How Cytochromes with Different Folds Control Heme Redox Potentials

Abstract

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)), alpha beta 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 E(m) 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.

Calculated Proton Uptake on Anaerobic Reduction of CytochromecOxidase: Is the Reaction Electroneutral?

“…On the basis of the pK a and E m studies of aquo-heme proteins (53), -0.74 ∆pK unit is added to ferric hydroxyl-heme and 0.5 ∆pK unit to ferrous waterheme. The heme propionic acids are treated as previously described with a pK a,sol of 4.9 (27 (76). The B3LYP method (77) with the LANL2DZ basis set (78) was used, and the CHELPG (79) algorithm was used to fit atomic charges.…”

“…A +0.3 charge is placed on the formyl group C and -0.3 on the electron-withdrawing formyl group of the a-type hemes. This simple metal-centered charge set has been used successfully in heme benchmark calculations for bis-His and His-aquo hemes (25,27,53).…”

“…MCCE (Multi-Conformation Continuum Electrostatics) calculates the shift in E m and pK a induced by the protein (27,(50)(51)(52). Given measured aqueous pK a s and E m s (pK a,sol and E m,sol ) for acidic and basic groups and redox cofactors, MCCE provides these values in the protein.…”

Electrostatic Environment of Hemes in Proteins: pKas of Hydroxyl Ligands

“…For example, sixcoordinate bis-His-hemes have E m s ranging from -410 to +360 mV with the redox differences being predominately due to the intraprotein electrostatic environment (1,(20)(21)(22). In these proteins the loss of solvation energy (15,23,24), interactions with the protein backbone and with other residues (15,(20)(21)(22)25), and local conformation changes on ionization changes (20, 26) determine the thermodynamic equilibrium measured by pK a s and E m s.Theoretical studies have analyzed hemes in different systems using a number of techniques. The large span of E m s in cytochromes has been subject to analysis by Protein Dipole Langevin Dipole (PDLD) (27,28), continuum electrostatics (CE) 1 (20,21,29,30), and other techniques (31-37).…”

“…In these proteins the loss of solvation energy (15,23,24), interactions with the protein backbone and with other residues (15,(20)(21)(22)25), and local conformation changes on ionization changes (20, 26) determine the thermodynamic equilibrium measured by pK a s and E m s.…”