How cytochromes with different folds control heme redox potentials

The electrochemical midpoint potentials (E-m's) of 13 cytochromes, in globin (c, c(2), c(551), c(553)), four-helix bundle (c', b(562)), alphabeta roll (b(5)), and beta sandwich (f) motifs, with E-m's spanning 450 mV were calculated with multiconformation continuum electrostatics (MCCE). MCCE calculates changes in oxidation free energy when a heme-axial ligand complex is moved from water into protein. Calculated and experimental E-m's are in good agreement for cytochromes with His-Met and bis-His ligated hemes, where microperoxidases provide reference E-m's. In all cytochromes, E-m's are raised by 130-260 mV relative to solvated hemes by the loss of reaction field (solvation) energy. However, there is no correlation between E-m and heme surface exposure. Backbone amide dipoles in loops or helix termini near the axial ligands raise E-m's, but amides in helix bundles contribute little. Heme propionates lower E-m's. If the propionic acids are partially protonated in the reduced cytochrome, protons are released on heme oxidation, contributing to the pH dependence of the E-m. In all cytochromes studied except b(5)'s and low potential globins, buried side chains raise E-m's. MCCE samples ionizable group protonation states, heme redox states, and side chain rotamers simultaneously. Globins show the largest structural changes on heme oxidation and four-helix bundles the least. Given the calculated protein-induced Em shift and measured cytochrome E-m the five-coordinate, His heme in c' is predicted to have a solution E-m between that of isolated bis-His and His-Met hemes, while the reference E-m for His-Ntr ligands in cytochrome f should be near that of His-Met hemes.