The effect of anions on azide binding to myoglobin: an unusual functional modulation.
The effect of increasing concentrations of several anions on the azide (N(-)(3)) binding properties of sperm whale and horse ferric myoglobin has been studied. Surprisingly, a number of anions may act as heterotropic effectors, decreasing the affinity of myoglobins for N(-)(3), in the following order: ClO(-)(4)=I(-)>Br(-)>Cl(-) and SO(2-)(4), which mirrors the increase in their charge density. The largest effects were measured using ClO(-)(4) and I(-), which produce a 4-fold and 8-fold reduction of the N(-)(3) binding affinity in horse and sperm whale myoglobins, respectively. A dissociation equilibrium constant (K(d)) ranging from 150 to 250 mM was estimated for ClO(-)(4) and I(-) binding to myoglobins. In order to analyse the molecular mechanism producing the reduction of the N(-)(3) binding affinity to ferric myoglobin, the potential anionic binding sites within ferric myoglobin were investigated by a molecular modelling study using the program Grid. Analysis of the theoretical results suggests two particularly favourable binding sites: the first, next to the distal side of the haem, whose occupancy might alter the electrostatic potential surrounding the bound N(-)(3); the second, involving residues of helices B and G which are far from the haem iron atom, thus implying a long range effect on the bound N(-)(3). Based on the evidence that no significant conformational changes are found in the three-dimensional structures of N(-)(3)-free and N(-)(3)-bound myoglobin and on previous results on N(-)(3) binding to ferric myoglobin mutants in CD3 positions, we favour the first hypothesis, suggesting that the functional heterotropic modulation of monomeric myoglobin is mainly depending on a decrease of the positive charge density induced by the binding of anions to the haem distal side.
De Rosa
M Cristina
MC
Institute of Chemistry and Clinical Chemistry, and C.N.R. Centre of Receptor Chemistry, Catholic University of Rome, Largo F. Vito 1, 00168 Rome, Italy.
Bertonati
Claudia
C
Giardina
Bruno
B
Di Stasio
Enrico
E
Brancaccio
Andrea
A
D000494:Allosteric Regulation; D000818:Animals; D000838:Anions; D001386:Azides / Q000737:chemistry*; D001665:Binding Sites; D002704:Chlorates / Q000737:chemistry; D006736:Horses; D007452:Iodates / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry*; D013053:Spectrophotometry; D014907:Whales
D000838:Anions; D001386:Azides; D002704:Chlorates; D007452:Iodates; D008024:Ligands; D009211:Myoglobin