Modulation of protein function by exogenous ligands in protein cavities: CO binding to a myoglobin cavity mutant containing unnatural proximal ligands.
A variety of heterocyclic ligands can be exchanged into the proximal cavity of sperm whale myoglobin mutant H93G, providing a simple method for introduction of the equivalent of unnatural amino acid side chains into a functionally critical location in this protein. These modified proteins bind CO on the distal side. 1H NMR data on H93G(Im)CO, where Im is imidazole, demonstrate that the structure of the distal heme pocket in H93G(Im)CO is very similar to that of wild type; thus, the effects of the proximal ligand's properties on CO binding can be studied with minimal perturbation of distal pocket structure. The exogenous proximal ligands used in this study include imidazole (Im), 4-methylimidazole (4-MeIm), 4-bromoimidazole (4-BrIm), N-methylimidazole (N-MeIm), pyridine (Pyr), and 3-fluoropyridine (3-FPyr). Substitution of the proximal ligand is found to produce substantial changes in the CO on and off rates, the equilibrium binding constant, and the vibrational stretch frequency of CO. Many of the changes are as large as those reported for distal pocket mutants prepared by site-directed mutagenesis. The ability to systematically vary the nature of the proximal ligand is exploited to test the effects of particular properties of the proximal ligand on CO binding. For example, 4-MeIm and 4-BrIm are similar in size and shape but differ significantly in pKa. The same relationship is true for Pyr and 3-FPyr. By comparison of the IR spectra and CO recombination kinetics of these complexes, the effects of proximal ligand pKa on the CO binding are assessed. Likewise, N-MeIm and 4-MeIm are similar in size and pKa but differ in their ability to hydrogen bond to amino acid residues in the proximal cavity. Comparisons of IR spectra and CO binding kinetics in these complexes reveal that proximal ligand conformation and hydrogen bonding affect the kinetics of CO binding. The mechanism of proximal ligand exchange between solution and the proximal cavity in CO complexes was investigated by obtaining the 19F NMR spectrum of H93G(3-FPyr)CO, whose 19F signal can be observed without interference from resonances of the protein. The proximal ligand is found to exchange within a few seconds by saturation transfer. This exchange rate is about 2 orders of magniture faster than what is observed for the isoelectronic metcyano complex [Decatur, S. M., & Boxer, S. G. (1995) Biochemistry 34, 2122-2129]; in both the ferrous CO and ferric cyano complexes, the proximal ligand exchange rate is independent of ligand concentration. These results suggest that the rate-limiting step in proximal ligand exchange is breakage of the iron-ligand bond, followed by rapid diffusion of the ligand through the protein to bulk solution.
Decatur
S M
SM
Department of Chemistry, Stanford University, Stanford, California 94305-5080, USA.
DePillis
G D
GD
Boxer
S G
SG
D000818:Animals; D001665:Binding Sites; D002248:Carbon Monoxide / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D014633:Valine; D014907:Whales
D008024:Ligands; D009211:Myoglobin; D011994:Recombinant Proteins; C018013:carboxymyoglobin; D006418:Heme; D002248:Carbon Monoxide; D014633:Valine