The binding of azide ion to sperm whale ferrimyoglobin has been measured at 25°C over the pH range 4.0-6.0 at ionic strengths 0.001, 0.002, 0.005, and 0.010 M. The pH dependence of the binding constant, kL, was analyzed in terms of the modified discrete charge electrostatic theory in three ways. First, an "independent-site" model was constructed in which the charged sites that interact with the charged heme iron, and which lose that interaction on neutralization of the iron charge in the azide complex, are considered not to interact with each other. This unrealistic analysis of the heme linkage fails to conform to the experimental results. The subsequent analyses employed the full electrostatic treatment in which each site is taken to interact with each other as well as with the charge borne by the iron. Second, the increase, Δν̄, in binding of hydrogen ions accompanying the azide binding was estimated from the experimental pH dependence of log kL and compared with that computed in terms of the theoretical predictions of the changes in pK values accompanying binding. The agreement was satisfactory under all conditions studied, with divergence of about 25% at most. Third, the observed pH dependence of log kL for each ionic strength was described within 0.10 unit in terms of the summed changes in electrostatic free energy for each charge site accompanying the azide binding as sensed at the iron binding site. The contributions attributed to the individual charged groups were most important where their static solvent accessibilities are low. In these terms, the effective electrostatic domain for interactions with the heme site corresponds to nearly the whole of the myoglobin molecule.
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