Abstract
Neuroglobin (Ngb) is a newly discovered oxygen-binding heme protein that is primarily expressed in the brain of humans and other vertebrates. To characterize the structure/function relationships of this new heme protein, we have used resonance Raman spectroscopy to determine the structure of the heme environment in Ngb from mice. In the Fe(2+)CO complex, two conformations of the Fe-CO unit are present, one of which arises from an open conformation of the heme pocket in which the CO is not interacting with any nearby residue, and the other arises from a closed conformation where a positively charged residue near the CO group stabilizes the complex. For the Fe(2+)O(2) complex, we detect a single nu(Fe-OO) stretching mode at a frequency similar to that of oxymyoglobins and oxyhemoglobins of vertebrates (571 cm(-1)). Based on the Fe-C-O frequencies of the closed conformation of Ngb, a highly polar distal environment is indicated from which the O(2) off-rate is predicted to be lower than that of Mb. In the absence of exogenous ligands, a heme pocket residue coordinates to the heme iron, forming a six-coordinate complex, thereby predicting a low on-rate for exogenous ligands. These structural properties of the heme pocket of Ngb are discussed with respect to its proposed in vivo oxygen delivery function.
Highlights
Neuroglobin (Ngb) is a newly discovered oxygen-binding heme protein that is primarily expressed in the brain of humans and other vertebrates
In the Fe2؉CO complex, two conformations of the Fe–CO unit are present, one of which arises from an open conformation of the heme pocket in which the CO is not interacting with any nearby residue, and the other arises from a closed conformation where a positively charged residue near the CO group stabilizes the complex
According to multiple sequence alignment, key amino acid residues that are required for the function of these proteins and are conserved among a great variety of globins are present in the primary amino acid sequence of Ngb: namely a proximal histidine residue that coordinates to the heme, a distal histidine residue that might be involved in interactions with heme-bound ligands (E7), and a phenylalanine residue at CD1 that is involved in - interactions with the heme [4]
Summary
Myoglobin; Hb, hemoglobin; Ngb, neuroglobin; CAPS, 3-(cyclohexylamino)propanesulfonic acid; HMP, flavohemoglobin; mW, milliwatts. The primary amino acid sequence of Ngb is only 21–25% identical to that of Mb and Hb [4]. According to multiple sequence alignment, key amino acid residues that are required for the function of these proteins and are conserved among a great variety of globins are present in the primary amino acid sequence of Ngb: namely a proximal histidine residue that coordinates to the heme (helical position F8), a distal histidine residue that might be involved in interactions with heme-bound ligands (E7), and a phenylalanine residue at CD1 that is involved in - interactions with the heme [4]. We present here a detailed characterization of the heme environment in Ngb using Raman spectroscopy, which is a very powerful technique that provides information about the axial ligands of the heme and the overall environment of the heme pocket. We discuss our results with respect to the kinetic properties of Ngb and the proposed function of this heme protein
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