Research Communication Neuroglobin Ligand Binding Kinetics Laurent Kiger 1 , Julien Uzan 1 , Sylvia Dewilde 2 , Thorsten Burmester 3 , Thomas Hankeln 4 , Luc Moens 2 , Djemel Hamdane 1 , Veronique Baudin-Creuza 1 and Michael C. Marden 1 1 Inserm U473, 94275 Le Kremlin-Bice ˆtre, France 2 Department of Biochemistry, University of Antwerp, B-2610 Antwerp, Belgium 3 Institute of Zoology, Johannes Gutenberg University of Mainz, D-55099 Mainz, Germany 4 Institute of Molecular Genetics, Biosafety Research and Consulting, Johannes Gutenberg University of Mainz, D-55099 Mainz, Germany Summary Neuroglobin, cytoglobin, and hemoglobins from Drosophila melanogaster and Arabidopsis thaliana were studied for their ligand binding properties versus temperature. These globins have a common feature of being hexacoordinated (via the distal histidine) under deoxy conditions, displaying an enhanced amplitude for the alpha absorption band at 560 nm. External ligands can bind, but the transition from the hexacoordinated form to the ligand (L) bound species is slow, as expected for a replacement reaction Fe-His $ Fe $ Fe-L. Histidine binding is on the order of 1 ms; dissociation times are variable, and may be as long as 1 s for the highest histidine affinities. Oxygen binds rapidly but dissociates slowly, requiring as much as 1 s. These rates would correspond to a very high affinity for the pentacoordinated form; however, competition with the distal histidine leads decreases the affinity for the external ligand. The observed oxygen affinity remains in the range of 1 to 10 mm Hg. The low oxygen dissociation indicates a stabilization via H-bonds as for certain globins from parasites (Ascaris, the trematodes). Other ligands such as CO, or CN for the ferric form, show a decreased affinity, since only the competition with the E7 histidine, but not the stabilizing H-bond, plays a role. In addition, the competitive internal ligand leads to a weaker observed temperature dependence of the ligand affinity, since the difference in equilibrium energy for the two ligands is much lower than that of ligand binding to pentacoordinated hemoglobin. This effect could be of biological relevance for certain organisms, since it would lead to an oxygen affinity that is nearly independent of temperature. IUBMB Life, 56: 709–719, 2004 Keywords Neuroglobin; myoglobin; oxygen binding; ligand ki- netics; activation energy. INTRODUCTION Neuroglobin (1) and cytoglobin (2 – 3) share a common feature with the hemoglobins (Hbs) from Drosophila melano- gaster and Arabidopsis thaliana. In the absence of external ligands, they display absorption spectra characteristic of a His-Fe-His hexacoordinated heme (Fig. 1), in which the protein forms two bonds with the iron atom, in addition to the four Fe-N bonds within the heme group. Unlike cytochrome c, the internal protein ligand can be replaced by the usual Hb ligands O 2 , CO, NO. Replacement of the E7 histidine changes the optical properties, confirming the crystallographic struc- tural studies that show the E7 distal histidine as the sixth ligand. Neuroglobin thus displays all of the ligand binding and oxidation reactions known for standard (pentacoordinated) hemoglobins (4 – 6). Many of the hexacoordinated globins show an oxygen affinity similar to that of Mb or Hb, suggesting a conserved function over the long period of genetic divergence. However the high rates of oxidation, and comparatively low concentrations, place some doubt concern- ing the standard role of oxygen transport or stockage (1 – 3, 7). Thus other possible functions of the hexacoordinated globins, such as a participation in redox cycles, are considered in parallel. Analysis of the ligand binding kinetics is more complicated than a simple two state system. In the simplest case, usually satisfied by for most myoglobins, the ligand binding can be described by the rates of transition between two states Fe$Fe- O 2 : liganded or ‘deoxy’ (which occurs as a pentacoordinated form). In this case the equilibrium is described as the ratio of the on and off rates; we will denote this equilibrium coefficient as K penta : k penta ¼ ko 2;on =ko 2;off ð1Þ For the hexacoordinated system, there will be three states involved: Fe-His$Fe$Fe-O 2. Received 15 October 2004; accepted 31 December 2004 Address correspondence to: Laurent Kiger, INSERM U473, 78 rue du General Leclerc, 94275 Le Kremlin-Bicetre Cedex, France. E-mail address: kiger@kb.inserm.fr Abbreviations: Ngb: neuroglobin, Mb: myoglobin. IUBMB Life, 56(11–12): 709–719, November/December 2004 ISSN 1521-6543 print/ISSN 1521-6551 online # 2004 IUBMB DOI: 10.1080/15216540500037711