Thermodynamic Investigation into the Mechanisms of Proton-Coupled Electron Transfer Events in Heme Protein Maquettes

To study the engineering requirements for proton pumping in energy-converting enzymes such as cytochrome c oxidase, the thermodynamics and mechanisms of proton-coupled electron transfer in two designed heme proteins are elucidated. Both heme protein maquettes chosen, heme b−[H10A24]2 and heme b−[Δ7-His]2, are four-α-helix bundles that display pH-dependent heme midpoint potential modulations, or redox-Bohr effects. Detailed equilibrium binding studies of ferric and ferrous heme b with these maquettes allow the individual contributions of heme−protein association, iron−histidine ligation, and heme−protein electrostatics to be elucidated. These data demonstrate that the larger, less well-structured [H10A24]2 binds heme b in both oxidation states tighter than the smaller and more well-structured [Δ7-His]2 due to a stronger porphyrin−protein hydrophobic interaction. The 66 mV (1.5 kcal/mol) difference in their heme reduction potentials observed at pH 8.0 is due mostly to stabilization of ferrous heme in [H10A24]2 relative to [Δ7-His]2. The data indicate that porphyrin−protein hydrophobic interactions and heme iron coordination are responsible for the K d value of 37 nM for the heme b−[Δ7-His]2 scaffold, while the affinity of heme b for [H10A24]2 is 20-fold tighter due to a combination of porphyrin−protein hydrophobic interactions, iron coordination, and electrostatic effects. The data also illustrate that the contribution of bis-His coordination to ferrous heme protein affinity is limited,