Evaluation of electron-withdrawing group effects on heme binding in designed proteins: implications for heme a in cytochrome c oxidase.

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 [Delta7-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 [Delta7-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)-[Delta7-H3m]2 maquette with the previously reported Fe(PPIX)-[Delta7-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 [Delta7-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 [Delta7-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 [Delta7-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.

Design of a five-coordinate heme protein maquette: a spectroscopic model of deoxymyoglobin.

The substitution of 1-methyl-l-histidine for the histidine heme ligands in a de novo designed four-alpha-helix bundle scaffold results in conversion of a six-coordinate cytochrome maquette into a self-assembled five-coordinate mono-(1-methyl-histidine)-ligated heme as an initial maquette for the dioxygen carrier protein myoglobin. UV-vis, magnetic circular dichroism, and resonance Raman spectroscopies demonstrate the presence of five-coordinate mono-(1-methyl-histidine) ligated ferrous heme spectroscopically similar to deoxymyoglobin. Thermodynamic analysis of the ferric and ferrous heme dissociation constants indicates greater destabilization of the ferric state than the ferrous state. The ferrous heme protein reacts with carbon monoxide to form a (1-methyl-histidine)-Fe(II)(heme)-CO complex; however, reaction with dioxygen leads to autoxidation and ferric heme dissociation. These results indicate that negative protein design can be used to generate a five-coordinate heme within a maquette scaffold.