family, which was discovered by Burmester et al. (1) in the brain of humans and mice. Interestingly, Ngb is predominantly expressed in specific areas of the brain, notably the frontal lobe, the subthalamic nucleus, and the thalamus (1, 2), although at a fairly low concentration (approximately micromolar) compared with, for example, myoglobin (Mb) in the heart (Ϸ0.2 mM) (3, 4). The unexpected discovery of Ngb raised considerable curiosity from the standpoints of evolutionary biology and pathophysiology alike. The interest in the structure and function of Ngb intensified considerably after the recent findings by Greenberg and collaborators (5, 6) that (i) its expression in neurons is up-regulated under conditions of hypoxia and (ii) the extent of ischemic damage after experimental stroke in the rat is reduced when the amount of Ngb expressed in the brain is enhanced and vice versa.Ngb was found to bind O 2 reversibly at the ferrous heme iron, with an affinity [P 50 Ϸ 2 torr (1 torr ϭ 133 Pa)] (1) comparable with that of Mb (P 50 Ϸ 1 torr) (7). This observation suggested that its function in the brain would mimic that of Mb in red muscles, i.e., storage, transport, and facilitated intracellular diffusion of O 2 . This hypothesis, however, is hardly tenable given the low average concentration of Ngb (8). Like other heme proteins, ferrous Ngb also binds CO and NO (9-13).The 3D structures of metNgb from humans (14) and mice (15) show the typical three-over-three ␣-helical globin fold.Over and above a number of interesting features that emerged from examination of the high resolution 3D structures, two findings stand out: namely (i) the ferric heme iron is hexacoordinated, with the distal His(E7)64 and the proximal His(F8)96 directly bound to the metal ion, and (ii) the protein contains a very large (Ϸ290