The structural characterization of de novo designed metalloproteins together with determination of chemical reactivity can provide a detailed understanding of the relationship between protein structure and functional properties. Toward this goal, using the basic scaffold of 1pbz (Rosenblatt et al. (2003) Proc Natl Acad Sci U S A;100:13140) we have designed cyclic DeltaF-containing heme-binding peptides. The alpha- and beta-bands in UV-Vis spectroscopy are indicative of bis-His-ligated heme complex. Most of our DeltaF-containing peptides have more affinity to cobalt(III)Coproporphyrinate-I than heme because cobalt(III)Coproporphyrinate-I contains two additional propionate groups which can have salt bridge interactions with the lysine residues in the peptide. Helicity induction in peptide by DeltaF and aromatic interaction of DeltaF with heme have increased the heme affinity of CP-6-12pbz (cyclic peptide with substitutions of Ala at positions 6 and 12 by DeltaF; 905/mm) compared with 1pbz (279/mm). The nuclear magnetic resonance spectra are indicative of overall helical structure for CP-6-12pbz and CP-6-12pbz in complex with cobalt (III)Coproporphyrinate-I. The descending order of heme affinity in peptides (CP-6-12pbz > CP-12pbz > CP-5-12pbz) indicates that DeltaF at i + 3 or i - 3 from the central H9 favors heme binding but disrupts the same when placed at i - 4.