The identity of the axial ligands provided by the protein to complete the coordination environment of the heme iron is one of the major structural determinants of heme protein functional properties. In the current work, the distal valine 68 residue in horse heart myoglobin has been replaced with a histidyl residue (Val68His), and the variant protein has been characterized by electronic absorption, MCD and EPR spectroscopies, and spectroelectro-chemistry. The electronic absorption spectrum of the oxidized form of the variant at ambient (25.0 degrees C) temperatures exhibits absorption maxima similar to those of cytochrome b(5). An additional high-spin component that is nest present at 77 K is also apparent. The low-temperature (77 K) spectrum of the reduced form of the variant exhibits the characteristic alpha and beta bands that characterize the spectrum of ferrocytochrome b(5). Near-IR MCD spectroscopy (300 K) of the oxidized derivative reveals an intense transition at 1607 nm that is similar to those observed for cytochrome b(5) and imidazole-Mb. The visible MCD spectrum (300 K) of reduced Val68His Mb is essentially identical to that of reduced cytochrome b(5), but it possesses an additional component at 592 nm that is absent at 77 K. The reduction potential of the variant (pH 7.0, mu = 0.10 M, 25.0 degrees C) is -110 +/- 0.1 mV vs SHE, compared to a value of 60.9 +/- 0.1 mV for wild-type Mb and 4 mV for cytochrome b(5). The overall spectroscopic properties of the variant are remarkably similar to those of cytochrome b(5), the classical bishitidine ligated heme protein, and direct coordination of His68 to the heme iron of the variant is proposed for both oxidation states in the absence of exogenous ligands (e.g., dioxygen).