Relative Binding Free Energies of Adenine and Guanine to Damaged and Undamaged DNA in Human DNA Polymerase η: Clues for Fidelity and Overall Efficiency

Human DNA polymerase η (Pol η) plays an essential protective role against skin cancer caused by cyclo­butane thymine–thymine dimers (TTDs), a frequent form of DNA damage arising from exposure to the sun. This enzyme rescues stalled replication forks at the TTDs by inserting bases opposite these DNA defects. Herein we calculate binding free energies for a free deoxy­ribose nucleotide triphosphate, dATP or dGTP, to Pol η complexed with undamaged or damaged DNA. The calculations indicate that the binding of dATP to the enzyme–DNA complex is thermodynamically favored for TTD-containing DNA over undamaged DNA, most likely because of more extensive hydrogen-bonding interactions between the TTD and the enzyme that hold the TTD more rigidly in place. The calculations also illustrate that dATP binding is thermodynamically favored over dGTP binding at both thymine positions of the TTD, most likely due to more persistent and stable hydrogen-bonding interactions between the TTD and dATP than between the TTD and dGTP. This free energy difference is slightly greater for binding at the 5′ thymine position than at the 3′ thymine position, presumably because of stabilization arising from the A:T base pair formed at the 3′ position of the TTD in the previous step of Pol η function. All of these trends in binding free energies are consistent with experimental measurements of binding strength, fidelity, processivity, and overall efficiency. The insights gained from this analysis have implications for drug design efforts aimed at modifying the binding properties of this enzyme for improving cancer chemotherapy treatments

Exploring the Role of the Third Active Site Metal Ion in DNA Polymerase η with QM/MM Free Energy Simulations

The enzyme human DNA polymerase η (Pol η) is critical for bypassing lesions during DNA replication. In addition to the two Mg²⁺ ions aligning the active site, experiments suggest that a third Mg²⁺ ion could play an essential catalytic role. Herein the role of this third metal ion is investigated with quantum mechanical/molecular mechanical (QM/MM) free energy simulations of the phosphoryl transfer reaction and a proposed self-activating proton transfer from the incoming nucleotide to the pyrophosphate leaving group. The simulations with only two metal ions in the active site support a sequential mechanism, with phosphoryl transfer followed by relatively fast proton transfer. The simulations with three metal ions in the active site suggest that the third metal ion may play a catalytic role through electrostatic interactions with the leaving group. These electrostatic interactions stabilize the product, making the phosphoryl transfer reaction more thermodynamically favorable with a lower free energy barrier relative to the activated state corresponding to the deprotonated 3′OH nucleophile, and also inhibit the subsequent proton transfer. The possibility that Mg²⁺-bound hydroxide acts as the base deprotonating the 3′OH nucleophile is also explored

Computational Study of Anomalous Reduction Potentials for Hydrogen Evolution Catalyzed by Cobalt Dithiolene Complexes

The design of efficient hydrogen-evolving catalysts based on earth-abundant materials is important for developing alternative renewable energy sources. A series of four hydrogen-evolving cobalt dithiolene complexes in acetonitrile–water solvent is studied with computational methods. Co­(mnt)₂ (mnt = maleonitrile-2,3-dithiolate) has been shown experimentally to be the least active electrocatalyst (i.e., to produce H₂ at the most negative potential) in this series, even though it has the most strongly electron-withdrawing substituents and the least negative Co^III/II reduction potential. The calculations provide an explanation for this anomalous behavior in terms of protonation of the sulfur atoms on the dithiolene ligands after the initial Co^III/II reduction. One fewer sulfur atom is protonated in the Co^II(mnt)₂ complex than in the other three complexes in the series. As a result, the subsequent Co^II/I reduction step occurs at the most negative potential for Co­(mnt)₂. According to the proposed mechanism, the resulting Co^I complex undergoes intramolecular proton transfer to form a catalytically active Co^III-hydride that can further react to produce H₂. Understanding the impact of ligand protonation on electrocatalytic activity is important for designing more effective electrocatalysts for solar devices

Effects of Active Site Mutations on Specificity of Nucleobase Binding in Human DNA Polymerase η

Human DNA polymerase η (Pol η) plays a vital role in protection against skin cancer caused by damage from ultraviolet light. This enzyme rescues stalled replication forks at cyclobutane thymine–thymine dimers (TTDs) by inserting nucleotides opposite these DNA lesions. Residue R61 is conserved in the Pol η enzymes across species, but the corresponding residue, as well as its neighbor S62, is different in other Y-family polymerases, Pol ι and Pol κ. Herein, R61 and S62 are mutated to their Pol ι and Pol κ counterparts. Relative binding free energies of dATP to mutant Pol η•DNA complexes with and without a TTD were calculated using thermodynamic integration. The binding free energies of dATP to the Pol η•DNA complex with and without a TTD are more similar for all of these mutants than for wild-type Pol η, suggesting that these mutations decrease the ability of this enzyme to distinguish between a TTD lesion and undamaged DNA. Molecular dynamics simulations of the mutant systems provide insights into the molecular level basis for the changes in relative binding free energies. The simulations identified differences in hydrogen-bonding, cation−π, and π–π interactions of the side chains with the dATP and the TTD or thymine–thymine (TT) motif. The simulations also revealed that R61 and Q38 act as a clamp to position the dATP and the TTD or TT and that the mutations impact the balance among the interactions related to this clamp. Overall, these calculations suggest that R61 and S62 play key roles in the specificity and effectiveness of Pol η for bypassing TTD lesions during DNA replication. Understanding the basis for this specificity is important for designing drugs aimed at cancer treatment

Substituent Effects on Cobalt Diglyoxime Catalysts for Hydrogen Evolution

The design of efficient, robust, and inexpensive hydrogen evolution catalysts is important for the development of renewable energy sources such as solar cells. Cobalt diglyoxime complexes, Co(dRgBF₂)₂ with substituents R, are promising candidates for such electrocatalysts. The mechanism for hydrogen production requires a series of reduction and protonation steps for various monometallic and bimetallic pathways. In this work, the reduction potentials and p<i>K</i>_a values associated with the individual steps were calculated for a series of substituents. The calculations revealed a linear relation between the reduction potentials and p<i>K</i>_a values with respect to the Hammett constants, which quantify the electron donating or withdrawing character of the substituents. Additionally, the reduction potentials and p<i>K</i>_a values are linearly correlated with each other. These linear correlations enable the prediction of reduction potentials and p<i>K</i>_a values, and thus the free energy changes along the reaction pathways, to assist in the design of more effective cobaloxime catalysts

Comparative Molecular Dynamics Studies of Human DNA Polymerase η

High-energy ultraviolet radiation damages DNA through the formation of cyclobutane pyrimidine dimers, which stall replication. When the lesion is a thymine–thymine dimer (TTD), human DNA polymerase η (Pol η) assists in resuming the replication process by inserting nucleotides opposite the damaged site. We performed extensive molecular dynamics (MD) simulations to investigate the structural and dynamical effects of four different Pol η complexes with or without a TTD and with either dATP or dGTP as the incoming base. No major differences in the overall structures and equilibrium dynamics were detected among the four systems, suggesting that the specificity of this enzyme is due predominantly to differences in local interactions in the binding regions. Analysis of the hydrogen-bonding interactions between the enzyme and the DNA and dNTP provided molecular-level insights. Specifically, the TTD was observed to engage in more hydrogen-bonding interactions with the enzyme than its undamaged counterpart of two normal thymines. The resulting greater rigidity and specific orientation of the TTD are consistent with the experimental observation of higher processivity and overall efficiency at TTD sites than at analogous sites with two normal thymines. The similarities between the systems containing dATP and dGTP are consistent with the experimental observation of relatively low fidelity with respect to the incoming base. Moreover, Q38 and R61, two strictly conserved amino acids across the Pol η family, were found to exhibit persistent hydrogen-bonding interactions with the TTD and cation-π interactions with the free base, respectively. Thus, these simulations provide molecular level insights into the basis for the selectivity and efficiency of this enzyme, as well as the roles of the two most strictly conserved residues

Probing Nonadiabaticity in the Proton-Coupled Electron Transfer Reaction Catalyzed by Soybean Lipoxygenase

Proton-coupled electron transfer (PCET) plays a vital role in many biological and chemical processes. PCET rate constant expressions are available for various well-defined regimes, and determining which expression is appropriate for a given system is essential for reliable modeling. Quantitative diagnostics have been devised to characterize the vibronic nonadiabaticity between the electron–proton quantum subsystem and the classical nuclei, as well as the electron–proton nonadiabaticity between the electrons and proton(s) within the quantum subsystem. Herein these diagnostics are applied to a model of the active site of the enzyme soybean lipoxygenase, which catalyzes a PCET reaction that exhibits unusually high deuterium kinetic isotope effects at room temperature. Both semiclassical and electronic charge density diagnostics illustrate vibronic and electron–proton nonadiabaticity for this PCET reaction, supporting the use of the Golden rule nonadiabatic rate constant expression with a specific form of the vibronic coupling. This type of characterization will be useful for theoretical modeling of a broad range of PCET processes