Nitric oxide binding properties of neuroglobin. A characterization by EPR and flash photolysis.
Neuroglobin is a recently discovered member of the globin superfamily. Combined electron paramagnetic resonance and optical measurements show that, in Escherichia coli cell cultures with low O(2) concentration overexpressing wild-type mouse recombinant neuroglobin, the heme protein is mainly in a hexacoordinated deoxy ferrous form (F8His-Fe(2+)-E7His), whereby for a small fraction of the protein the endogenous protein ligand is replaced by NO. Analogous studies for mutated neuroglobin (mutation of E7-His to Leu, Val, or Gln) reveal the predominant presence of the nitrosyl ferrous form. After sonication of the cells wild-type neuroglobin oxidizes rapidly to the hexacoordinated ferric form, whereas NO ligation initially protects the mutants from oxidation. Flash photolysis studies of wild-type neuroglobin and its E7 mutants show high recombination rates (k(on)) and low dissociation rates (k(off)) for NO, indicating a high intrinsic affinity for this ligand similar to that of other hemoglobins. Since the rate-limiting step in ligand combination with the deoxy-hexacoordinated wild-type form involves the dissociation of the protein ligand, NO binding is slower than for the related mutants. Structural and kinetic characteristics of neuroglobin and its mutants are analyzed. NO production in rapidly growing E. coli cell cultures is discussed.
Van Doorslaer
Sabine
S
Department of Physics, University of Antwerp, B-2610 Antwerp, Belgium. Sabine.VanDoorslaer@ua.ac.be
Dewilde
Sylvia
S
Kiger
Laurent
L
Nistor
Sergiu V
SV
Goovaerts
Etienne
E
Marden
Michael C
MC
Moens
Luc
L
D000818:Animals; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D005914:Globins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D000078787:Neuroglobin; D009569:Nitric Oxide / Q000378:metabolism*; D010782:Photolysis; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism
D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D011994:Recombinant Proteins; D009569:Nitric Oxide; D005914:Globins