Microperoxidase-8 (MP8) is the heme octapeptide derived from enzymatic proteolysis of horse-heart cytochrome c. Not only is MP8 a functional peroxidase (it catalyzes the oxidation of various substrates by hydrogen peroxide), it has also served as a useful calibration for the interpretation of the electronic spectroscopic properties of heme proteins. NMR structural characterization of MP8 has been difficult, owing to extensive aggregation at millimolar concentrations. We have obtained well-resolved H-1 and C-13 NMR spectra of monomeric ferric MP8-CN in mixed aqueous-organic solvent mixtures containing excess cyanide. Most peptide resonances were assigned by through-bond correlations using TOCSY spectra; heme resonances were identified largely by through-space correlations using NOESY spectra, HMQC spectra were interpreted with the aid of proton assignments to identify C-13 resonances. Most peptide resonances appear within the diamagnetic region and are very sharp, the exceptions being resonances associated with the His18 residue. Protons on the His18 imidazole ring exhibit very broad resonances, reflecting efficient relaxation. The signals of heme substituents are shifted outside of the diamagnetic envelope, with heme methyl resonances appearing between 10 and 25 ppm. The pattern of MP8-CN heme methyl resonances bears a striking resemblance to those of intact cyanoferric heme c protein derivatives. Large amide-proton/a-proton coupling constants and interresidue NOE contacts were found between residues 14 and 18, while moderate amide-a coupling constants were found between residues 19 and 21. The imidazole group of His18 remains coordinated to the ferric center in MP8; and the pattern of heme methyl resonances confirms that a fixed axial imidazole orientation is preserved in the isolated heme active site. The observed interresidue NOE's and amide-H/H-alpha proton coupling constants indicate that His18 is part of a rigid loop of five residues anchored to the heme that serves to orient the axial imidazole, while residues 19-21 form a flexible C-terminal domain.