Heme a, the metalloporphyrin cofactor unique to cytochrome c oxidases, differs from the more common heme b by two chemical modifications, a C-2 hydroxyethylfarnesyl group and a C-8 formyl group. To elucidate a role of the C-8 formyl group, we compare the heme affinity, spectroscopy, and electrochemistry of a heme a mimic, Fe( diacetyldeuterioporphyrin IX) or Fe(DADPIX), with heme b, Fe(protoporphryrin IX) or Fe(PPIX), incorporated into a designed heme protein. The [Delta 7-H3m](2) protein ligand, or maquette, selected for this study contains two equivalent bis-(3-methyl-L-histidine) heme binding sites within a four-alpha-helix bundle scaffold. The spectroscopic data on Fe( PPIX) and Fe(DADPIX) bound to [Delta 7-H3m](2) demonstrate that these complexes are excellent synthetic analogues for natural cytochromes b and a, respectively. Comparison of the spectroscopic, electrochemical, and equilibrium thermodynamic data measured for the Fe(PPIX)-[Delta 7- H3m](2) maquette with the previously reported Fe(PPIX)-[Delta 7-His] (2) complex demonstrates that changing the heme axial ligands to 3-methyl-L-histidine from L-histidine does not alter the resulting heme protein properties significantly in either oxidation state. Heme binding studies demonstrate that [Delta 7-H3m](2) binds two ferrous Fe(DADPIX) or Fe(PPIX) moieties with similar dissociation constant values. However, in the ferric state, the data show that [Delta 7-H3m](2) only binds a single Fe(DADPIX) and that one 2500-fold weaker than oxidized Fe(PPIX). The data demonstrate that the 4.6 kcal mol(-1) weakened affinity of [Delta 7-H3m](2) for oxidized Fe(DADPIX) results in the majority of the 160 mV, 3.7 kcal mol(-1), positive shift in the heme reduction potential relative to Fe( PPIX). These data indicate that a role of the formyl group on heme a is to raise the iron reduction potential, thus making it a better electron acceptor, but that it does so by destabilizing the affinity of bis- imidazole sites for the ferric state.