Quantum Mechanical Analysis of Nonenzymatic Nucleotidyl Transfer Reactions: Kinetic and Thermodynamic Effects of β–γ Bridging Groups of dNTP Substrates

Rate (<i>k</i>) and equilibrium (<i>K</i>) constants for the reaction of tetrahydrofuranol with a series of Mg²⁺ complexes of methyl triphosphate analogues, CH₃O-P­(O₂)-O-P­(O₂)-X-PO₃^4–, X = O, CH₂, CHCH₃, C­(CH₃)₂, CFCH₃, CHF, CHCl, CHBr, CFCl, CF₂, CCl₂, and CBr₂, forming phosphate diester and pyrophosphate or bisphosphonate in aqueous solution were evaluated by B3LYP/TZVP//HF/6-31G* quantum chemical calculations and Langevin dipoles and polarized continuum solvation models. The calculated log <i>k</i> and log <i>K</i> values were found to depend linearly on the experimental p<i>K</i>_a4 of the conjugate acid of the corresponding pyrophosphate or bisphosphonate leaving group. The calculated slopes of these Brønsted linear free energy relationships were β_lg = −0.89 and β_eq = −0.93, respectively. The studied compounds also followed the linear relationship Δlog <i>k</i> = 0.8Δlog <i>K</i>, which became less steep, Δlog <i>k</i> = 0.6Δlog <i>K</i>, after the range of studied compounds was extended to include analogues that were doubly protonated on γ-phosphate, CH₃O-P­(O₂)-O-P­(O₂)-X-PO₃H₂^2–. The scissile P_α–O_lg bond length in studied methyl triphosphate analogues slightly increases with decreasing p<i>K</i>_a of the leaving group; concomitantly, the CH₃OP_α(O₂) moiety becomes more positive. These structural effects indicate that substituents with low p<i>K</i>_a can facilitate both P_α–O_lg bond breaking and the P_α–O_nuc bond forming process, thus explaining the large negative β_lg calculated for the transition state geometry that has significantly longer P_α–O_nuc distance than the P_α–O_lg distance

Membrane-Anchored Cytochrome P450 1A2–Cytochrome <i>b</i>₅ Complex Features an X‑Shaped Contact between Antiparallel Transmembrane Helices

Eukaryotic cytochromes P450 (P450) are membrane-bound enzymes oxidizing a broad spectrum of hydrophobic substrates, including xenobiotics. Protein–protein interactions play a critical role in this process. In particular, the formation of transient complexes of P450 with another protein of the endoplasmic reticulum membrane, cytochrome <i>b</i>₅ (cyt <i>b</i>₅), dictates catalytic activities of several P450s. To lay a structural foundation for the investigation of these effects, we constructed a model of the membrane-bound full-length human P450 1A2–cyt <i>b</i>₅ complex. The model was assembled from several parts using a multiscale modeling approach covering all-atom and coarse-grained molecular dynamics (MD). For soluble P450 1A2–cyt <i>b</i>₅ complexes, these simulations yielded three stable binding modes (sA_I, sA_II, and sB). The membrane-spanning transmembrane domains were reconstituted with the phospholipid bilayer using self-assembly MD. The predicted full-length membrane-bound complexes (mA_I and mB) featured a spontaneously formed X-shaped contact between antiparallel transmembrane domains, whereas the mA_II mode was found to be unstable in the membrane environment. The mutual position of soluble domains in binding mode mA_I was analogous to the sA_I complex. Featuring the largest contact area, the least structural flexibility, the shortest electron transfer distance, and the highest number of interprotein salt bridges, mode mA_I is the best candidate for the catalytically relevant full-length complex

Uniform Free-Energy Profiles of the P–O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ

Human X-family DNA polymerases β (Polβ) and λ (Polλ) catalyze the nucleotidyl-transfer reaction in the base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free-energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3′-OH group of the primer DNA strand, and the subsequent formation and cleavage of P–O bonds in four Polβ, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal mol^–1 for Polβ, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P–O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in the overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest p<i>K</i>_a of the 3′-OH group as a predictor of the catalytic rate of X-family DNA polymerases

Spectroscopic Evidence of Work Function Alterations Due to Photoswitchable Monolayers on Gold Surfaces

Taking advantage of surfaces’ response to interfacial dipoles, a class of photochromophores (dihydroindolizine) is demonstrated to alter the work function of the underlying substrate (∼170 meV). This same molecule also provides spectroscopic signatures for correlating the change in molecular structure to the induced change in the surfaces’ electronic properties. Polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) allows analysis of the characteristic dihydroindolizine CC (1559 cm^–1) and pyridinium (1643 cm^–1) stretch as a function of photoexcitation. Structural assignments of this photochromophore are corroborated to density function theory calculations. Conformational changes in the monolayers appear in parallel with work function changes and are consistent with both its rate and magnitude

DNA Polymerase λ Active Site Favors a Mutagenic Mispair between the Enol Form of Deoxyguanosine Triphosphate Substrate and the Keto Form of Thymidine Template: A Free Energy Perturbation Study

Human DNA polymerase λ is an intermediate fidelity member of the X family, which plays a role in DNA repair. Recent X-ray diffraction structures of a ternary complex of a loop-deletion mutant of polymerase λ, a deoxyguanosine triphosphate analogue, and a gapped DNA show that guanine and thymine form a mutagenic mispair with an unexpected Watson–Crick-like geometry rather than a wobble geometry. Hence, there is an intriguing possibility that either thymine in the DNA or guanine in the deoxyguanosine triphosphate analogue may spend a substantial fraction of time in a deprotonated or enol form (both are minor species in aqueous solution) in the active site of the polymerase λ mutant. The experiments do not determine particular forms of the nucleobases that contribute to this mutagenic mispair. Thus, we investigate the thermodynamics of formation of various mispairs between guanine and thymine in the ternary complex at a neutral pH using classical molecular dynamics simulations and the free energy perturbation method. Our free energy calculations, as well as a comparison of the experimental and computed structures of mispairs, indicate that the Watson–Crick-like mispair between the enol tautomer of guanine and the keto tautomer of thymine is dominant. The wobble mispair between the keto forms of guanine and thymine and the Watson–Crick-like mispair between the keto tautomer of guanine and the enol tautomer of thymine are less prevalent, and mispairs that involve deprotonated guanine or thymine are thermodynamically unlikely. These findings are consistent with the experiment and relevant for understanding mechanisms of spontaneous mutagenesis

Empirical Valence Bond Simulations of the Chemical Mechanism of ATP to cAMP Conversion by Anthrax Edema Factor

The two-metal catalysis by the adenylyl cyclase domain of the anthrax edema factor toxin was simulated using the empirical valence bond (EVB) quantum mechanical/molecular mechanical approach. These calculations considered the energetics of the nucleophile deprotonation and the formation of a new P–O bond in aqueous solution and in the enzyme–substrate complex present in the crystal structure models of the reactant and product states of the reaction. Our calculations support a reaction pathway that involves metal-assisted transfer of a proton from the nucleophile to the bulk aqueous solution followed by subsequent formation of an unstable pentavalent intermediate that decomposes into cAMP and pyrophosphate (PP_i). This pathway involves ligand exchange in the first solvation sphere of the catalytic metal. At 12.9 kcal/mol, the barrier for the last step of the reaction, the cleavage of the P–O bond to PP_i, corresponds to the highest point on the free energy profile for this reaction pathway. However, this energy is too close to the value of 11.4 kcal/mol calculated for the barrier of the nucleophilic attack step to reach a definitive conclusion about the rate-limiting step. The calculated reaction mechanism is supported by reasonable agreement between the experimental and calculated catalytic rate constant decrease caused by the mutation of the active site lysine 346 to arginine

Flexible Docking-Based Molecular Dynamics/Steered Molecular Dynamics Calculations of Protein–Protein Contacts in a Complex of Cytochrome P450 1A2 with Cytochrome <i>b</i>₅

Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome <i>b</i>₅ (cyt <i>b</i>₅), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt <i>b</i>₅. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2–cyt <i>b</i>₅ complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2–cyt <i>b</i>₅ binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450–cyt <i>b</i>₅ interactions, indicated that the negatively charged convex surface of cyt <i>b</i>₅ binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt <i>b</i>₅. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex

Intramolecular Base Stacking of Dinucleoside Monophosphate Anions in Aqueous Solution

Time-dependent motions of 32 deoxyribodinucleoside and ribodinucleoside monophosphate anions in aqueous solution at 310 K were monitored during 40 ns using classical molecular dynamics (MD). In all studied molecules, spontaneous stacking/unstacking transitions occurred on a time-scale of 10 ns. To facilitate the structural analysis of the sampled configurations we defined a reaction coordinate for the nucleobase stacking that considers both the angle between the planes of the two nucleobases and the distance between their mass-centers. Additionally, we proposed a physically meaningful transient point on this coordinate that separates the stacked and unstacked states. We applied this definition to calculate free energies for stacking of all pairwise combinations of adenine, thymine (uracil), cytosine and guanine moieties embedded in studied dinucleosides monophosphate anions. The stacking equilibrium constants decreased in the order 5′-AG-3′ > GA ∼ GG ∼ AA > GT ∼ TG ∼ AT ∼ GC ∼ AC > CG ∼ TA > CA ∼ TC ∼ TT ∼ CT ∼ CC. The stacked conformations of AG occurred 10 times more frequently than its unstacked conformations. On the other hand, the last five base combinations showed a greater preference for the unstacked than the stacked state. The presence of an additional 2′-OH group in the RNA-based dinucleoside monophosphates increased the fraction of stacked complexes but decreased the compactness of the stacked state. The calculated MD trajectories were also used to reveal prevailing mutual orientation of the nucleobase dipoles in the stacked state

Metal Ion Complexes of <i>N,N</i>′‑Bis(2-Pyridylmethyl)-<i>trans</i>-1,2-Diaminocyclohexane-<i>N,N</i>′‑Diacetic Acid, H₂bpcd: Lanthanide(III)–bpcd^2– Cationic Complexes

The synthesis and characterization of <i>N,N</i>′-bis­(2-pyridylmethyl)-<i>trans</i>-1,2-diaminocyclohexane-<i>N,N</i>′-diacetic acid (H₂bpcd) cationic complexes of La­(III), Nd­(III), and Sm­(III) are reported. The Ln­(III)–bpcd^2– complex ions, where bpcd^2– stands for <i>N,N</i>′-bis­(2-pyridylmethyl)-<i>trans</i>-1,2-diaminocyclohexane-<i>N,N</i>′-diacetate, were isolated as PF₆^– salts. These salts were characterized by elemental analysis, X-ray crystallography, IR, and ¹H and ¹³C NMR spectroscopy. Binuclear [La₂(bpcd)₂(H₂O)₂]²⁺ crystallized from an aqueous solution in the monoclinic <i>P</i>2₁/<i>c</i> space group as a cocrystallate with Na₂bpcd and NaPF₆, nominally Na_2.34[La_1.22(C₂₂H₂₆N₄O₄)₂(H₂O)₂]­[PF₆]₂·2H₂O, with <i>a</i> = 11.3343(6) Å, <i>b</i> = 17.7090(9) Å, <i>c</i> = 15.0567(8) Å, β = 110.632(3)°, and <i>Z</i> = 4 (<i>Z</i>′ = 2). La is eight-coordinate with distorted dodecahedral coordination geometry provided by a N₄O₄ donor atom set. In addition to four N atoms from the bpcd^2– ligand, La’s coordination sphere includes O atoms from a water molecule and three acetate groups (one O atom from singly bound acetate and two O atoms from acetate groups that bridge the La centers). The ¹H and ¹³C assignments for H₂bpcd and the metal–bpcd^2– complexes were made on the basis of 2D COSY and HSQC experiments, which established ¹H–¹H and ¹H–¹³C correlations. The NMR spectral data were used to establish the symmetry of the cationic complexes present in aqueous solution. The data indicate that the La­(III)–bpcd^2– and Sm­(III)–bpcd^2– complexes are present in solution as a single species with <i>C</i>₂ symmetry. The ¹H NMR spectrum of [Nd­(bpcd)]­PF₆ in D₂O consists of eight considerably line-broadened, paramagnetic-shifted singlets. The ab initio quantum mechanical calculations at the PCM/MP2/SDD//HF/SDD level, which were established previously for determining isomerization energies for octahedral M­(III)–bp<i>a</i>d^2– complex ions, were used to determine the relative free energies of the geometric isomers possible for eight- and nine-coordinate La­(III)–bpcd^2– cationic aqua complexes in aqueous solution, i.e., [La­(bpcd)­(H₂O)₂]⁺ and La­(bpcd)­(H₂O)₃]⁺

Metal Ion Complexes of <i>N,N</i>′‑Bis(2-Pyridylmethyl)-1,3-Diaminopropane-<i>N,N</i>′‑Diacetic Acid, H₂bppd

A higher yield synthesis of <i>N,N</i>′-bis­(2-pyridylmethyl)-1,3-diaminopropane-<i>N,N</i>′-diacetic acid (H₂bppd) and its complexation of trivalent metal ions (Al­(III), Ga­(III), In­(III)) and selected lanthanides (Ln­(III)) are reported. H₂bppd and the metal–bppd^2– complexes, isolated as hexafluorophosphate salts, were characterized by elemental analysis, mass spectrometry, IR, and ¹H and ¹³C NMR spectroscopy. [Ga­(bppd)]­PF₆, [Ga­(C₁₉H₂₂N₄O₄)]­PF₆, was crystallized as colorless needles by slow evaporation from anhydrous methanol; its molecular structure was solved by direct X-ray crystallography methods. The compound crystallized in the monoclinic space group <i>P</i>2₁/<i>c</i>, with <i>a</i> = 9.6134(2) Å, <i>b</i> = 20.2505(4) Å, <i>c</i> = 11.6483(3) Å, β = 97.520(1)^o, and <i>Z</i> = 4. Ga is coordinated in a distorted octahedral geometry provided by a N₄O₂ donor atom set with cis-monodentate acetate groups and <i>cis</i>-2-pyridylmethyl N atoms. Quantum mechanical calculations were performed for the three possible geometric isomers of a pseudo-octahedral metal–bppd^2– complex with five different metal ions. The results indicate, that in aqueous solution, the stability of the <i>trans</i>-O,O isomer is similar to that of the <i>cis</i>-O,O; <i>cis</i>-N_py,N_py isomer but is greater than that of the <i>trans</i>-N_py,N_py isomer. Calculations for a six-coordinate La­(III)-bppd^2– complex converge to a structure with a very large N_py–La–N_py bond angle (146.4°), a high metal charge (2.28 au), and a high solvation free energy (−79.4 kcal/mol). The most stable geometric arrangement for bppd^2– around the larger La­(III) is best described as an open nestlike structure with space available for additional ligands. IR spectroscopy was used to investigate the nature of the H₂bppd–metal complexes isolated in the solid state and the binding modes of the carboxylate functionalities. The spectra indicate that fully deprotonated [M­(bppd)]⁺ complexes as well as partially protonated complexes [M­(Hbppd)­Cl]⁺ were isolated. The ¹H and ¹³C assignments for H₂bppd and metal–bppd^2– complexes were made on the basis of 2D COSY, NOESY, and ¹H–¹³C HSQC experiments, which were used to differentiate among the cis (<i>C</i>₁ symmetry) and the two trans (<i>C</i>₂ symmetry) isomers