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A reversed phase high-performance liquid chromatography method (HPLC) with coulometric electrochemical detection has been applied and validated for the simultaneous analysis of pyridoxamine (PM), pyridoxal (PL), and pyridoxine (PN) in cereal products. Isocratic separation was achieved using a C18 column with a mobile phase consisting of methanol-phosphate buffer (10:90) and 0.018 M trimethylamine adjusted to pH 3.55 with 85% orthophosphoric acid. The limits of detection for PM, PL, and PN were 0.28, 0.36, and 0.43 ng mL−1, respectively, with vitamins recoveries ranging from 90.4 to 98.1%. The applied method for the analysis of B6 vitamins naturally present in grain products, offers a simple and fast sample preparation without derivatization. To understand vitamer separation further, methods of computational chemistry were employed. Specifically, density functional theory (DFT) was used to determine electrostatic potentials, as well as the charges of each vitamer. A number of correlations were established between these properties and elution order.

A detailed computational analysis employing density functional theory (DFT), atoms in molecules, and quantum mechanics/molecular mechanics (QM/MM) tools has been performed to investigate the primary coordination environment of cob(I)alamin (Co(+)Cbx), which is a ubiquitous B(12) intermediate in methyltransferases and ATP:corrinoid adenosyltransferases. The DFT calculations suggest that the simplified (Co(+)Cbl) as well as the complete (Co(+)Cbi) complexes can adapt to the square pyramidal or octahedral coordination geometry owing to the unconventional H-bonding between the Co(+) ion and its axial ligands. These Co(+)-H bonds contain appreciable amounts of electrostatic, charge transfer, long-range correlation, and dispersion components. The computed reduction potentials of the Co(2+)/Co(+) couple imply that the Co(+)-H(H(2)O) interaction causes a greater anodic shift [5-98 mV vs. the normal hydrogen electrode (NHE) in chloroform solvent] than the analogous Co(+)-H(imidazole) interaction (1 mV vs. NHE) in the reduction potential of the Co(2+)/Co(+) couple. This may explain why a β-axial H(2)O ligand has specifically been found in the active sites of certain methyltransferases. The QM/MM analysis of methionine synthase bound Co(+)Cbx (Protein Data Bank ID 1BMT, resolution 3.0 Å) indicates that the enzyme-bound Co(+)Cbx can also form a Co(+)-H bond, but can only exist in square pyramidal form because of the steric constraints imposed by the cellular environment. The present calculations thus support a recently proposed alternate mechanism for the enzyme-bound Co(2+)/Co(+) reduction that involves the conversion of square pyramidal Co(2+)Cbx into square pyramidal Co(+)Cbx (Kumar and Kozlowski in Angew. Chem. Int. Ed. 50:8702-8705, 2011).

Density functional theory has been applied to investigate the methyl transfer from methylcobalamin (MeCbl) cofactor to homocysteine (Hcy) as catalyzed by methionine synthase (MetH). Specifically, the S(N)2 and the reductive elimination pathways have been probed as the possible mechanistic pathways for the methyl transfer reaction. The calculations indicate that the activation barrier for the reductive elimination reaction (24.4 kcal mol(-1)) is almost four times higher than that for the S(N)2 reaction (7.3 kcal mol(-1)). This high energy demand of the reductive elimination pathway is rooted in the structural distortion of the corrin ring that is induced en route to the formation of the triangular transition state. Furthermore, the reductive elimination reaction demands the syn accommodation of the methyl group and the substrate over the upper face of the corrin ring, which also accounts for the high energy demand of the reaction. Consequently, the reductive elimination pathway for MetH-catalyzed methyl transfer from MeCbl to Hcy cannot be considered as one of the possible mechanistic routes.

The full configuration interaction method in the space of fractionally occupied unrestricted natural orbitals (UNO-CAS method) is extended to excited states as well as to strongly correlated and reactive systems with large active spaces. This is accomplished by␣using restricted active space (RAS) wave functions introduced by Olsen et al. [(1988) J Chem Phys 89: 2185] and using the UNOs without the expensive orbital optimization step. In RAS, the space of active orbitals is subdivided into three groups: a group with essentially doubly occupied orbitals (RAS1), the usual CAS space (RAS2), and a space with weakly occupied active orbitals (RAS3). We select these spaces on the basis of the occupation numbers of the UNOs. All possible electron distributions are allowed in the usual CAS space, but the number of vacancies is limited in RAS1 and the number of electrons is limited in RAS3. We discuss an efficient algorithm for generating a RAS wave function. This is based on the Handy-Knowles determinantal expansion with an addressing scheme adopted for the restricted expansion. Results for both ground and excited states of azulene and free base porphyrin are presented.

We present the results of a comprehensive study on the inner-hydrogen migration in free base porphyrin, using density functional theory with the hybrid B3-LYP exchange-correlation functional, and both the 6-31G(d ) and a triple-zeta double-polarization (TZ2P) basis set. The latter computations, involving 726 contracted functions, are the largest calculations on this system to date. Full geometry optimization was carried out for the cis and trans minima, the transition state for trans-cis isomerization, and the symmetric stationary point for the synchronous trans-trans isomerization. All stationary points were characterized by vibrational analysis. Our results strongly support the conclusion, reached by earlier workers, that trans-trans hydrogen transfer occurs in a two-step process via a cis intermediate. With the TZ2P basis and including zero-point effects for the -h 2 isotopomer, the trans-cis barrier height is 13.1 kcal/mol, the cis-trans energy difference is 8.1 kcal/mol and the reverse cis-trans barrier height is 5.0 kcal/mol. The trans-cis barrier height agrees well with the value of Braun et al. (J Am Chem Soc (1996) 118: 7231) obtained from NMR line shapes and a modified Bell tunneling model, but our cis-trans energy difference is higher, and the reverse barrier is lower, than the values of Braun et al. Tunneling precludes the existence of -h 2 cis-porphyrin as an observable species, but the -d 2 and, especially, -t 2 isotopomers might be observable at low temperatures if the reverse barrier is higher than our calculated value. We predict the theoretical vibrational spectrum of cis-porphyrin and suggest that IR active modes at 566 cm−1 and 2333 cm−1 in the -d 2 isotopomer may be used to detect the presence of the cis intermediate.

The paradigm that nature protects us from CO poisoning by forcing the bound CO to bend over in heme proteins, thereby reducing its binding affinity, is now in textbooks, but is nevertheless problematic. Results from vibrational spectroscopy give no evidence for bent CO, although X-ray crystallography continues to indicate appreciable distortions in myoglobin. However, the energetic significance of the discrepancy is doubtful, since new Density Functional Theory calculations indicate that much less energy is required to distort the CO than had been thought, perhaps 2 kcal/mol or less. Binding studies on site-directed mutants of myoglobin suggest that steric hindrance by the distal histidine is worth ca. 1 kcal/mol. While the distal histidine does account for the discrimination by Mb against CO and in favor of O2, most of the effect is due to its H-bond with bound O2.

An explicit relation between the density matrix and its s-state part is analyzed for electron closed shells moving in a bare Coulomb potential. The density matrix has a simple separable form in terms ofr1 +r2 and |r1 −r2|. It is demonstrated that for an arbitrary number of closed shells, the off-diagonal dependence is simply polynomial in the |r1 −r2| coordinate.