Computational insights into the mechanism of radical generation in B12-dependent methylmalonyl-CoA mutase

ONIOM calculations have provided novel insights into the mechanism of homolytic Co-C5' bond cleavage in the 5'-deoxyadenosylcobalamin cofactor catalyzed by methylmalonyl-CoA mutase. We have shown that it is a stepwise process in which conformational changes in the 5'-deoxyadenosine moiety precede the actual homolysis step. In the transition state structure for homolysis, the Co-C5' bond elongates by approximately 0.5 Angstroms from the value found in the substrate-bound reactant complex. The overall barrier to homolysis is approximately 10 kcal/mol, and the radical products are approximately 2.5 kcal/mol less stable than the initial ternary complex of enzyme, substrate, and cofactor. The movement of the deoxyadenosine moiety during the homolysis step positions the resulting 5'-deoxyadenosyl radical for the subsequent hydrogen atom transfer from the substrate, methylmalonyl-CoA.     

Solvent effects on the photodissociation of formic acid: a theoretical study

Photodissociation of aqueous formic acid has been investigated with the CASSCF, DFT, and MR-CI methods. Solvent effects are considered as a combination of the hydrogen-bonding interaction from explicit H2O molecules and the effects from the bulk surrounding H2O molecules using the polarizable continuum model. It is found that the hydrogen-bonding effect from the explicit water in the complex is the major factor to influence properties of aqueous formic acid, while the bulk surrounding H2O molecules has a noticeable influence on the structures of the complex. The direct C-O bond fission along the S1 pathway is predicted to be an important channel upon photolysis of aqueous formic acid at 200 nm, which is consistent with experimental observation that aqueous formic acid dissociates predominantly into fragments of HCO and OH. The existence of a dark channel upon photolysis of aqueous formic acid at 200 nm is assigned as fast relaxation from the S1 Franck-Condon geometry to the T1/S1 intersection and subsequent S1-->T1 intersystem crossing process. S1-->S0 internal conversion followed by molecular elimination to CO+H2O is the most probable primary process for formation of carbon monoxide, which was observed with considerable yield upon photolysis of aqueous formic acid at 253.7 nm.     

Co-C bond activation in methylmalonyl-CoA mutase by stabilization of the post-homolysis product Co2+ cobalamin

Despite decades of research, the mechanism by which coenzyme B12 (adenosylcobalamin, AdoCbl)-dependent enzymes promote homolytic cleavage of the cofactor's Co-C bond to initiate catalysis has continued to elude researchers. In this work, we utilized magnetic circular dichroism spectroscopy to explore how the electronic structure of the reduced B12 cofactor (i.e., the post-homolysis product Co2+ Cbl) is modulated by the enzyme methylmalonyl-CoA mutase. Our data reveal a fairly uniform stabilization of the Co 3d orbitals relative to the corrin pi/pi*-based molecular orbitals when Co2+ Cbl is bound to the enzyme active site, particularly in the presence of substrate. Contrastingly, our previous studies (Brooks, A. J.; Vlasie, M.; Banerjee, R.; Brunold, T. C. J. Am. Chem. Soc. 2004, 126, 8167-8180.) showed that when AdoCbl is bound to the MMCM active site, no enzymatic perturbation of the Co3+ Cbl electronic structure occurs, even in the presence of substrate (analogues). Collectively, these observations provide direct evidence that enzymatic Co-C bond activation involves stabilization of the post-homolysis product, Co2+ Cbl, rather than destabilization of the Co3+ Cbl "ground" state.     

Optical absorption of a green fluorescent protein variant: environment effects in a density functional study

We present an ab initio study of the optical absorption properties of a particularly interesting fluorescent protein (E2GFP), whose complex photophysics still escapes elucidation. In particular, we focus on the role of the protein environment, showing that the effects of both nearby residues and the external field due to residues not accounted for explicitly are needed to properly reproduce the experimental data. The spectra calculated taking such contributions into account provide for the first time a robust identification of the states relevant for the photophysics of this system.     

A combined density functional theory and molecular mechanics study of the relationship between the structure of coenzyme B12 and its binding to methylmalonyl-CoA mutase

A combined density functional theory (DFT) and molecular mechanics (MM) approach was applied to investigate the relationship between the structure of a free coenzyme B12, and bound to methylmalonyl-CoA mutase. It was found that, upon coenzyme binding to apoenzyme, the Co-C bond remains intact, while the C-Naxial bond becomes slightly elongated and labilized. The labilization of the Co-Naxial bond that takes place in coenzyme B12-dependent enzymes is most likely necessary for fine-tuning of the cobalt-nitrogen (axial base) distance. The controlling of this distance is important to inhibit abiological site reaction involving heterolysis of the Co-C bond but is not important for biologically relevant Co-C bond homolysis.     

The Co(I) induced methylmalonyl-succinyl rearrangement in a model for the coenzyme B12 dependent methylmalonyl-CoA mutase

The rearrangement of 2-bromomethyl-2-methylmonothiomalonates to succinyl derivatives was found to take place in quantitative yields in the presence of one molar equivalent of Co(I) generated by the reduction of heptamethyl Co(II)yrinate perchlorate with NaBH4 or electrochemically. The chiral thiomalonate gave racemic succinate.     

A theoretical study on the substrate deacylation mechanism of class C beta-lactamase

The whole reaction of the deacylation of class C beta-lactamase was investigated by performing quantum chemical calculations under physiological conditions. In this study, the X-ray crystallographic structure of the inhibitor moxalactam-bound class C beta-lactamase (Patera et al. J. Am. Chem. Soc. 2000, 122, 10504-10512.) was utilized and moxalactam was changed into the substrate cefaclor. A model for quantum chemical calculations was constructed using an energy-minimized structure of the substrate-bound enzyme obtained by molecular mechanics calculation, in which the enzyme was soaked in thousands of TIP3P water molecules. It was found that the deacylation reaction consisted of two elementary processes. The first process was formation of a tetrahedral intermediate, which was initiated by the activation of catalytic water by Tyr150, and the second process was detachment of the hydroxylated substrate from the enzyme, which associated with proton transfer from the side chain of Lys67 to Ser64O(gamma). The first process is a rate-determining process, and the activation energy was estimated to be 30.47 kcal/mol from density functional theory calculations considering electron correlation (B3LYP/6-31G**). The side chain of Tyr150 was initially in a deprotonated state and was stably present in the active site of the acyl-enzyme complex, being held by Lys67 and Lys315 cooperatively.     

Spectroscopic and computational studies on the adenosylcobalamin-dependent methylmalonyl-CoA mutase: evaluation of enzymatic contributions to Co-C bond activation in the Co3+ ground state

Methylmalonyl-CoA mutase (MMCM) is an enzyme that utilizes the adenosylcobalamin (AdoCbl) cofactor to catalyze the rearrangement of methylmalonyl-CoA to succinyl-CoA. Despite many years of dedicated research, the mechanism by which MMCM and related AdoCbl-dependent enzymes accelerate the rate for homolytic cleavage of the cofactor's Co-C bond by approximately 12 orders of magnitude while avoiding potentially harmful side reactions remains one of the greatest subjects of debate among B(12) researchers. In this study, we have employed electronic absorption (Abs) and magnetic circular dichroism (MCD) spectroscopic techniques to probe cofactor/enzyme active site interactions in the Co(3+)Cbl "ground" state for MMCM reconstituted with both the native cofactor AdoCbl and its derivative methylcobalamin (MeCbl). In both cases, Abs and MCD spectra of the free and enzyme-bound cofactor are very similar, indicating that replacement of the intramolecular base 5,6-dimethylbenzimidazole (DMB) by a histidine residue from the enzyme active site has insignificant effects on the cofactor's electronic properties. Likewise, spectral perturbations associated with substrate (analogue) binding to holo-MMCM are minor, arguing against substrate-induced enzymatic Co-C bond activation. As compared to the AdoCbl data, however, Abs and MCD spectral changes for the sterically less constrained MeCbl cofactor upon binding to MMCM and treatment of holoenzyme with substrate (analogues) are much more substantial. Analysis of these changes within the framework of time-dependent density functional theory calculations provides uniquely detailed insight into the structural distortions imposed on the cofactor as the enzyme progresses through the reaction cycle. Together, our results indicate that, although the enzyme may serve to activate the cofactor in its Co(3+)Cbl ground state to a small degree, the dominant contribution to the enzymatic Co-C bond activation presumably comes through stabilization of the Co(2+)Cbl/Ado. post-homolysis products.     

Substituent effects on the redox potentials of dihydroxybenzenes: theoretical and experimental study

The redox reactions of p-hydroquinone and pyrocatechol undergo a two-proton-two-electron process in aqueous solution. We calculated their redox potentials at the B3LYP/6-311+G(d,p) level, and verified the values by employing cyclic voltammetry experiments. Then we selected seven substituent groups (–F, –Cl, –OH, –COOH, –CN, –NH₂, and –NO₂ groups) to systematically investigate the substituent effect, including the sort, position, and number of the substituent, on the redox potentials of p-hydroquinone and pyrocatechol. The calculated results show that –NH₂ and –OH groups can decrease the redox potentials, while –F, –Cl, –COOH, –CN, and –NO₂ groups increase the potential values of p-hydroquinone and pyrocatechol. The calculations can accurately predict the substituent effects on the redox potentials of pyrocatechol and p-hydroquinone. We would expect that the accurate calculation results for the model systems could be applied in the prediction of electrode potentials of other molecules.     

Solvent,substituent, and dimerization effects on the ring-opening mechanisms of monosilacyclopropylidenoids: a theoretical study

Density functional theory and ab initio computations elucidated the ring-opening of substituted (R = –CF₃, –CN, –CH₃, –H, –NH₂, –OCH₃, –OH, –SiH₃) 1-bromo–1-lithiosilirane 1 and 2-bromo–2-lithiosilirane 2 to LiBr complexes of 2-silaallene and 1-silaallene, respectively. Formally, two competitive pathways can be considered. The ring-opening reaction can take place through a concerted manner via TS3. Alternatively, the reaction may proceed in a stepwise fashion with the intermediacy of a free silacyclopropylidene–LiBr complex 7. In both cases, the position of the substituents determines the kinetic of the reactions. The structures with an electron-donating group are generally unstable, whereas the silacyclopropylidenoids bearing electron-withdrawing substituents are particularly stable species. Here, we propose the ring-opening of 5a–h to corresponding LiBr complexes of 2-silaallenes can proceed in both concerted and stepwise mechanism except for –H, –CH₃, and –SiH₃. The obtained activation energies for the ring-openings of 5a–h to related 2-silaallenes are too high for a reaction at room temperature with up to 61.4 kcal/mol. In contrast, the activation energy barriers for the isomerization of 6a–h to the LiBr complexes of 1-silaallenes was determined to be relatively low at the B3LYP/6-31+G(d,p), M06/6-31+G(d,p), and MP2/6-31+G(d,p) levels. Moreover, we have also investigated the solvent effect on the unsubstituted models using both implicit and explicit solvation models. The energy barriers of the solvated models are found to be slightly higher than the results of gas phase calculations. Additionally, the ring-opening of dimer 6 (6–Dim) is also calculated for the ring-opening mechanism with the energy barrier of 3.7 kcal/mol at B3LYP/6-31+G(d,p) level of theory.     

Unusual pi-donating effects of pi-accepting substituents on the stabilities of benzylic cations: a theoretical study

The pi-donating effects of pi-accepting X-substituents in substituted benzylic cations, X-C(6)H(5)-CHR(+) where R = CF(3), H and OCH(3), and X = p-NH(2), p-OCH(3), p-CH(3), H, p-F, p-Cl, p-CHO, m-CN, p-CN, m-NO(2) or p-NO(2), have been studied theoretically by using isodesmic hydride transfer reactions at various levels of theory. It might be difficult to determine the pi-donating effects of pi-acceptors using the simple Hammett-type linear equation, because it is not sensitive enough to include small pi-donating effects. Therefore, this effect was estimated using the NBO deletion energy (DeltaE(D)) of the second-order charge-transfer interaction (DeltaE(ct)) between the pi-orbitals (or lone pair orbitals) of the X-substituent and the pi-orbitals of phenyl ring. The extents of pi-donating effects increased in the order X = p-NO(2) < p-CHO < p-CN < p-Cl for both neutral and cationic species, and these effects were found to be more important for para- than for meta-substituents. Moreover, this could represent a general trend for pi-donation by pi-acceptors. On the other hand, the effects of R-substituents on this pi-donating effect were found to be in the order R = OCH(3) < H congruent with CF(3), as predicted by natural resonance theory (NRT) analyses.     

Ligand effects on the stereochemical outcome of Suzuki-Miyaura couplings

The ligands associated with various Pd catalysts play a crucial role in determining the stereochemistry of cross-couplings between boronic acids and Z-alkenyl halides. A ligand on palladium has been found that leads to the desired products under mild conditions and in high yields that, in most cases, retain their Z-olefin geometry.     

Mirror "base-off" conformation of coenzyme B12 in human adenosyltransferase and its downstream target, methylmalonyl-CoA mutase

Human adenosyltransferase synthesizes coenzyme B12, for the target mitochondrial B12 enzyme, methylmalonyl-CoA mutase. It binds B12 in the "base-off" conformation in both the Co2+ and Co3+ oxidation states as revealed by UV-visible and EPR spectroscopy although it lacks the signature DXHXXG motif found in other B12 proteins that bind the cofactor in this conformation. The "base-off" conformation, which is rare at physiological pH, mirrors that in the target enzyme, methylmalonyl-CoA mutase, which utilizes the product, AdoCbl. However, the coordination environment for cobalt in the two proteins is distinct, which is reflected in an approximately 40-fold difference in their affinity for the cofactor.     

Solvent effects on the thioamide rotational barrier: an experimental and theoretical study.

The solvent effect on the C-N rotational barriers of N,N-dimethylthioformamide (DMTF) and N,N-dimethylthioacetamide (DMTA) has been investigated using ab initio theory and NMR spectroscopy. Selective inversion recovery NMR experiments were used to measure rotational barriers in a series of solvents. These data are compared to ab initio results at the G2(MP2) theoretical level. The latter are corrected for large amplitude vibrational motions to give differences in free energy. The calculated gas phase barriers are in very good agreement with the experimental values. Solvation effects were calculated using reaction field theory. This approach has been found to give barriers that are in good agreement with experiment for many aprotic, nonaromatic solvents that do not engage in specific interactions with the solute molecules. The calculated solution-phase barriers for the thioamides using the above solvents are also in good agreement with the observed barriers. The solvent effect on the thioamide rotational barrier is larger than that for the amides because the thioamides have a larger ground-state dipole moment, and there is a larger change in dipole moment with increasing solvent polarity. The transition-state dipole moments for the amides and thioamides are relatively similar. The origin of the C-N rotational barrier and its relation to the concept of amide "resonance" is examined.     

Entropic and electrostatic effects on the folding free energy of a surface-attached biomolecule: an experimental and theoretical study

Surface-tethered biomolecules play key roles in many biological processes and biotechnologies. However, while the physical consequences of such surface attachment have seen significant theoretical study, to date this issue has seen relatively little experimental investigation. In response we present here a quantitative experimental and theoretical study of the extent to which attachment to a charged-but otherwise apparently inert-surface alters the folding free energy of a simple biomolecule. Specifically, we have measured the folding free energy of a DNA stem loop both in solution and when site-specifically attached to a negatively charged, hydroxylalkane-coated gold surface. We find that whereas surface attachment is destabilizing at low ionic strength, it becomes stabilizing at ionic strengths above ~130 mM. This behavior presumably reflects two competing mechanisms: excluded volume effects, which stabilize the folded conformation by reducing the entropy of the unfolded state, and electrostatics, which, at lower ionic strengths, destabilizes the more compact folded state via repulsion from the negatively charged surface. To test this hypothesis, we have employed existing theories of the electrostatics of surface-bound polyelectrolytes and the entropy of surface-bound polymers to model both effects. Despite lacking any fitted parameters, these theoretical models quantitatively fit our experimental results, suggesting that, for this system, current knowledge of both surface electrostatics and excluded volume effects is reasonably complete and accurate.     

Solvent effects on tamoxifen molecule interacting with a single-walled carbon nanotube: a theoretical NMR study

Quantum chemical calculations of the electronic structure of tamoxifen molecule interacting with an open end of a single-walled carbon nanotube (SWCNT) were carried out and the effects of solvents (water, methanol, DMSO, acetone) on the ¹H, ¹³C, ¹⁵N, and ¹⁷O NMR parameters were studied by the GIAO-HF/STO-3G, GIAO-HF/3-21G, and GIAO/B1LYP/3-21G methods using the GAUSSIAN-98 program. The largest σ_iso value was obtained for acetone, whereas the smallest one for water. The opposite trend was obtained for the shielding asymmetry η. According to calculations, atoms at interaction site bear negative charges. The O(43) and N(38) atoms produce negative charge because they have high electron affinities. The dipole moment of tamoxifen molecule in different solvents increases with increasing the dielectric constant of the solvent. The largest dipole moment value was obtained for water by the B1LYP/3-21G method.     

Substituent effects on tandem alkenyl migration/electrophilic aromatic substitution reactions: a theoretical study

Quantum mechanical calculations (B3LYP/6-31G(d)) were used to study the substituent effects and the concertedness of the alkenyl migration/electrophilic aromatic substitution reactions recently reported by Oshima and co-workers. Our calculations suggest that these systems prefer stepwise mechanisms with their aryl attack steps having the highest energy transition structures, but that in some highly substituted cases, effectively concerted but very asynchronous processes may occur.     

Relevance of torsional effects to the conformational equilibria of 1,5-diaza-cis-decalins: a theoretical and experimental study

1,5-Diaza-cis-decalin populates two conformations in which the nitrogen atoms are either gauche (N-in) or anti (N-out) to one another. The equilibrium mixture of the two conformers depends on the substituents at the nitrogen atom, as well as the reaction conditions. Ab initio (HF/6-31G, B3LYP/6-31+G) and molecular mechanics (Amber) calculations have been performed to examine the possible role of stereoelectronics and steric effects in controlling the equilibrium of substituted 1,5-diaza-cis-decalins. In the present study, N,N'-diethyl- and N,N'-bistrifluoroethyl-1,5-diaza-cis-decalins have been synthesized, and the equilibrium mixtures have been measured using 1H and 13C NMR experiments. Steric effects appear to control the equilibria between the two conformational isomers of 1,5-diaza-cis-decalin while torsional effects appear to dominate the equilibria for the N,N'-dialkyl derivatives.     

Excited-state properties and environmental effects for protonated schiff bases: a theoretical study.

Complete active space self-consistent field (CASSCF), multireference configuration interaction (MRCI), density functional theory (DFT), time dependent DFT (TDDFT) and the singles and doubles coupled-cluster (CC2) methodologies have been used to study the ground state and excited states of protonated and neutral Schiff bases (PSB and SB) as models for the retinal chromophore. Systems with two to four conjugated double bonds are investigated. Geometry relaxation effects are studied in the excited pipi* state using the aforementioned methods. Taking the MRCI results as reference we find that CASSCF results are quite reliable even though overshooting of geometry changes is observed. TDDFT does not reproduce bond alternation well in the pipi* state. CC2 takes an intermediate position. Environmental effects due to solvent or protein surroundings have been studied in the excited states of the PSBs and SBs using a water molecule and solvated formate as model cases. Particular emphasis is given to the proton transfer process from the PSB to its solvent partner in the excited state. It is found that its feasibility is significantly enhanced in the excited state as compared to the ground state, which means that a proton transfer could be initiated already at an early step in the photodynamics of PSBs.