Multiconformation continuum electrostatics (MCCE) explores different conformational degrees of freedom in Monte Carlo calculations of protein residue and ligand pKas. Explicit changes in side chain conformations throughout a titration create a position dependent, heterogeneous dielectric response giving a more accurate picture of coupled ionization and position changes. The MCCE2 methods for choosing a group of input heavy atom and proton positions are described. The pKas calculated with different isosteric conformers, heavy atom rotamers and proton positions, with different degrees of optimization are tested against a curated group of 305 experimental pKas in 33 proteins. QUICK calculations, with rotation around Asn and Gln termini, sampling His tautomers and torsion minimum hydroxyls yield an RMSD of 1.34 with 84% of the errors being <1.5 pH units. FULL calculations adding heavy atom rotamers and side chain optimization yield an RMSD of 0.90 with 90% of the errors <1.5 pH unit. Good results are also found for pKas in the membrane protein bacteriorhodopsin. The inclusion of extra side chain positions distorts the dielectric boundary and also biases the calculated pKas by creating more neutral than ionized conformers. Methods for correcting these errors are introduced. Calculations are compared with multiple X-ray and NMR derived structures in 36 soluble proteins. Calculations with X-ray structures give significantly better pKas. Results with the default protein dielectric constant of 4 are as good as those using a value of 8.

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.

Residue ionization states were calculated in nine crystal structures of bacteriorhodopsin trapped in bR, early M, and late M states by multiconformation continuum electrostatics. This combines continuum electrostatics and molecular mechanics, deriving equilibrium distributions of ionization states and polar residue and water positions. The three central cluster groups [retinal Schiff base (SB), Asp 85 and Asp 212] are ionized in bR structures while a proton has transferred from SB(+) to Asp 85(-) in late M structures matching experimental results. The proton shift in M is due to weaker SB(+)-ionized acid and more favorable SB(0)-ionized acid interactions following retinal isomerization. The proton release cluster (Glu 194 and Glu 204) binds one proton in bR, which is lost to water by pH 8 in late M. In bR the half-ionized state is stabilized by charge-dipole interactions while full ionization is disallowed by charge-charge repulsion between the closely spaced acids. In M the acids move apart, permitting full ionization. Arg 82 movement connects the proton shifts in the central and proton release clusters. Changes in total charge of the two clusters are coupled by direct long-range interactions. Separate calculations consider continuum or explicit water in internal cavities. The explicit waters and nearby polar residues can reorient to stabilize different charge distributions. Proton release to the low-pH, extracellular side of the protein occurs in these calculations where residue ionization remains at equilibrium with the medium. Thus, the key changes distinguishing the intermediates are indeed trapped in the structures.

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A protein structure should provide the information needed to understand its observed properties. Significant progress has been made in developing accurate calculations of acid/base and oxidation/reduction reactions in proteins. Current methods and their strengths and weaknesses are discussed. The distribution and calculated ionization states in a survey of proteins is described, showing that a significant minority of acidic and basic residues are buried in the protein and that most of these remain ionized. The electrochemistry of heme and quinones are considered. Proton transfers in bacteriorhodopsin and coupled electron and proton transfers in photosynthetic reaction centers, 5-coordinate heme binding proteins and cytochrome c oxidase are highlighted as systems where calculations have provided insight into the reaction mechanism.