Electrostatic Environment of Hemes in Proteins: pKas of Hydroxyl Ligands

Abstract

The pK_as of ferric aquo−heme and aquo−heme electrochemical midpoints (E_ms) at pH 7 in sperm whale myoglobin, Aplysia myoblogin, hemoglobin I, heme oxygenase 1, horseradish peroxidase and cytochrome c oxidase were calculated with Multi-Conformation Continuum Electrostatics (MCCE). The pK_as span 3.3 pH units from 7.6 in heme oxygenase 1 to 10.9 in peroxidase, and the E_ms range from −250 mV in peroxidase to 125 mV in Aplysia myoglobin. Proteins with higher in situ ferric aquo−heme pK_as tend to have lower E_ms. Both changes arise from the protein stabilizing a positively charged heme. However, compared with values in solution, the protein shifts the aquo−heme E_ms more than the pK_as. Thus, the protein has a larger effective dielectric constant for the protonation reaction, showing that electron and proton transfers are coupled to different conformational changes that are captured in the MCCE analysis. The calculations reveal a breakdown in the classical continuum electrostatic analysis of pairwise interactions. Comparisons with DFT calculations show that Coulomb's law overestimates the large unfavorable interactions between the ferric water−heme and positively charged groups facing the heme plane by as much as 60%. If interactions with Cu_B in cytochrome c oxidase and Arg 38 in horseradish peroxidase are not corrected, the pK_a calculations are in error by as much as 6 pH units. With DFT corrected interactions calculated pK_as and E_ms differ from measured values by less than 1 pH unit or 35 mV, respectively. The in situ aquo−heme pK_a is important for the function of cytochrome c oxidase since it helps to control the stoichiometry of proton uptake coupled to electron transfer [Song, Michonova-Alexova, and Gunner (2006) Biochemistry 45, 7959-7975].