The effect of anions on azide binding to myoglobin: an unusual functional modulation

The effect of increasing concentrations of several anions on the azide (N3 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 N3 3 , in the following order: ClO3 4 =I3 sBr3 sCl3 and SO23 4 , which mirrors the increase in their charge density. The largest effects were measured using ClO3 4 and I3, which produce a 4-fold and 8-fold reduction of the N3 3 binding affinity in horse and sperm whale myoglobins, respectively. A dissociation equilibrium constant (Kd) ranging from 150 to 250 mM was estimated for ClO3 4 and I3 binding to myoglobins. In order to analyse the molecular mechanism producing the reduction of the N3 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 N3 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 N3 3 . Based on the evidence that no significant conformational changes are found in the three-dimensional structures of N3 3 -free and N3 3 -bound myoglobin and on previous results on N3 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.