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, <3.0 kcal/mol. The 1H⁺/1e^- redox-Bohr effect of heme b−[H10A24]₂ is due to the greater absolute stabilization of the ferric heme (4.1 kcal/mol) compared to the ferrous heme (1.4 kcal/mol) binding upon glutamic acid deprotonation, i.e., an electrostatic response mechanism. The 2H⁺/1e^- redox-Bohr effect observed for heme b−[Δ7-His]₂ is due to histidine protonation and histidine dissociation of ferrous heme b upon reduction, i.e., a ligand loss mechanism. These results indicate that the contribution of porphyrin−protein hydrophobic interactions to heme affinity is critical to maintaining the heme bound in both oxidation states and eliciting an electrostatic response from these designed heme protein scaffolds.