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Cobalamins are of widespread importance in biology. Both of the cofactors essential for human metabolism, the organocobalamins coenzyme B12 and methylcobalamin, are highly photolabile, as are other alkylcobalamins. The alkynylcobalamin phenylethynylcobalamin (PhEtyCbl) and the arylcobalamin 4-ethylphenylcobalamin (EtPhCbl) with “atypical” Co–C-bonds to unsaturated carbons, were recently designed as metabolically inert cobalamins, classified as “antivitamins B12”. The further development of an ideal light-activated or “conditional” antivitamin B12 would require it to be readily converted by light into an active B12 vitamin form. Very photolabile “antivitamins B12” would also represent particularly useful scaffolds for therapeutic light-activated reagents. Here, the photoactive arylcobalamin EtPhCbl and the remarkably photostable alkynylcobalamin PhEtyCbl are examined using femtosecond to picosecond UV–visible transient absorption spectroscopy. PhEtyCbl undergoes internal conversion to the ground state with...

The His143, Glu170, and Asp335 residues at the substrate-binding site of diol dehydratase, a calcium–metalloenzyme, are shown by a computational mutation study to play important roles in OH group m...

Riboswitches are RNA elements that bind specific metabolites in order to regulate the gene expression involved in controlling the cellular concentration of the respective molecule or ion. Ligand recognition is mostly facilitated by Mg2+ mediated pre-organization of the riboswitch to an active tertiary fold. To predict these specific Mg2+ induced tertiary interactions of the btuB riboswitch from E. coli, we here report Mg2+ binding pockets in its aptameric part in both, the ligand-free and the ligand-bound form. An ensemble of weak and strong metal ion binding sites distributed over the entire aptamer was detected by terbium(III) cleavage assays, Tb3+ being an established Mg2+ mimic. Interestingly many of the Mn+ (n = 2 or 3) binding sites involve conserved bases within the class of coenzyme B12-binding riboswitches. Comparison with the published crystal structure of the coenzyme B12 riboswitch of S. thermophilum aided in identifying a common set of Mn+ binding sites that might be crucial for tertiary interactions involved in the organization of the aptamer. Our results suggest that Mn+ binding at strategic locations of the btuB riboswitch indeed facilitates the assembly of the binding pocket needed for ligand recognition. Binding of the specific ligand, coenzyme B12 (AdoCbl), to the btuB aptamer does however not lead to drastic alterations of these Mn+ binding cores, indicating the lack of a major rearrangement within the three-dimensional structure of the RNA. This finding is strengthened by Tb3+ mediated footprints of the riboswitch's structure in its ligand-free and ligand-bound state indicating that AdoCbl indeed induces local changes rather than a global structural rearrangement.

Vitamin B(12) and its biologically active counterparts possess the only examples of carbon-cobalt bonds in living systems. The role of such motifs as radical reservoirs has potential application in future catalytic and electronic nanodevices. To fully understand radical generation in coenzyme B(12) (dAdoCbl)-dependent enzymes, however, major obstacles still need to be overcome. In this work, we have used Car-Parrinello molecular dynamics (CPMD) simulations, in a mixed quantum mechanics/molecular mechanics (QM/MM) framework, to investigate the initial stages of the methylmalonyl-CoA-mutase-catalyzed reaction. We demonstrate that the 5'-deoxyadenosyl radical (dAdo(•)) exists as a distinct entity in this reaction, consistent with the results of extensive experimental and some previous theoretical studies. We report free energy calculations and first-principles trajectories that help understand how B(12) enzymes catalyze coenzyme activation and control highly reactive radical intermediates.

Two new coenzyme B12 analogues, 2′,5′-dideoxycytidylcobalamin (2a) and 2′,5′-dideoxyuridinylcobalamin (2b), and two others, 2′,5′-dideoxyadenosylcobalamin (2c), and 5′-deoxythymidylcobalamin (2d) were prepared by an improved method. All the four analogues were investigated by UV-vis and 2D 1H NMR spectroscopy. The comparisons and discussion about their spectroscopic properties were done.

Enzymes accelerate chemical reactions with an exceptional selectivity that makes life itself possible. Understanding the factors responsible for this efficient catalysis is of utmost importance in our quest to harness the tremendous power of enzymes. Computational chemistry has emerged as an important adjunct to experimental chemistry and biochemistry in this regard, because it provides detailed insights into the relationship between structure and function in a systematic and straightforward manner. In this Account, we highlight our recent high-level theoretical investigations toward this end in studying the radical-based reactions catalyzed by enzymes dependent on coenzyme B(12) (or adenosylcobalamin, AdoCbl). In addition to their fundamental position in biology, the AdoCbl-dependent enzymes represent a valuable framework within which to understand Nature's method of efficiently handling high-energy species to execute very specific reactions. The AdoCbl-mediated reactions are characterized by the interchange of a hydrogen atom and a functional group on adjacent carbon atoms. Our calculations are consistent with the conclusion that the main role of AdoCbl is to provide a source of radicals, thus moving the 1,2-rearrangements onto the radical potential energy surface. Our studies also show that the radical rearrangement step is facilitated by partial proton transfer involving the substrate. Specifically, we observe that the energy requirements for radical rearrangement are reduced dramatically with appropriate partial protonation or partial deprotonation or sometimes (synergistically) both. Such interactions are particularly relevant to enzyme catalysis, because it is likely that the local amino acid environment in the active site of an enzyme can function in this capacity through hydrogen bonding. Finally, our calculations indicate that the intervention of a very stable radical along the reaction pathway may inactivate the enzyme, demonstrating that sustained catalysis depends on a delicate energy balance. Radical-based enzyme reactions are often difficult to probe experimentally, so theoretical investigations have a particularly valuable role to play in their study. Our research demonstrates that a small-model approach can provide important and revealing insights into the mechanism of action of AdoCbl-dependent enzymes.

The formation of the Co(II)-substrate radical pair catalytic intermediate in coenzyme B12 (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been studied by using time-resolved continuous-wave electron paramagnetic resonance (EPR) spectroscopy in a cryosolvent system. The 41% v/v DMSO/water cryosolvent allows mixing of holoenzyme and substrate, (S)-2-aminopropanol, at 230 K under conditions of kinetic arrest. Temperature step from 230 to 234-248 K initiates the cleavage of the cobalt-carbon bond and the monoexponential rise (rate constant, k(obs) = tau(obs)(-1)) of the EPR-detected Co(II)-substrate radical pair state. The detection deadtime: tau(obs) ratio is reduced by10(2), relative to millisecond rapid mixing experiments at ambient temperatures. The EPR spectrum acquisition time istau(obs), which allows continuous acquisition of spectra during progress of the reaction. The k(obs) values and Co(II)-substrate radical pair amplitudes are independent of substrate concentration at each temperature. Therefore, the reaction occurs from the enzyme x coenzyme x substrate ternary complex. The constant value of the Co(II)-substrate radical pair amplitude at reaction times5tau(obs), the approximately 10(2)-fold slower rate of the substrate radical rearrangement reaction relative to k(obs), and the reversible temperature dependence of the amplitude indicate that the Co(II)-substrate radical pair and ternary complex are essentially at equilibrium. The reaction is thus treated as a relaxation to equilibrium by using a linear two-step, three-state mechanism. The intermediate state in this mechanism, the Co(II)-5'-deoxyadenosyl radical pair, is not detected by EPR at signal-to-noise ratios of 10(3), which indicates that the free energy of the Co(II)-5'-deoxyadenosyl radical pair state is3.3 kcal/mol, relative to the Co(II)-substrate radical pair. Van't Hoff analysis yields DeltaH13 = 10.8 +/- 0.8 kcal/mol and DeltaS13 = 45 +/- 3 cal/mol/K for the transition from the ternary complex to the Co(II)-substrate radical pair state. The free energy difference, DeltaG13, is zero to within one standard deviation over the temperature range 234-248 K. The extrapolated value of DeltaG13 at 298 K is -2.6 +/- 1.2 kcal/mol. The estimated EAL protein-associated contribution to the free energy difference is DeltaG(EAL) = -24 kcal/mol at 240 K, and DeltaH(EAL) = -13 kcal/mol and DeltaS(EAL) = 38 cal/mol/K. The results show that the EAL protein makes both strong enthalpic and entropic contributions to overcome the large, unfavorable cobalt-carbon bond dissociation energy, which biases the reaction in the forward direction of Co-C bond cleavage and Co(II)-substrate radical pair formation.

Computational modeling of the enzymatic activity of B12-dependent enzymes requires a detailed understanding of the factors that influence the strength of the CoC bond and the limits associated with a particular level of theory. To address this issue, a systematic analysis of the electronic and structural properties of coenzyme B12 models has been performed to establish the performance of three different functionals including B3LYP, BP86, and revPBE. In particular the cobalt–carbon bond dissociation energies, axial bond lengths, and selected stretching frequencies have been analyzed in detail. Current analysis shows that widely used B3LYP functional significantly underestimates the strength of the CoC bond while the nonhybrid BP86 functional produces very consistent results in comparison to experimental data. To explain such different performance of these functionals molecular orbital analysis associated with axial bonds has been performed to show differences in axial bonding provided by hybrid and nonhybrid functionals. © 2006 Wiley Periodicals, Inc. J Comput Chem 27: 1429–1437, 2006

The crystal and molecular structures of alkylcobaloxime/ g -cyclodextrin ( g -CD) inclusion complexes: butyl(aqua)cobaloxime/ g -CD ( 1 ). hexyl(aqua)cobaloxime/ g -CD ( 2 ) and cyclohexyl(aqua)cobaloxime/ g -CD ( 3 ) were determined by X-ray diffraction analyses. Crystal data for 1 ·14H 2 O: P 2 1 2 1 2 1 , a =15.1335(3), b =18.9630(2), c =28.1545(5) A, Z =4; 2 ·12H 2 O: P 2 1 2 1 2 1 , a =15.1135(4), b =19.1477(5), c =28.2715(7) A, Z =4; 3 ·10H 2 O: P 2 1 2 1 2 1 , a =15.6898(2), b =16.9094(2), c =28.9762(2) A, Z =4. The structural and conformational comparisons for the three g -CD inclusion complexes with their guest molecules as well as other CD inclusion analogs are discussed.

Coenzyme B12 (AdoCbl) is one of the most biologically active forms of vitamin B12, and continues to be a topic of active research interest. The mechanism of Co-C bond cleavage in AdoCbl, and the corresponding enzymatic reactions are however, not well understood at the molecular level. In this work, time-dependent density functional theory (TD-DFT) has been applied to investigate the photodissociation of coenzyme B12. To reduce computational cost, while retaining the major spectroscopic features of AdoCbl, a truncated model based on ribosylcobalamin (RibCbl) was used to simulate Co-C photodissociation. Equilibrium geometries of RibCbl were obtained by optimization at the DFT/BP86/TZVP level of theory, and low-lying excited states were calculated by TD-DFT using the same functional and basis set. The calculated singlet states, and absorption spectra were simulated in both the gas phase, and water, using the polarizable continuum model (PCM). Both spectra were in reasonable agreement with experimental data, and potential energy curves based on vertical excitations were plotted to explore the nature of Co-C bond dissociation. It was found that a repulsive (3)(σCo-C → σ(*) Co-C) triplet state became dissociative at large Co-C bond distance, similar to a previous observation for methylcobalamin (MeCbl). Furthermore, potential energy surfaces (PESs) obtained as a function of both Co-CRib and Co-NIm distances, identify the S1 state as a key intermediate generated during photoexcitation of RibCbl, attributed to a mixture of a metal-to-ligand charge transfer (MLCT) and a σ bonding-ligand charge transfer (SBLCT) states.

The research in our laboratory focuses on the role of metal ions and their complexes in structure formation and folding of nucleic acids. Large catalytic RNAs, like group II introns and some riboswitches, as well as shorter RNAs and DNAs containing modified nucleotides for the assembly of nanodevices are examined. Abundant metal ions like Mg2+ or natural metabolites like coenzyme B12 are in the center of interest, but also other metal ions, complexes thereof and B12 derivatives are applied with the aim to understand the largely unknown and manifold non-covalent interactions with nucleic acids. We apply a multitude of techniques, including potentiometric pH titrations, NMR spectroscopy, X-ray crystallography, gel electrophoresis and single molecule FRET experiments. Here we briefly summarize each of our research topics emphasizing the interaction of coenzyme B12 and its derivatives with the btuB riboswitch of E. coli. This highly conserved sequence, found in the 5'-untranslated region (5'-UTR) of the btuB mRNA, is involved in the regulation of the btuB protein expression. After a summary on the historical discovery of such riboswitches and their mechanism of action, we shortly focus on our own contributions to understand the structural equilibrium, high affinity and selectivity of the interaction between this specific RNA sequence and the largest and most complex cellular metabolite, coenzyme B12.

With CoI corrins as supernucleophiles, methylation by methanol is achieved when the leaving group is activated by Zn2+ ions at elevated temperatures (see reaction below). This methylation reaction is the first model for the Co-dependent methyltransferase.

Ab initio molecular orbital calculations have been used to study the mechanism of suicide inactivation of ethanolamine ammonia-lyase induced by three different substrate analogues. Analysis of the normal catalytic mechanism with 2-aminoethanol (ethanolamine) as substrate predicts that both the hydrogen-abstraction and hydrogen-reabstraction steps involving the B(12)-cofactor are likely to be exothermic. On the other hand, the proposed inactivation mechanism for the first substrate analogue, glycolaldehyde, leads to a highly stabilized radical that results in a very endothermic (by ca. 90 kJ mol(-)(1)) hydrogen-reabstraction step, which is thought to halt the normal function of the enzyme. Curiously, the energy requirements for a catalytically imposed mechanism in the case of the second substrate analogue, 2-hydroxyethylhydrazine (HEH), parallel those for the catalytic substrate, despite the fact that HEH is found to inactivate EAL experimentally. However, further analysis reveals the presence of a lower energy pathway for HEH that leads to the formation of the highly stabilized hydrazinium radical cation. In a manner similar to when glycolaldehyde is the substrate analogue, this results in an endothermicity for the hydrogen-reabstraction step that is prohibitively large. In contrast to these related inactivation mechanisms, the third substrate analogue, 2-aminoacetaldehyde, apparently accomplishes the inactivation of EAL in an entirely different manner. A pathway for the experimentally observed formation of acetic acid and ammonium cation has been identified and appears catalytic in the sense that 5'-deoxyadenosyl radical is regenerated. However, mechanisms to account for the subsequent formation of 4',5'-anhydroadenosine and degradation of the corrinoid ring of the cofactor have not been elucidated.

High-level ab initio calculations have been used to study the mechanism for the inactivation of diol dehydratase (DDH) by glycolaldehyde or 2-chloroacetaldehyde. As in the case of the catalytic substrates of DDH, e.g., ethane-1,2-diol, the 5'-deoxyadenosyl radical (Ado*) is able to abstract a hydrogen atom from both substrate analogues in the initial step on the reaction pathway, as evidenced by comparable energy barriers. However, in subsequent step(s), each substrate analogue produces the highly stable glycolaldehyde radical. The barrier for hydrogen atom reabstraction by the glycolaldehyde radical is calculated to be too high ( approximately 110 kJ mol-1) to allow Ado* to be regenerated and recombine with the cob(II)alamin radical, the latter therefore remaining tightly bound to DDH. As a consequence, the catalytic pathway is disrupted, and DDH becomes an impotent enzyme. Interconversion of equivalent structures of the glycolaldehyde radical via the symmetrical cis-ethanesemidione radical is calculated to require 38 kJ mol-1. EPR indications of a symmetrical cis-ethanesemidione structure are likely to be the result of formation of an equilibrium mixture of glycolaldehyde radical structures, this equilibration being facilitated by partial deprotonation of the glycolaldehyde radical by the carboxylate of an amino acid residue within the active site of DDH.

Ab initio molecular orbital calculations are used to examine the mechanism of action of B(12)-dependent ethanolamine ammonia-lyase involving the conversion of 2-aminoethanol to acetaldehyde plus ammonia. We attempt to elucidate the mechanism by which the enzyme facilitates this reaction through interactions between active-site residues and the substrate. Our calculations suggest a preferred pathway involving a 1,2-shift in the associated radical and also suggest that interactions between the enzyme and the migrating group of the substrate that afford an almost fully protonated migrating group will lead to the most efficient catalysis. However, this criterion on its own is insufficient to fully understand the rearrangement. Additional synergistic interactions between the spectator hydroxyl group in the substrate and active-site residues on the enzyme are required to lower the barrier height to a value consistent with experimental observations.

A new cobaloxime-type model for coenzyme B12 has been synthesized. For this model strong steric repulsion between its dioxymethylene bridge and (one of) the axial ligands on cobalt presages enhanced rates of axial ligand exchange.

The title compound, [Co(C11H19N4O2)(C5H11)(H2O)]ClO4, is one of the coenzyme B12 models with the equatorial ligand having the same −1 formal charge as B12 corrin. In the complex, the Co atom has a distorted octahedral coordination, with the n-pentyl and water ligands in axial positions.

The degenerate rearrangement of the 3-propanal radical (as a model for the methylmalonyl-CoA-mutase-catalyzed reaction) is an example of a reaction whose barrier is lowered substantially by protonation. Application of the idea of partial proton transfer to this reaction demonstrates that complete protonation is not necessary to obtain a significant amount of proton-induced barrier lowering. We believe that these findings may provide some clue as to the ability of enzymes to catalyze certain categories of reactions. If a reaction is facilitated by protonation and the proton-accepting site is H-bonded to the enzyme, there will be a contribution to the lowering of the barrier made by partial proton transfer. It is possible to view protonation as a “source” of energy for those reactions whose barriers are lowered by it. It would seem curious under such circumstances for nature not to tap this source, at least to some degree. We submit that such a tapping mechanism is provided by partial proton transfer.

Air oxidation of a CoII(salen) derivative, whose ethanediyl moiety carries a methylene-linked 4-hydroxypent-1-en-3-yl substituent, yields an intramolecularly bridged (tetrahydrofurfuryl)CoIII(salen) complex of which the crystal structure has been determined; this B12 model is very resistant to Co–C bond homolysis, even in the presence of a large excess of the radical trap TEMPO.

A novel B12 derivative 4 containing a peripheral thymine group is alkylated under reductive conditions with 2-bromo- methyl-2-methylmonothiomalonate 8 bearing the complementary adenine; photolysis in chloroform–acetonitrile gives the rearranged succinate 12 and dimethylmalonate 13 (2 : 1), whereas in MeOH–H2O only the dimethylmalonate 13 is formed.

This article illustrates how the combined computational/spectroscopic methodology used in our studies of metal centers in proteins and cofactors can be applied to vitamin B12 and its biologically relevant derivatives. The B12 cofactors have long fascinated chemists because of their complex structures and unusual reactivities in biological systems; however, their electronic absorption (Abs) spectra have remained largely unassigned. In this study, Abs, circular dichroism (CD), magnetic CD (MCD), and resonance Raman spectroscopic techniques are used to probe the electronic excited states of various Co3+ Cbl species that differ with respect to their upper axial ligand. Spectroscopic data for each species are analyzed within the framework of time-dependent density functional theory (TD-DFT) to assign the major spectral features and to generate experimentally validated bonding descriptions. A simple model is presented that explains why the identity of the upper axial ligand has a major effect on the Co–Nax bond strength, whereas the lower axial ligand does not appreciably modulate the nature of the Co–C bond. Impli- cations of these results with respect to enzymatic Co–C bond activation are discussed.

The radical cleavage of the carbon-cobalt bond of 1-phenyl-2-oxocyclopentylmethyl cobaloxime (1) and 1-ethoxycarbonyl-2-oxocyclopentylmethyl cobaloxime (2) gave only 3-phenylcyclohex-2-enone (7) and 3-ethoxycarbonylcyclohex-2-enone (8), respectively, by acyl migration. This rearrangement may be a reasonable mimicry of the ester migration mediated by coenzyme-B12.

Organic radicals generated by the oxidation of aldehydes, alcohols and ethers under reducing conditions are trapped by vitamin B12r to yield substituted organocobalamins. From higher n-alkyl aldehydes, acylcobalamins are formed. With acetaldehyde, a mixture of acetylcobalamin and of methylcobalamin is obtained due to the spontaneous decarbonylation of the CH3CO· radical. From saturated alcohols, w-hydroxyalkylcobalamins are produced, while w-alkoxyalkylcobalamins are formed in the corresponding reactions with radicals generated from ethers. Maximum yields of the organocobalamins are obtained if reducing conditions are maintained during the oxidation of the organic substrates. This is conveniently achieved by using V(III) salts as the reductants and the slow addition of oxidants (e.g. of O2, H2O2, Fenton reagent, or of electrochemically generated oxidizing equivalents). With 5′-deoxyadenosine, 5′-deoxyadenosylcobalamin is formed.

Coenzyme B12 (5′-deoxyadenosylcobalamin, abbreviated [CoIIIR−]) serves as a cofactor for several enzymic reactions, a common feature of which involves the 1,2-interchange of a H atom and another group (X = OH, NH2, CH(NH2)COOH, etc.) on adjacent carbon atoms, i.e., A widely accepted mechanistic interpretation of these reactions, supported by a variety of evidence from studies on the enzymic processes as well as on model systems, is, At the present stage it appears that the principal, if not the only, role of coenzyme B12 is to serve as the precursor for an organic free radical which triggers the substrate rearrangement. This role implies and utilizes a very weak Coalkyl bond, susceptible to homolytic cleavage under mild conditions, for which there has until recently been no convincing evidence or precedent. We have developed the first reliable systematic methods for the determination of transition metalalkyl bond dissociation energies (BDE) and have applied these to the determination of such BDE's in a variety of organocobalt compounds (notably containing dioxime or Schiff base ligands) related to coenzyme B12. The CoC BDE's yielded by such measurements are in the range 15–25 kcal/mol, i.e., low enough to be consistent with the proposed role of coenzyme B12 depicted in Eqn. (1). The influences of electronic and steric factors on CoC bond dissociation energies, revealed by these studies, are such as to suggest that coenzyme B12 is an ideal candidate for such a role and that steric factors probably are responsible for the enzyme-induced CoC bond weakening and dissociation. An analogy is developed between the role of coenzyme B12 in biological systems as a reversible ‘free radical carrier’ (i.e., according to Eqn. (1)) and the role of hemes as reversible dioxygen carriers, i.e., [FeIIIO2−] ⇄ [FeII] + O2

Simple corrins such as vitamin B12 and vitamin B12 coenzyme catalyze a variety of unusual enzymatic reactions of which some are still without analogy in organic or organometallic chemistry. The mechanisms of these reactions are currently the subject of lively discussion. The present review focuses attention on new ideas about the mode of action of vitamin B12 coenzymes in enzymatic reactions.