The effect of electrostatic substitutions in the heme pocket of myoglobin on the ligand binding and electrochemical properties of three variants of horse heart myoglobin (S92D, V67R, and V68H) have been examined. Introduction of a negative charge on the proximal side of the heme (S92D variant) has no effect on the ability of the protein to bind an exogenous cyanide ligand (K-d = 0.99(4) mu M compared to the value for the wild-type protein of 2.31(4) mu M (pH 7.0, 20.0(1) degrees C, mu = 0.10 M)). The formal potential of the cyano-met derivative of this variant, E degrees' vs SHE, is decreased by 27 mV relative to the potential of the wild-type protein (-412 and -385 mV, respectively (pH 7.0, 20(1) degrees C, mu = 0.10 M)). Introduction of a positive charge on the distal side of the heme resulted in K-d values for cyanide binding of 0.110(9) mu M for the V67R variant and 140(20) mu M for the V68H variant (pH 7.0, 20.0(1) OC, re = 0.10 M). The formal potentials for the cyano-met derivatives of the V67R and V68H variants were -392 and -257 mV, respectively (pH 7.0, 20(1) degrees C, mu = 0.10 M). These results are discussed in terms of charge compensation and alterations in electrostatic potential as a consequence of the mutations.