Shining light on the mechanism of photochemical alkene formation in vitamin B12.

Vitamin B12 is a complex organometallic molecule, the derivatives of which such as adenosylcobalamin (AdoCbl) and methylcobalamin (CH3Cbl) act as a cofactor in numerous enzymatic reactions. These two biologically active cofactors contain a unique organometallic Co-C σ bond. Important feature of this Co-C bond is that it can be activated by both thermally and photolytically inside the enzymatic environment as well as in the solution. In the case of enzymatic reactions where AdoCbl molecule act as a cofactor, the cleavage of the Co-C bond constitutes the key catalytic step. The most intriguing features of this cleavage is that upon binding with a substrate the cleavage of the Co-C bond is 1012-fold rate accelerated inside enzyme compared to the isolated cofactor. There are number of factors responsible behind this trillion-fold rate acceleration which are still under investigation. Alternatively, the Co-C bond in AdoCbl can also be cleaved by light to generate the same radical pair (RP) as in the case of enzymatic catalysis. The light sensitivity of these cobalamins was known for more than five decades only until recently it has been associated with controlled reactivity, namely optogenetic regulation and light-activated drug delivery. Moreover, the ability to probe photolytic cleavage of the Co−C bond for enzyme bound AdoCbl is of particular relevance in enzymatic catalysis. Herein this study the photolysis and native catalysis mechanism for vitamin B12-dependent enzymes will be investigated using a combined quantum mechanics/molecular mechanics (QM/MM) approach. According to our studies, it appears that the enzymatic environment controls the cleavage of Co-C bond by exerting an electrostatic interaction. In addition, the enzymatic environment controls the generated Ado radical path by providing a cage. This ultimately effects the formation of the product in native catalysis as well as in photolysis.Finally, I will be discussing a plausible connection between photolysis and native catalysis mechanism in vitamin B12-dependent enzymes based on the idea that the protein environment reduces the cofactor. The reaction mechanism based on the reduced cofactor in native catalysis was found to be similar with the photo-dissociation mechanism.

Sonolysis of aqueous solutions produces H. and HO. that lead to Co-C bond cleavage in methylcob-(III)alamin (CH3-CblIII) and 2-[4-[4'-[bis(2-chloroethyl)amino]phenyl]butyroxy]ethylcob (III)alamin (Chl-HE-CblIII). Under anaerobic conditions, H. reduces CH3-CblIII to the unstable 19 e-CH3-CblII that dissociates to the alkane and CblII. Under aerobic conditions, O2 scavenges H. and Co-C bond cleavage occurs via a HO.-mediated process along with modification of the corrin ring by HO.. When H. and HO. are scavenged, there is no evidence of Co-C bond cleavage. This suggests no direct sonolysis of the Co-C bond occurs, in spite of the fact that the Co-C bond is 80 kcal/mol weaker than the H-OH bond. A bioconjugate of cob(III)alamin and the alkylating agent chlorambucil has been synthesized to give 2-[4-[4'-[bis(2-chloroethyl)amino]phenyl]butyroxy]ethylcob(I II)alamin. The chlorambucil-cobalamin complex also undergoes Co-C bond cleavage in a manner similar to that of methylcob-(III)alamin. Sonorelease of an active alkylating agent from the bioconjugate may provide a new method for the selective release of anticancer drugs and thus potentially reduce systemic toxicity.

Adenosylcobinamide methyl phosphate, a novel analog of adenosylcobalamin lacking the nucleotide loop moiety, was synthesized. It did not show detectable coenzymic activity but behaved as a strong competitive inhibitor against AdoCbl with relatively high affinity (Ki = 2.5 microM). When apoenzyme was incubated at 37 degrees C with this analog in the presence of substrate, the Co-C bond of the analog was almost completely and irreversibly cleaved within 10 min, forming an enzyme-bound Co(II)-containing species. The cleavage was not observed in the absence of substrate. The Co-C bond cleavage in the presence of substrate was not catalytic but stoichiometric, implying that the Co-C bond of the analog undergoes activation when the analog binds to the active site of the enzyme. 5'-Deoxyadenosine was the only product derived from the adenosyl group of the analog upon the Co-C bond cleavage. Apoenzyme did not undergo modification during this process. Therefore, it seems likely that adenosylcobinamide methyl phosphate acts as a pseudocoenzyme or a potent suicide coenzyme. Since adenosylcobinamide neither functions as coenzyme nor binds tightly to apoenzyme, it can be concluded that the phosphodiester moiety of the nucleotide loop of adenosylcobalamin is essential for tight binding to apoenzyme and therefore for subsequent activation of the Co-C bond and catalysis. It is also evident that the nucleotide loop is obligatory for the normal progress of catalytic cycle.

The United Nations and the U.S. Environmental Protection Agency have identified a variety of chlorinated aromatics that constitute a significant health and environmental risk as "priority organic pollutants," the so-called "dirty dozen." Microbes have evolved the ability to utilize chlorinated aromatics as terminal electron acceptors in an energy-generating process called dehalorespiration. In this process, a reductive dehalogenase (CprA), couples the oxidation of an electron donor to the reductive elimination of chloride. We have characterized the B12 and iron-sulfur cluster-containing 3-chloro-4-hydroxybenzoate reductive dehalogenase from Desulfitobacterium chlororespirans. By defining the substrate and inhibitor specificity for the dehalogenase, the enzyme was found to require an hydroxyl group ortho to the halide. Inhibition studies indicate that the hydroxyl group is required for substrate binding. The carboxyl group can be replaced by other functionalities, e.g. acetyl or halide groups, ortho or meta to the chloride to be eliminated. The purified D. chlororespirans enzyme could dechlorinate an hydroxylated PCB (3,3',5,5'-tetrachloro-4,4'-biphenyldiol) at a rate about 1% of that with 3-chloro-4-hydroxybenzoate. Solvent deuterium isotope effect studies indicate that transfer of a single proton is partially rate-limiting in the dehalogenation reaction.

The vitamin B12 derivates, otherwise known as cobalamin (Cbl), are ubiquitous organometallic cofactors. The biologically active forms of Cbl, such as methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), act as cofactors in different physiological reactions for both prokaryotes and eukaryotes. A crucial aspect of the Cbl-mediated systems is the activation of the organometallic Co-C bond that plays a critical role in its catalytic activity. One of the most remarkable features of this Co-C bond is its unusual activation in AdoCbl-dependent enzymatic reactions, where a trillion-fold rate acceleration of the Co-C bond cleavage is observed inside the enzyme compared to the isolated AdoCbl. Although several hypotheses have been proposed previously, none can fully explain the trillion-fold rate acceleration that is observed for the Co-C bond cleavage. Thus, the factor(s) responsible for the unusual activation of the Co-C bond in the AdoCbl-dependent enzyme remains elusive. Nonetheless, this Co-C bond of MeCbl and AdoCbl cofactors is also known for its unique ability to be activated both thermally and photolytically within the enzymatic environment as well as in solutions. Even though the photochemistry of Cbl-dependent systems has been known for almost five decades, it has recently received a lot of attention due to its potential role in light-activated drug delivery, biomimetic catalysis, and a variety of other applications. Therefore, with these applications in mind, understanding the mechanistic insight into the activation of the Co-C bond is paramount for gaining a comprehensive knowledge of these reactions. In this dissertation, the mechanistic details of the activation of the Co-C in the photolysis and native catalysis of MeCbl and AdoCbl-dependent systems have been investigated using hybrid quantum mechanics/molecular mechanics (QM/MM) simulations, density functional theory (DFT), and time-dependent density functional theory (TD-DFT) methodologies. Overall, this dissertation is divided into six chapters. Chapter one gives an introduction, a historical overview of B12 chemistry, and possible applications in therapeutics and optogenetics. Chapters two and three discuss the photoactivation of the Co-C bond in MeCbl-dependent methionine synthase (MetH) and explore the role of the enzymatic environment on photoreaction. The photochemical data of isolated MeCbl cofactor in solution were also discussed and compared with the enzymatic environment to understand the effect of protein binding on the photolysis of Co-C bonds. The influence of mutation on the photolysis of Co-C is discussed in chapter three. Overall, in these two chapters, it was shown that the enzymatic environment affects the photolysis of the Co-C bond by modulating the electronically excited state. Chapter four provides an in-depth insight into the aerobic photolysis of MeCbl, with emphasis placed on the mechanistic details of the insertion of O2 in the activated Co-C bond. It was shown that the photochemical properties of MeCbl can also be modulated in the presence of molecular oxygen, i.e., in aerobic conditions. While chapters two to four cover the light-activation of the Co-C bond, chapter five focused on the activation of the Co-C bond during the native catalysis of AdoCbl-dependent methylmalonyl CoA mutase (MCM). The QM/MM methodology has been used to investigate the factor(s) responsible for the unusual activation of the Co-C bond that is observed in the enzyme as compared to AdoCbl in solution. While there are at least three previously reported hypotheses for the activation of the Co-C5ʹ bond including, substrate-induced conformational changes, electrostatic interaction between the Ado group and the enzyme, and involvement of tyrosine residue, none of these can explain this unusual activation. Thus, how the arrival of the substrate triggers the activation of the Co-C5ʹ bond remains an open issue.

The CASSCF geometry optimization of the adenosylcobalamin cofactor dependent processes common models with 12 orbitals and 12 electrons in the active space has been performed. The MCSCF geometry optimization shows a strong HOMO-LUMO coupling during the CASSCF orbitals mixing process. The HOMO-LUMO coupling causes an electronic density transfer from the HOMO, which at the beginning of the optimization process is constituted from the substrate atoms orbitals, to the LUMO, which is constituted from the adenosylco(III)balamin structure atomic orbitals. The Co-C bond cleavage reaction is starting from the beginning of the geometry optimization process due to the intermolecular transferred electronic density from the substrates to the adenosylco(III)balamin cofactor compound. Then, the HOMO and LUMO of the calculated models are converting into a bonding and an antibonding pair of orbitals with a central atom plus σ-axial ligands orbitals contribution and with a corrin ring plus axial ligands orbitals contribution, respectively. The HOMO-LUMO mixing process in the CASSCF procedure causes the intermolecular charge transfer process that converts into intramolecular charge transfer process, which is increasing up to about 1e- at the Co-C bond cleavage distance. The substrates of the adenosylcobalamin cofactor dependent bio-processes from one side and the 5'- deoxy-5’-adenosyl radical from another side are permanently growing their direct interactions along with the Co-C bond rupture process up to a strong direct interaction at the Co-C bond cleavage distance. Evidently, this is allowing for a hydrogen atom transfer between them. Altogether, the total energy barrier of the hydrogen transfer reaction from the substrate to the 5'- deoxy-5’-adenosyl radical reaction, the CASSCF HOMO and LUMO surface orbitals of the substrate and 5'-adenosyl radical interaction common model before and after the hydrogen transfer and a strong Pseudo-Jahn-Teller effect for only direct reaction demonstrate that the hydrogen transfer is an irreversible tunneling process, which certainly leads to the final products. All these results are pointing out to the Co-C bond cleavage and hydrogen transfer from substrate to 5'- deoxy-5’-adenosyl ligand concerted reactions in full agreement with the experimental data.

The adenosylcobalamin (AdoCbl)-dependent enzyme ethanolamine ammonia-lyase (EAL) catalyzes the conversion of ethanolamine to acetaldehyde and ammonia. As is the case for all AdoCbl-dependent isomerases, the catalytic cycle of EAL is initiated by homolytic cleavage of the cofactor's Co-C bond, producing CoIIcobalamin (CoIICbl) and an adenosyl radical that serves to abstract a hydrogen atom from the substrate. Remarkably, in the presence of substrate, the rate of Co-C bond homolysis of enzyme-bound AdoCbl is increased by 12 orders of magnitude. For Class I AdoCbl-dependent isomerases, an important contribution to this rate acceleration stems from a stabilization of the CoIICbl posthomolysis product by the axially coordinated histidine residue that displaces the pendant base from the Co ion upon AdoCbl binding to these enzymes. However, EAL and other Class II isomerases bind AdoCbl in the so-called "base-on" conformation and must therefore employ a different mechanism of Co-C bond activation. In the present study, we have used a combined spectroscopic and computational approach to probe the conformational changes and enzyme/cofactor/substrate interactions that contribute to the rate acceleration of Co-C bond homolysis in EAL. Spectroscopic data of AdoCbl and CoIICbl show minimal perturbations upon cofactor binding to EAL in both the absence and presence of substrate. Structural models of free and EAL-bound AdoCbl were constructed using molecular dynamics and quantum mechanics/molecular mechanics computations. By carrying out relaxed potential energy scans for Co-C bond cleavage of free and EAL-bound AdoCbl, we identified key cofactor/enzyme interactions that contribute to the Co-C bond activation by EAL and obtained Co-C bond dissociation energies that agree well with published experimental data.

Glutamate mutase (GLM) is a coenzyme B12-dependent enzyme that catalyzes the conversion of S-glutamate to (2 S,3 S)-3-methyl aspartate. The initial step in the catalytic process is the homolytic cleavage of the coenzyme's Co-C bond upon binding of a substrate. Alternatively, the Co-C bond can be cleaved using light. To investigate the photolytic cleavage of the Co-C bond in GLM, we applied a combined density functional theory/molecular mechanics (DFT/MM) and time-dependent-DFT/MM method to scrutinize the ground and the low-lying excited states. Potential energy surfaces (PESs) were generated as a function of axial bond lengths to describe the photodissociation mechanism. The S1 PES was characterized as the crossing of two electronic states, metal-to-ligand charge transfer (MLCT), and ligand field (LF). In GLM, radical pairs generate from the LF state. Two pathways, path A and path B, were identified as possible channels to connect the MLCT and LF electronic states. The S1 PES in GLM was compared with the S1 PES for coenzyme B12-dependent ethanolamine ammonia lyase as well as the isolated AdoCbl cofactor. Finally, the theoretical insights related to the photodissociation mechanism were compared with transient absorption spectroscopy, electron paramagnetic resonance, and resonance Raman spectroscopy.

ChemInformVolume 18, Issue 22 Physical Organic Chemistry ChemInform Abstract: New Insights into the Solution Behavior of Cobalamins. Studies of the Base-Off Form of Coenzyme B12 Using Modern Two-Dimensional NMR Methods. A. BAX, A. BAX Lab. Mol. Biophys., Natl. Inst. Environ. Health Sci., Research Triangle Park, NC 27709, USASearch for more papers by this authorL. G. MARZILLI, L. G. MARZILLI Lab. Mol. Biophys., Natl. Inst. Environ. Health Sci., Research Triangle Park, NC 27709, USASearch for more papers by this authorM. F. SUMMERS, M. F. SUMMERS Lab. Mol. Biophys., Natl. Inst. Environ. Health Sci., Research Triangle Park, NC 27709, USASearch for more papers by this author A. BAX, A. BAX Lab. Mol. Biophys., Natl. Inst. Environ. Health Sci., Research Triangle Park, NC 27709, USASearch for more papers by this authorL. G. MARZILLI, L. G. MARZILLI Lab. Mol. Biophys., Natl. Inst. Environ. Health Sci., Research Triangle Park, NC 27709, USASearch for more papers by this authorM. F. SUMMERS, M. F. SUMMERS Lab. Mol. Biophys., Natl. Inst. Environ. Health Sci., Research Triangle Park, NC 27709, USASearch for more papers by this author First published: June 2, 1987 https://doi.org/10.1002/chin.198722039AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume18, Issue22June 2, 1987 RelatedInformation

Optically active a carbon-chiral 1-substituted ethyl and 1, 2-disubstituted ethyl bis (dimethylglyoximato) cobalt complexes were prepared by resolution of chiral axial base-coordinated complexes followed by ligand displacement of the chiral axial base with achiral base. An unprecedented type of chiral octahedral cobalt complexes, trans- [Co (AB) 2 (X) (Y)], have also been synthesized enantioselectively. By taking advantage of the features of these chiral complexes, some precise information was obtained on the mechanism of Co-C bond formation and cleavage of the alkyl cobaloxime complexes, and Co-C bond dissociation energies were estimated which, in turn, made it possible to estimate steric and electronic effects on the Co-C bond cleavage quantita-tively.Unexpectedly, these complexes lead to the findings of X-ray-induced racemization of α carbon-chiral cobalt complexes in the crystalline-state, solid state-specific phenomena in the solid state photoracemization, unidirectional β to α (solid-state specific) photoisomerization. The mechanisms of these reactions are elucidated based on the X-ray structural analyses.

Recently developed two-dimensional (2D) NMR methods are used to assign completely the IH and I3C NMR spectra of the base-off form of coenzyme BI2 (5'-deoxyadenosylcobalamin, M, = 1580). The NMR data, including coupling constants, chemical shifts, 2D proton-carbon multiple-bond peak intensities, and 2D NOE peak intensities, are compared with those of the base-on form of coenzyme B12 and with the structure recently determined by neutron diffraction. The NOE signal intensities between protons of the corrin and adenosyl indicate a dynamic equilibrium between at least two states: in one state, the orientation of the adenosyl with respect to the corrin is similar to that observed in the crystal structure, and in the other state, the adenosyl is rotated ca. 50 counterclockwise (when viewed from the adenosyl) about the Co-C bond. Furthermore, we find no evidence for a change in corrin ring pucker on dissociation of the 5,6-dimethylbenzimidazole (DMBz). For the base-off species, the DMBz is repositioned upside down with the B7, B1 1, and R1 protons in the vicinity of the C20 methyl group. Previously unexplainable shift trends in the base-on to base-off conversion are more readily rationalized by use of the new data since, in earlier studies, errors were made both in assigning some of the signals of the base-on form and in following the shift of some of the signals with changes in pH. The protonated nucleotide loop in the base-off form has I3C chemical shifts similar to those of the protonated nucleotide, ribazole-3'-phosphate. The changes in chemical shifts and coupling constants in the ribose moiety of the loop suggest a change in conformation on formation of the base-off form. The primary effects on the side-chain signals of the base-on to base-off conversion are for the signals of the e-propionamide side chain. Relatively little effect is found for the A ring side-chain signals, and intermediate effects are found for the B and D ring side chains. Thus, from the base-off I3C data, there appears to be no reason for concluding that the eastern half of the corrin (rings B and C) is more flexible. 5'-Deoxyadenosylcobalamin (coenzyme BI2, M, = 1580) is a cofactor in over a dozen enzymatic reactions, two of which are known to be important in humans.' It is now generally believed that an essential early step in the catalytic cycle is the homolysis of the bond between Co and the A15 carbon of deoxyadenosyl (Chart I).2-4 The coenzyme is relatively stable to thermolytic homolysis, and it is clear that Co-C bond cleavage is induced by or electronic effects or a combination of both. In a recent review, several mechanisms were discussed in terms of their possible importance in the triggering of Co-C bond homolysi~.~ Four of these included, in some regard, interactions between the 5'-deoxyadenosyl moiety and the corrin ring. In two putative mechanisms, interactions were affected by a repositioning of the 5,6-dimethylbenzimidazole (DMBz) ligand. In this regard, UV data have been interpreted to indicate that, during catalysis, the cobalt-to-DMBz bond is br~ken.~ In addition, analysis of I3C NMR chemical shifts has led to speculation that the DMBz experiences steric compression with side chains of the corrin ring6*' and that the eastern half of the molecule (rings B and C) is more flexible than the western half.*

Biorelevant Chemistry of Cobalamin

See accompanying article by Yeounjoo Ko et al. DOI 10.1002/biot.201400221 Vitamin B12 is one of the most complex small molecules acting as a co-factor in processes that are crucial to many living organisms. Vitamin B12 can be produced by fermentation of native bacterial producers with up to 10000 kilogram commercial production of vitamin B12 annually [1]. Because native bacterial producers are not the usual industrial producer strains, many attempts have been made to heterologously produce vitamin B12 in non-native microbial hosts. Since the de novo biosynthesis of vitamin B12 involves 30 enzyme-mediated reactions, those efforts have been unsuccessful until the recent study made by Ko et al. [2], which is published in this issue of Biotechnology Journal. The structure of vitamin B12 is so complex that Dorothy Crowfoot Hodgkin, who resolved the three-dimensional structure of vitamin B12 (cyanocobalamin) [3] and other important biochemical substances, was awarded the Nobel Prize in Chemistry in 1964. Chemical synthesis of vitamin B12 is a highly complicated process involving at least 60 steps, which is technically challenging and economically unviable. In nature, vitamin B12 is unique in the sense that its de novo biosynthesis appears to be limited to some bacteria and archaea [1]. This includes aerobi biosynthesis in Rhodobacter sphaeroides and Pseudomonas denitrificans, and anaerobic biosynthesis in Bacillus megaterium, Propionibacterium shermanii, Salmonella typhimurium and Lactobacillus reuteri [4]. Transferring a metabolic pathway from its native producer into heterologous microbial hosts has become a common practice in metabolic engineering. Nevertheless, assembling a functional metabolic pathway comprising more than 30 biochemical reactions remains a great challenge. Successful assembly of such a complex metabolic pathway requires that all of the genes be cloned accurately and expressed correctly, and that all of the introduced enzymes be functional so that the carbon flux can be transferred to the final target product. Very often, a single nucleotide mutation would result in failure of the entire pathway, and trouble-shooting would be very difficult and time-consuming. Ko et al. [2] use three compatible plasmids to clone the Pseudomonas denitrificans vitamin B12 pathway, which is encoded by 25 genes located in six different operons [2]. This strategy successfully resulted in the production of vitamin B12 in Escherichia coli, demonstrating that vitamin B12, which has the most complex structure amongst all vitamins, can be produced heterologously. Interestingly, although the synthesis of vitamin B12 in P. denitrificans is strictly oxygen-dependent, Ko et al. [2] show that the recombinant E. coli can produce vitamin B12 under anaerobic conditions. This suggests that the biosynthetic route towards vitamin B12 may not be dependent on molecular oxygen; the oxygen-dependant vitamin B12 biosynthesis in P. denitrificans might be due to some regulatory mechanisms in those native hosts, which is only effective under aerobic conditions. Biosynthesis of vitamin B12 appears to be restricted to a few representative bacteria and archaea, while vitamin B12-dependent enzymes are widespread throughout all domains of life [5]. Generally, vitamin B12-dependent reactions are described to catalyze methyl transfers and carbon backbone rearrangements. Besides the well-known diol and glycerol dehydratase, some additional examples of enzymes that require vitamin B12 include methionine synthase, methylmalonyl-CoA mutase, glutamate mutase, isobutyryl-CoA mutase, and reductive dehalogenases. For those organisms that do not possess vitamin B12 biosynthetic ability, addition of vitamin B12 to the medium is a necessity to initiate vitamin B12-dependent reactions. Production of vitamin B12 in recombinant E. coli enables scientists to consider vitamin B12-dependent biochemical reactions when designing new pathways, thus expanding the availability of enzymes. Previously, production of vitamin B12 in its native producers can be improved by optimization of the culture medium and process, mutagenesis of the producing strain, overexpression of the gene cluster involved in biosynthesis of vitamin B12, or optimizing promoters, ribosomal-binding sites and terminators [1]. Production of vitamin B12 in recombinant E. coli opens new possibilities for further improvement. Recently, engineered E. coli strains which are capable of producing some amino acids, organic acids, and alcohols have outcompeted their native producers. This demonstrates the power of applying synthetic biology approaches in strain improvement. With this rapid progress in E. coli cell factories, it can be optimistically predicted that production of vitamin B12 by engineered E. coli will have a bright future and lead to many more complex applications of metabolic engineering and synthetic biology.

5'-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) serves as the cofactor for several enzymes that play important roles in fermentation and catabolism. All of these enzymes initiate catalysis by promoting homolytic cleavage of the cofactor's Co-C bond in response to substrate binding to their active sites. Despite considerable research efforts, the role of the lower axial ligand in facilitating Co-C bond homolysis remains incompletely understood. In the present study, we characterized several derivatives of AdoCbl and its one-electron reduced form, Co(II)Cbl, by using electronic absorption and magnetic circular dichroism spectroscopies. To complement our experimental data, we performed computations on these species, as well as additional Co(II)Cbl analogues. The geometries of all species investigated were optimized using a quantum mechanics/molecular mechanics method, and the optimized geometries were used to compute absorption spectra with time-dependent density functional theory. Collectively, our results indicate that a reduction in the basicity of the lower axial ligand causes changes to the cofactor's electronic structure in the Co(II) state that replicate the effects seen upon binding of Co(II)Cbl to Class I isomerases, which replace the lower axial dimethylbenzimidazole ligand of AdoCbl with a protein-derived histidine (His) residue. Such a reduction of the basicity of the His ligand in the enzyme active site may be achieved through proton uptake by the catalytic triad of conserved residues, DXHXGXK, during Co-C bond homolysis.

The roles of the D-ribosyl moiety and the bulky axial ligand of the nucleotide loop of adenosylcobalamin in coenzymic function have been investigated using two series of coenzyme analogs bearing various artificial bases. The 2-methylbenzimidazolyl trimethylene analog that exists exclusively in the base-off form was a totally inactive coenzyme for diol dehydratase and served as a competitive inhibitor. The benzimidazolyl trimethylene analog and the benzimidazolylcobamide coenzyme were highly active for diol dehydratase and ethanolamine ammonia-lyase. The imidazolylcobamide coenzyme was 59 and 9% as active as the normal coenzyme for diol dehydratase and ethanolamine ammonia-lyase, respectively. The latter analog served as an effective suicide coenzyme for both enzymes, although the partition ratio (k(cat)/k(inact)) of 630 for ethanolamine ammonia-lyase is much lower than that for diol dehydratase. Suicide inactivation was accompanied by the accumulation of a cob(II)amide species, indicating irreversible cleavage of the coenzyme Co-C bond during the inactivation. It was thus concluded that the bulkiness of a Co-coordinating base of the nucleotide loop is essential for both the initial activity and continuous catalytic turnovers. Since the k(cat)/k(inact) value for the imidazolylcobamide in diol dehydratase was 27-times higher than that for the imidazolyl trimethylene analog, it is clear that the ribosyl moiety protects the reaction intermediates from suicide inactivation. Stopped-flow measurements indicated that the rate of Co-C bond homolysis is essentially unaffected by the bulkiness of the Co-coordinating base for diol dehydratase. Thus, it seems unlikely that the Co-C bond is labilized through a ground state mechanochemical triggering mechanism in diol dehydratase.