Fluoride binding in hemoproteins: the importance of the distal cavity structure.

The electronic absorption and resonance Raman spectra of the fluoride complexes of various peroxidases and selected site-directed mutants have been studied at pH 5.0, and compared to the spectra obtained for the myoglobin-F adduct. It is shown that the electronic absorption maxima depend on the degree of conjugation between the porphyrin macrocycle and the vinyl substituents. Moreover, it is confirmed that the wavelength of the CT1 band is a sensitive probe of axial ligand polarity and of its interaction with the distal protein residues. The results highlight the different mechanism of stabilization of the fluoride ligand exerted by the distal residues in myoglobin and peroxidases. In peroxidases, the Arg is determinant in controlling the ligand binding via a strong hydrogen bond between the positively charged guanidinium group and the anion. Mutation of Arg to Leu decreases the stability of the complex by 900-fold, suggesting that this interaction stabilizes the complex by 4 kcal/mol. The distal His also contributes to the stability of the fluoride complex, presumably by accepting a proton from HF and hydrogen-bonding, through a water molecule, to the anion. Mutation of His to Leu decreases the stability of the fluoride complex by 30-fold, suggesting that this interaction is much weaker than the interaction with the distal Arg. For Mb, the distal His is solely responsible for stabilization of the exogenous ligand.