Ligand binding, reactivity and biological activity of a distal pocket mutant of neuroglobin.
We have generated the Lys67Glu mutant form of neuroglobin. Experimental spectral studies are consistent with a six coordinate heme in which the distal histidine bond is stretched compared to the wild type protein. Carbon monoxide binding to the ferrous form of the mutant follows a hyperbolic concentration dependence limiting at the histidine dissociation rate of 0.7 s(-1). Further analysis indicates a significantly lowered histidine binding constant. Oxygen binding kinetic studies confirm the higher heme ligand dissociation level and indicate a p50 value for oxygen binding<1 mmHg. The ferrous form of the protein yields an oxygenated intermediate on reaction with oxygen. The rate of oxidation, by oxygen, follows a complex concentration dependence, consistent with the presence of two distinct oxidation mechanisms. A quantitative model for the two oxidation processes has been developed, which is consistent with a lowered distal histidine binding constant in the mutant form of the protein. These data suggest that the protein structure surrounding the heme site in neuroglobin limits access to external ligands and provides an energy barrier to the structural changes following ligand binding in this protein. However, the mutation does not appear to affect reactivity with cytochrome c and the anti-apoptotic activity of the mutant in human cells of neuronal origin is increased as compared to the wild type protein.
School of Biological Sciences, University of Auckland, Auckland, New Zealand.
J
J
Skommer
S
S
Helbo
K
K
Henty
T
T
Brittain
D017209:Apoptosis / Q000187:drug effects; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005914:Globins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D006418:Heme / Q000737:chemistry; D006801:Humans; D008024:Ligands; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D000078787:Neuroglobin; D010084:Oxidation-Reduction / Q000187:drug effects; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
D008024:Ligands; D050505:Mutant Proteins; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D006418:Heme; D002248:Carbon Monoxide; D005914:Globins; D045304:Cytochromes c; D053453:Caspase 9; D010100:Oxygen