Structural dynamics in the active site of murine neuroglobin and its effects on ligand binding.
We have examined the effects of active site residues on ligand binding to the heme iron of mouse neuroglobin using steady-state and time-resolved visible spectroscopy. Absorption spectra of the native protein, mutants H64L and K67L and double mutant H64L/K67L were recorded for the ferric and ferrous states over a wide pH range (pH 4-11), which allowed us to identify a number of different species with different ligands at the sixth coordination, to characterize their spectroscopic properties, and to determine the pK values of active site residues. In flash photolysis experiments on CO-ligated samples, reaction intermediates and the competition of ligands for the sixth coordination were studied. These data provide insights into structural changes in the active site and the role of the key residues His64 and Lys67. His64 interferes with exogenous ligand access to the heme iron. Lys67 sequesters the distal pocket from the solvent. The heme iron is very reactive, as inferred from the fast ligand binding kinetics and the ability to bind water or hydroxyl ligands to the ferrous heme. Fast bond formation favors geminate rebinding; yet the large fraction of bimolecular rebinding observed in the kinetics implies that ligand escape from the distal pocket is highly efficient. Even slight pH variations cause pronounced changes in the association rate of exogenous ligands near physiological pH, which may be important in functional processes.
Nienhaus
Karin
K
Department of Biophysics, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany.
Kriegl
Jan M
JM
Nienhaus
G Ulrich
GU
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D005914:Globins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000078787:Neuroglobin; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D008024:Ligands; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D005914:Globins