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Hemoglobin-mediated transport of dioxygen (O(2)) critically depends on the stability of the reduced (Fe(2+)) form of the heme cofactors. Some protein mutations stabilize the oxidized (Fe(3+)) state (methemoglobin, Hb M), causing methemoglobinemia, and can be lethal above 30%. The majority of the analyses of factors influencing Hb oxidation are retrospective and give insights only for inner-sphere mutations of heme (His58, His87). Herein, we report the first all-atom molecular dynamics simulations on both redox states and calculations of the Marcus electron transfer (ET) parameters for the α chain Hb oxidation and reduction rates for Hb M. The Hb wild-type (WT) and most of the studied α chain variants maintain globin structure except the Hb M Iwate (H87Y). The mutants forming Hb M tend to have lower redox potentials and thus stabilize the oxidized (Fe(3+)) state (in particular, the Hb Miyagi variant with K61E mutation). Solvent reorganization (λ(solv) 73–96%) makes major contributions to reorganization free energy, whereas protein reorganization (λ(prot)) accounts for 27–30% except for the Miyagi and J-Buda variants (λ(prot) ∼4%). Analysis of heme-solvent H-bonding interactions among variants provide insights into the role of Lys61 residue in stabilizing the Fe(2+) state. Semiclassical Marcus ET theory-based calculations predict experimental k(ET) for the Cyt b5-Hb complex and provide insights into relative reduction rates for Hb M in Hb variants. Thus, our methodology provides a rationale for the effect of mutations on the structure, stability, and Hb oxidation reduction rates and has potential for identification of mutations that result in methemoglobinemia.

The free energy profile for the first step of formamide hydrolysis in alkaline aqueous solution is computed using Car–Parrinello molecular dynamics simulation combined with umbrella sampling. An activation free energy of 22.3 kcal/mol and a reaction free energy of 17.3 kcal/mol are estimated from the potential of mean force in unprecedented good agreement with the corresponding free enthalpies obtained from experiment, 21.2 and 16.5 kcal/mol, respectively. The errors introduced by the BLYP density functional and pseudopotentials are investigated as well as the approximations made to relate the potential of mean force to the free enthalpies measured in experiment.

A recently developed ab initio molecular dynamics method for the simulation of electrochemical half reactions is placed in the context of commonly accepted pictures of electrode processes and the related Marcus theory of heterogeneous electron transfer. Viewing our computational approach from this perspective we comment on a number of more technical aspects of the method.