Ligand binding and protein dynamics in neuroglobin

Abstract

Neuroglobin (Ngb) is a recently discovered protein in vertebrate brain tissue that belongs to the globin family of proteins. It has been implicated in the neuronal response to hypoxia or ischemia, although its physiological role has been hitherto unknown. Ngb is hexacoordinate in the ferrous deoxy form under physiological conditions. To bind exogenous ligands like O₂ and CO, the His E7 endogenous ligand is displaced from the sixth coordination. By using infrared spectroscopy and nanosecond time-resolved visible spectroscopy, we have investigated the ligand-binding reaction over a wide temperature range (3–353 K). Multiple, intrinsically heterogeneous distal heme pocket conformations exist in NgbCO. Photolysis at cryogenic temperatures creates a five-coordinate deoxy species with very low geminate-rebinding barriers. The photodissociated CO is observed to migrate within the distal heme pocket even at 20 K. Flash photolysis near physiological temperature (275–353 K) exhibits four sequential kinetic features: (i) geminate rebinding (t < 1 μs); (ii) extremely fast bimolecular exogenous ligand binding (10 μs < t < 1 ms) with a nontrivial temperature dependence; (iii) endogenous ligand binding (100 μs < t < 10 ms), which can be studied by using flash photolysis on deoxy Ngb; and (iv) displacement of the endogenous by the exogenous ligand (10 ms < t < 10 ks). All four processes are markedly nonexponential, suggesting that Ngb fluctuates among different conformations on surprisingly long time scales. Globins are proteins that bind dioxygen and other small ligands at the central iron of a heme prosthetic group embedded in a highly conserved α-helical “globin” fold. Hemoglobin (Hb) and myoglobin (Mb) are the most prominent members of this protein family (1). Interrelations among structure, dynamics, and function in globins have been investigated in great detail, most likely more thoroughly than for any other protein family. Mb especially has, for a long time, served as a paradigm in the biological physics of proteins (2, 3).Recently, two new members have joined the globin family, cytoglobin (4) and neuroglobin (Ngb) (5). The latter consists of a single chain of 151 aa. Ngb, which occurs in neurons, has less than 25% sequence identity with Mb or Hb but nevertheless displays all key determinants of the globin fold (6). It has moderate oxygen affinity and seems most closely related to the globin found in the glial cells of the annelid Aphrodite (7). The discovery of a six-coordinate heme in the deoxy form of Ngb (5), with the His E7 side chain occupying the sixth coordination, came somewhat as a surprise because it was believed for a long time that a pentacoordinate heme iron, with a vacant ligand-binding site, was a common characteristic of globins. In recent years, however, hexacoordinate globins also have been isolated from bacteria, unicellular eukaryotes, and plants [nonsymbiotic Hbs, nsHbs (8, 9), and truncated Hbs, trHbs (10)]. In these proteins, the exogenous ligand has to compete with an intramolecular side chain for the binding site, and therefore, hexacoordination appears as a novel mechanism for regulating ligand-binding affinity. As of yet, no precise physiological role has been assigned to hexacoordinate globins. A recent study has reported an up-regulation of Ngb under conditions of hypoxia or ischemia, thus promoting neuronal survival (11). To understand the function of Ngb in the vertebrate brain and that of hexacoordinate globins in general, a thorough investigation of the structure, dynamics and ligand-binding (equilibrium and kinetic) properties is a prerequisite. First kinetic studies on Ngb at room temperature have reported extremely fast CO and O₂ association to the pentacoordinate deoxy form (12, 13). The two papers disagreed, however, by a factor of 1,000 in the His dissociation rate from the hexacoordinate deoxy form, which essentially governs exogenous ligand binding at physiological temperature. Here, we present a ligand-binding study of NgbCO in the infrared and visible spectral regions over a wide range of temperature (3–353 K). The results are quite peculiar when compared with HbCO or MbCO and provide important insights into both structural and dynamic properties in Ngb's reaction with ligands.