Biochemical Characterization and Ligand Binding Properties of Neuroglobin, a Novel Member of the Globin Family

Abstract

Neuroglobin is a recently discovered member of the globin superfamily that is suggested to enhance the O 2 supply of the vertebrate brain. Spectral measurements with human and mouse recombinant neuroglobin provide evidence for a hexacoordinated deoxy ferrous (Fe 2؉ ) form, indicating a His-Fe 2؉ -His binding scheme. O 2 or CO can displace the endogenous protein ligand, which is identified as the distal histidine by mutagenesis. The ferric (Fe 3؉ ) form of neuroglobin is also hexacoordinated with the protein ligand E7-His and does not exhibit pH dependence. Flash photolysis studies show a high recombination rate (k on ) and a slow dissociation rate (k off ) for both O 2 and CO, indicating a high intrinsic affinity for these ligands. However, because the ratelimiting step in ligand combination with the deoxy hexacoordinated form involves the dissociation of the protein ligand, O 2 and CO binding is suggested to be slow in vivo. Because of this competition, the observed O 2 affinity of recombinant human neuroglobin is average (1 torr at 37°C). Neuroglobin has a high autoxidation rate, resulting in an oxidation at 37°C by air within a few minutes. The oxidation/reduction potential of mouse neuroglobin (E o ‫؍‬ ؊129 mV) lies within the physiological range. Under natural conditions, recombinant mouse neuroglobin occurs as a monomer with disulfidedependent formation of dimers. The biochemical and kinetic characteristics are discussed in view of the possible functions of neuroglobin in the vertebrate brain.In addition to the well known hemoglobins (Hbs) 1 and myoglobins (Mbs), a third type of globin has recently been described in vertebrates that is predominantly expressed in the brain and other nerve tissues (1). These neuroglobins (NGBs) consist of single chains with 151 amino acids (M r ϭ ϳ17,000) that share only little sequence similarity with the vertebrate globins (Mb Ͻ 21%; Hb Ͻ 25%). Nevertheless, all key determinants of genuine globins are conserved (2). Although NGB was initially discovered in mouse and man, recent data show its presence in many different mammalian species as well as in fish, suggesting the universal occurrence of NGB in vertebrate brains. Nerve-specific globins have been sporadically observed in mollusc, annelid, arthropod, and nemertean species (3-5). These invertebrate nerve globins reach high local concentrations up to the millimolar range, which may be sufficient to facilitate O 2 diffusion or store O 2 that supports cell function during temporary hypoxia (5). The latter assumption is supported by the observation that the nervous function in the mollusc Tellina alternata under anoxic conditions depends on the oxygenation of a nerve globin (6, 7). However, the estimated amount of NGB in the vertebrate brain under nonpathological conditions is only in the micromolar range and thus is much lower than that of a typical invertebrate nerve globin (1). The physiological role of such lowly expressed globins is not well understood. Wittenberg (8) proposed that cytoplasmic globins at low concentrat...

Cytoglobin, a Novel Member of the Globin Family, Protects Kidney Fibroblasts against Oxidative Stress under Ischemic Conditions

“…In neuroglobin, mutation of His to Leu in the E helix increases the ligand-binding constants, indicating that the endogenous ligand can be replaced by external ligands such as oxygen and carbonic monoxide. 46 Based on their spectroscopic analysis, Sawai et al 47 also suggested that His residue is responsible for the globin conformer structure and heme-ligand binding. These findings support experiments demonstrating that the heme domain is an important contributor to radical scavenging.…”

Exploring three different expression systems for recombinant expression of globins: Escherichia coli, Pichia pastoris and Spodoptera frugiperda

“…The CH-IQ-AB domain was PCR-amplified for further cloning from the pENTR4 ADGB vector. The NGB domain was PCR-amplified from pET3a NGB construct, which was already available in our laboratory [15]. For the cloning of CH-IQ-AB in the pPicZα vector, the cDNA of the fragment was synthesized by Invitrogen GeneArt, with optimized codons for expression in P.pastoris.…”

“…The estimated amount of Ngb in the vertebrate brain under non-pathological conditions is only in the micromolar range. It was possible to perform a biochemical and structural characterization of NGB subsequent to the recombinant expression of the protein in E.coli [15].…”

Slow Ligand Binding Kinetics Dominate Ferrous Hexacoordinate Hemoglobin Reactivities and Reveal Differences between Plants and Other Species

“…While hxHbs can bind oxygen and other exogenous ligands, kinetic and equilibrium analysis of the reactions is much more complex than the bimolecular reactions observed with pentacoordinate Hbs due to competition from intramolecular coordination of the binding site. While many functions have been proposed for hxHbs from different organisms, and they have the biochemical capabilities to perform many reactions (4), no clear role has yet been supported to the exclusion of others (6,(8)(9)(10)(11)(12).…”

“…Flash photolysis and rapid mixing have been used to examine ligand binding kinetics in many hxHbs, and both techniques have revealed multiphasic time courses for oxygen and carbon monoxide binding that cannot be accounted for using simple reaction models. In an effort to extract useful binding constants, it has been necessary to disregard heterogeneity in reaction time courses (6,11,12,16,17). For this reason, different binding constants have been reported for some hxHbs that are dependent on how the reactions are initiated or on what time scales they are analyzed (16,17).…”