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.     

Spectroscopic and computational studies of Co2+corrinoids: spectral and electronic properties of the biologically relevant base-on and base-off forms of Co2+cobalamin

Co(2+)cobalmain (Co(2+)Cbl) is implicated in the catalytic cycles of all adenosylcobalamin (AdoCbl)-dependent enzymes, as in each case catalysis is initiated through homolytic cleavage of the cofactor's Co-C bond. The rate of Co-C bond homolysis, while slow for the free cofactor, is accelerated by 12 orders of magnitude when AdoCbl is bound to the protein active site, possibly through enzyme-mediated stabilization of the post-homolysis products. As an essential step toward the elucidation of the mechanism of enzymatic Co-C bond activation, we employed electronic absorption (Abs), magnetic circular dichroism (MCD), and resonance Raman spectroscopies to characterize the electronic excited states of Co(2+)Cbl and Co(2+)cobinamide (Co(2+)Cbi(+), a cobalamin derivative that lacks the nucleotide loop and 5,6-dimethylbenzimazole (DMB) base and instead binds a water molecule in the lower axial position). Although relatively modest differences exist between the Abs spectra of these two Co(2+)corrinoid species, MCD data reveal that substitution of the lower axial ligand gives rise to dramatic changes in the low-energy region where Co(2+)-centered ligand field transitions are expected to occur. Our quantitative analysis of these spectral changes within the framework of time-dependent density functional theory (TD-DFT) calculations indicates that corrin-based pi --> pi transitions, which dominate the Co(2+)corrinoid Abs spectra, are essentially insulated from perturbations of the lower ligand environment. Contrastingly, the Co(2+)-centered ligand field transitions, which are observed here for the first time using MCD spectroscopy, are extremely sensitive to alterations in the Co(2+) ligand environment and thus may serve as excellent reporters of enzyme-induced perturbations of the Co(2+) state. The power of this combined spectroscopic/computational methodology for studying Co(2+)corrinoid/enzyme active site interactions is demonstrated by the dramatic changes in the MCD spectrum as Co(2+)Cbi(+) binds to the adenosyltransferase CobA.     

The enzymatic activation of coenzyme B12

The enzymatic "activation" of coenzyme B12 (5'-deoxyadenosylcobalamin, AdoCbl), in which homolysis of the carbon-cobalt bond of the coenzyme is catalyzed by some 10(9)- to 10(14)-fold, remains one of the outstanding problems in bioinorganic chemistry. Mechanisms which feature the enzymatic manipulation of the axial Co-N bond length have been investigated by theoretical and experimental methods. Classical mechanochemical triggering, in which steric compression of the long axial Co-N bond leads to increased upward folding of the corrin ring and stretching of the Co-C bond is found to be feasible by molecular modeling, but the strain induced in the Co-C bond seems to be too small to account for the observed catalytic power. The modeling study shows that the effect is a steric one which depends on the size of the axial nucleotide base, as substitution of imidazole (Im) for the normal 5,6-dimethylbenzimidazole (Bzm) axial base decreases the Co-C bond labilization considerably. An experimental test was thus devised using the coenzyme analog with Im in place of Bzm (Ado(Im)Cbl). Studies of the enzymatic activation of this analog by the B12-dependent ribonucleoside triphosphate reductase from Lactobacillus leichmannii coupled with studies of the non-enzymatic homolytic lability of the Co-C bond of Ado(Im)Cbl show that the enzyme is only slightly less efficient (3.8-fold, 0.8 kcal mol(-1)) at activating Ado(Im)Cbl than at activating AdoCbl itself. This suggests, in agreement with the modeling study, that mechanochemical triggering can make only a small contribution to the enzymatic activation of AdoCbl. Another possibility, electronic stabilization of the Co(II) homolysis product by compression of the axial Co-N bond, requires that enzymatic activation be sensitive to the basicity of the axial nucleotide. Preliminary studies of the enzymatic activation of a coenzyme analog with a 5-fluoroimidazole axial nucleotide suggest that the catalysis of Co-C bond homolysis may indeed be significantly slowed by the decrease in basicity.     

Spectroscopic and computational studies of Co1+cobalamin: spectral and electronic properties of the "superreduced" B12 cofactor

The 4-coordinate, low-spin cob(I)alamin (Co1+Cbl) species, which can be obtained by heterolytic cleavage of the Co-C bond in methylcobalamin or the two-electron reduction of vitamin B12, is one of the most powerful nucleophiles known to date. The supernucleophilicity of Co1+Cbl has been harnessed by a number of cobalamin-dependent enzymes, such as the B12-dependent methionine synthase, and by enzymes involved in the biosynthesis of B12, including the human adenosyltransferase. The nontoxic nature of the Co1+Cbl supernucleophile also makes it an attractive target for the in situ bioremediation of halogenated waste. To gain insight into the geometric, electronic, and vibrational properties of this highly reactive species, electronic absorption, circular dichroism (CD), magnetic CD, and resonance Raman (rR) spectroscopies have been employed in conjunction with density functional theory (DFT), time-dependent DFT, and combined quantum mechanics/molecular mechanics computations. Collectively, our results indicate that the supernucleophilicity of Co1+Cbl can be attributed to the large destabilization of the Co 3dz2-based HOMO and its favorable orientation with respect to the corrin macrocycle, which minimizes steric repulsion during nucleophilic attack. An intense feature in the CD spectrum and a prominent peak in the rR spectra of Co1+Cbl have been identified that may serve as excellent probes of the nucleophilic character, and thus the reactivity, of Co1+Cbl in altered environments, including enzyme active sites. The implications of our results with respect to the enzymatic formation and reactivity of Co1+Cbl are discussed, and spectroscopic trends along the series from Co3+Cbls to Co2+Cbl and Co1+Cbl are explored.     

Spectroscopic and computational studies of the ATP:corrinoid adenosyltransferase (CobA) from Salmonella enterica: insights into the mechanism of adenosylcobalamin biosynthesis

CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo biosynthesis of adenosylcobalamin (AdoCbl), catalyzing the formation of the essential Co-C bond by transferring the adenosyl group from a molecule of ATP to a transient Co(1+)corrinoid species generated in the enzyme active site. A particularly fascinating aspect of this reaction is that the flavodoxin in vivo reducing agent that serves as the electron donor to CobA possesses a reduction potential that is considerably more positive than that of the Co(2+/1+) couple of the corrinoid substrate. To explore how CobA may overcome this challenge, we have employed electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to probe the interaction between Co(3+)- and Co(2+)corrinoids and the enzyme active site. Our data reveal that while Co(3+)corrinoids interact only weakly with CobA, Co(2+)corrinoids undergo partial conversion to a new paramagnetic species that can be obtained in nearly quantitative yield when CobA is preincubated with the co-substrate ATP. This "activated" species is characterized by a distinct set of ligand field transitions in the near-IR spectral region and EPR parameters that are unprecedented for Co(2+)corrinoids. Analysis of these data on the basis of qualitative spectral correlations and density functional theory computations reveals that this unique Co(2+)corrinoid species possesses an essentially square-planar Co(2+) center that lacks any significant axial bonding interactions. Possible implications of these findings for the mechanism of Co(2+) --> Co(1+) reduction employed by CobA and Co-C bond-forming enzymes in general are explored.     

Influence of environment on the electronic structure of Cob(III)alamins: time-resolved absorption studies of the S(1) state spectrum and dynamics

Transient absorption spectroscopy has been used to elucidate the nature of the S1 intermediate state populated following excitation of cob(III)alamin (Cbl(III)) compounds. This state is sensitive both to axial ligation and to solvent polarity. The excited-state lifetime as a function of temperature and solvent environment is used to separate the dynamic and electrostatic influence of the solvent. Two distinct types of excited states are identified, both assigned to pi3d configurations. The spectra of both types of excited states are characterized by a red absorption band (ca. 600 nm) assigned to Co 3d --> 3d or Co 3d --> corrin pi* transitions and by visible absorption bands similar to the corrin pi-->pi* transitions observed for ground state Cbl(III) compounds. The excited state observed following excitation of nonalkyl Cbl(III) compounds has an excited-state spectrum characteristic of Cbl(III) molecules with a weakened bond to the axial ligand (Type I). A similar excited-state spectrum is observed for adenosylcobalamin (AdoCbl) in water and ethylene glycol. The excited-state spectrum of methyl, ethyl, and n-propylcobalamin is characteristic of a Cbl(III) species with a sigma-donating alkyl anion ligand (Type II). This Type II excited-state spectrum is also observed for AdoCbl bound to glutamate mutase. The results are discussed in the context of theoretical calculations of Cbl(III) species reported in the literature and highlight the need for additional calculations exploring the influence of the alkyl ligand on the electronic structure of cobalamins.     

Structural Insights into the Very Low Activity of the Homocoenzyme B12 Adenosylmethylcobalamin in Coenzyme B12-Dependent Diol Dehydratase and Ethanolamine Ammonia-Lyase

The X-ray structures of coenzyme B₁₂ (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co−C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states. The impacts of incorporating an extra CH₂ group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.     

Time-resolved measurements of the photolysis and recombination of adenosylcobalamin bound to glutamate mutase

Femtosecond to nanosecond transient absorption spectroscopy is used to investigate the photolysis of 5'-deoxyadenosylcobalamin (coenzyme B12, AdoCbl) bound to glutamate mutase. The photochemistry of AdoCbl is found to be inherently dependent upon the environment of the cofactor. Excitation of AdoCbl bound to glutamate mutase results in formation of a metal-to-ligand charge transfer intermediate state which decays to form cob(II)alamin with a time constant of 105 ps. This observation is in contrast to earlier measurements in water where the photohomolysis proceeds through an intermediate state in which the axial dimethylbenzimidazole ligand appears to have dissociated, and measurements in ethylene glycol where prompt bond homolysis is observed (Yoder, L. M.; Cole, A. G.; Walker, L. A., II; Sension, R. J. J. Phys. Chem. B 2001, 105, 12180-12188). The quantum yield for formation of stable radical pairs in the enzyme is found to be phi = 0.05 +/- 0.03, and the resulting intrinsic rate constants for geminate recombination and "cage escape" are 1.0 +/- 0.1 and 0.05 +/- 0.03 ns(-1), respectively. The rate constant for geminate recombination is 30% less than that observed for AdoCbl in water or ethylene glycol. This reduction is insufficient to account for the 10(12)-fold increase in the homolysis rate observed when substrate is bound to the protein. Finally, the protein provides a cage to prevent diffusive loss of the adenosyl radical; however, the ultimate yield for long-lived radicals is determined by the evolution from a singlet to a triplet radical pair as proposed for AdoCbl in ethylene glycol.     

Spectroscopic characterization of active-site variants of the PduO-type ATP:corrinoid adenosyltransferase from Lactobacillus reuteri: insights into the mechanism of four-coordinate Co(II)corrinoid formation

The PduO-type adenosine 5'-triphosphate (ATP):corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) catalyzes the transfer of the adenosyl-group of ATP to Co(1+)cobalamin (Cbl) and Co(1+)cobinamide (Cbi) substrates to synthesize adenosylcobalamin (AdoCbl) and adenosylcobinamide (AdoCbi(+)), respectively. Previous studies revealed that to overcome the thermodynamically challenging Co(2+) → Co(1+) reduction, the enzyme drastically weakens the axial ligand-Co(2+) bond so as to generate effectively four-coordinate (4c) Co(2+)corrinoid species. To explore how LrPduO generates these unusual 4c species, we have used magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopic techniques. The effects of active-site amino acid substitutions on the relative yield of formation of 4c Co(2+)corrinoid species were examined by performing eight single-amino acid substitutions at seven residues that are involved in ATP-binding, an intersubunit salt bridge, and the hydrophobic region surrounding the bound corrin ring. A quantitative analysis of our MCD and EPR spectra indicates that the entire hydrophobic pocket below the corrin ring, and not just residue F112, is critical for the removal of the axial ligand from the cobalt center of the Co(2+)corrinoids. Our data also show that a higher level of coordination among several LrPduO amino acid residues is required to exclude the dimethylbenzimidazole moiety of Co(II)Cbl from the active site than to remove the water molecule from Co(II)Cbi(+). Thus, the hydrophilic interactions around and above the corrin ring are more critical to form 4c Co(II)Cbl than 4c Co(II)Cbi(+). Finally, when ATP analogues were used as cosubstrate, only "unactivated" five-coordinate (5c) Co(II)Cbl was observed, disclosing an unexpectedly large role of the ATP-induced active-site conformational changes with respect to the formation of 4c Co(II)Cbl. Collectively, our results indicate that the level of control exerted by LrPduO over the timing for the formation of the 4c Co(2+)corrinoid intermediates is even more exquisite than previously anticipated.     

Reductive cleavage mechanism of methylcobalamin: elementary steps of Co-C bond breaking

Density functional theory has been applied to the investigation of the reductive cleavage mechanism of methylcobalamin (MeCbl). In the reductive cleavage of MeCbl, the Co-C bond is cleaved homolytically, and formation of the anion radical ([MeCbl]*-) reduces the dissociation energy by approximately 50%. Such dissociation energy lowering in [MeCbl]*- arises from the involvement of two electronic states: the initial state, which is formed upon electron addition, has dominant pi*corrin character, but when the Co-C bond is stretched the unpaired electron moves to the sigma*Co-C state, and the final cleavage involves the three-electron (sigma)2(sigma*)1 bond. The pi*corrin-sigma*Co-C states crossing does not take place at the equilibrium geometry of [MeCbl]*- but only when the Co-C bond is stretched to 2.3 A. In contrast to the neutral cofactor, the most energetically efficient cleavage of the Co-C bond is from the base-off form. The analysis of thermodynamic and kinetic data provides a rationale as to why Co-C cleavage in reduced form requires prior departure of the axial base. Finally, the possible connection of present work to B12 enzymatic catalysis and the involvement of anion-radical-like [MeCbl]*- species in relevant methyl transfer reactions is discussed.     

Characterization of protein contributions to cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase by using photolysis in the ternary complex

Protein contributions to the substrate-triggered cleavage of the cobalt-carbon (Co-C) bond and formation of the cob(II)alamin-5'-deoxyadenosyl radical pair in the adenosylcobalamin (AdoCbl)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium have been studied by using pulsed-laser photolysis of AdoCbl in the EAL-AdoCbl-substrate ternary complex, and time-resolved probing of the photoproduct dynamics by using ultraviolet-visible absorption spectroscopy on the 10(-7)-10(-1) s time scale. Experiments were performed in a fluid dimethylsulfoxide/water cryosolvent system at 240 K, under conditions of kinetic competence for thermal cleavage of the Co-C bond in the ternary complex. The static ultraviolet-visible absorption spectra of holo-EAL and ternary complex are comparable, indicating that the binding of substrate does not labilize the cofactor cobalt-carbon (Co-C) bond by significantly distorting the equilibrium AdoCbl structure. Photolysis of AdoCbl in EAL at 240 K leads to cob(II)alamin-5'-deoxyadenosyl radical pair quantum yields of <0.01 at 10(-6) s in both holo-EAL and ternary complex. Three photoproduct states are populated following a saturating laser pulse, and labeled, P(f), P(s), and P(c). The relative amplitudes and first-order recombination rate constants of P(f) (0.4-0.6; 40-50 s(-1)), P(s) (0.3-0.4; 4 s(-1)), and P(c) (0.1-0.2; 0) are comparable in holo-EAL and in the ternary complex. Time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy shows that visible irradiation alters neither the kinetics of thermal cob(II)alamin-substrate radical pair formation, nor the equilibrium between ternary complex and cob(II)alamin-substrate radical pair, at 246 K. The results indicate that substrate binding to holo-EAL does not "switch" the protein to a new structural state, which promptly stabilizes the cob(II)alamin-5'-deoxyadenosyl radical pair photoproduct, either through an increased barrier to recombination, a decreased barrier to further radical pair separation, or lowering of the radical pair state free energy, or a combination of these effects. Therefore, we conclude that such a change in protein structure, which is independent of changes in the AdoCbl structure, and specifically the Co-C bond length, is not a basis of Co-C bond cleavage catalysis. The results suggest that, following the substrate trigger, the protein interacts with the cofactor to contiguously guide the cleavage of the Co-C bond, at every step along the cleavage coordinate, starting from the equilibrium configuration of the ternary complex. The cleavage is thus represented by a diagonal trajectory across a free energy surface, that is defined by chemical (Co-C separation) and protein configuration coordinates.     

Solution structure and thermolysis of Cobeta-5'-deoxyadenosylimidazolylcobamide, a coenzyme B12 analogue with an imidazole axial nucleoside

The solution structure of Cobeta-5'-deoxyadenosylimidazolylcobamide, Ado(Im)Cbl, the coenzyme B(12) analogue in which the axial 5,6-dimethylbenzimidazole (Bzm) ligand is replaced by imidazole, has been determined by NMR-restrained molecular modeling. A two-state model, in which a conformation with the adenosyl moiety over the southern quadrant of the corrin and a conformation with the adenosyl ligand over the eastern quadrant of the corrin are both populated at room temperature, was required by the nOe data. A rotation profile and molecular dynamics simulations suggest that the eastern conformation is the more stable, in contrast to AdoCbl itself in which the southern conformation is preferred. Consensus structures of the two conformers show that the axial Co-N bond is slightly shorter and the corrin ring is less folded in Ado(Im)Cbl than in AdoCbl. A study of the thermolysis of Ado(Im)Cbl in aqueous solution (50-125 degrees C) revealed competing homolytic and heterolytic pathways as for AdoCbl but with heterolysis being 9-fold faster and homolysis being 3-fold slower at 100 degrees C than for AdoCbl. Determination of the pK(a)'s for the Ado(Im)Cbl base-on/base-off reaction and for the detached imidazole ribonucleoside as a function of temperature permitted correction of the homolysis and heterolysis rate constants for the temperature-dependent presence of the base-off species of Ado(Im)Cbl. Activation analysis of the resulting rate constants for the base-on species show that the entropy of activation for Ado(Im)Cbl homolysis (13.7 +/- 0.9 cal mol(-1) K(-1)) is identical with that of AdoCbl (13.5 +/- 0.7 cal mol(-1) K(-1)) but that the enthalpy of activation (34.8 kcal mol(-1)) is 1.0 +/- 0.4 kcal mol(-1) larger. The opposite effect is seen for heterolysis, where the enthalpies of activation are identical but the entropy of activation is 5 +/- 1 cal mol(-1) K(-1) less negative for Ado(Im)Cbl. Extrapolation to 37 degrees C provides a rate constant for Ado(Im)Cbl homolysis of 2.1 x 10(-9) s(-1), 4.3-fold smaller than for AdoCbl. Combined with earlier results for the enzyme-induced homolysis of Ado(Im)Cbl by the ribonucleoside triphosphate reductase from Lactobacillus leichmannii, the catalytic efficiency of the enzyme for homolysis of Ado(Im)Cbl at 37 degrees C can be calculated to be 4.0 x 10(8), 3.8-fold, or 0.8 kcal mol(-1), smaller than for AdoCbl. Thus, the bulky Bzm ligand makes at best a <1 kcal mol(-1) contribution to the enzymatic activation of coenzyme B(12).     

DFT study of Co-C bond cleavage in the neutral and one-electron-reduced alkyl-cobalt(III) phthalocyanines

Density functional theory (DFT) has been applied to the analysis of the structural and electronic properties of the alkyl-cobalt(III) phthalocyanine complexes, [CoIIIPc]-R (Pc = phthalocyanine, R = Me or Et), and their pyridine adducts. The BP86/6-31G(d) level of theory shows good reliability for the optimized axial bond lengths and bond dissociation energies (BDEs). The mechanism of the reductive cleavage was probed for the [CoIIIPc]-Me complex which is known as a highly effective methyl group donor. In the present analysis, which follows a recent study on the reductive Co-C bond cleavage in methylcobalamin (J. Phys. Chem. B 2007, 111, 7638-7645), it is demonstrated that addition of an electron and formation of the pi-anion radical [CoIII(Pc*)]-Me- significantly lowers the energetic barrier required for homolytic Co-C bond dissociation. Such BDE lowering in [CoIII(Pc*)]-Me- arises from the involvement of two electronic states: upon electron addition, a quasi-degenerate pi*Pc state is initially formed, but when the cobalt-carbon bond is stretched, the unpaired electron moves to a sigma*Co-C state and the final cleavage involves the three-electron (sigma)2(sigma*)1 bond. As in corrin complexes, the pi*Pc-sigma*Co-C states crossing does not take place at the equilibrium geometry of [CoIII(Pc*)]-Me- but only when the Co-C bond is stretched to approximately 2.3 A. The DFT computed Co-C BDE of 23.3 kcal/mol in the one-electron-reduced phthalocyanine species, [CoIII(Pc*)]-Me-, is lowered by approximately 37% compared to the neutral Py-[CoIIIPc]-Me complex where BDE = 36.8 kcal/mol. A similar comparison for the corrin-containing complexes shows that a DFT computed BDE of 20.4 kcal/mol for [CoIII(corrin*)]-Me leads to approximately 45% bond strength reduction, in comparison to 37.0 kcal/mol for Im-[CoIII(corrin)]-Me+. These results suggest some preference by the alkylcorrinoids for the reductive cleavage mechanism.     

How the Co-C bond is cleaved in coenzyme B12 enzymes: a theoretical study

The homolytic cleavage of the organometallic Co-C bond in vitamin B12-dependent enzymes is accelerated by a factor of approximately 10(12) in the protein compared to that of the isolated cofactor in aqueous solution. To understand this much debated effect, we have studied the Co-C bond cleavage in the enzyme glutamate mutase with combined quantum and molecular mechanics methods. We show that the calculated bond dissociation energy (BDE) of the Co-C bond in adenosyl cobalamin is reduced by 135 kJ/mol in the enzyme. This catalytic effect can be divided into four terms. First, the adenosine radical is kept within 4.2 angstroms of the Co ion in the enzyme, which decreases the BDE by 20 kJ/mol. Second, the surrounding enzyme stabilizes the dissociated state by 42 kJ/mol using electrostatic and van der Waals interactions. Third, the protein itself is stabilized by 11 kJ/mol in the dissociated state. Finally, the coenzyme is geometrically distorted by the protein, and this distortion is 61 kJ/mol larger in the Co(III) state. This deformation of the coenzyme is caused mainly by steric interactions, and it is especially the ribose moiety and the Co-C5'-C4' angle that are distorted. Without the polar ribose group, the catalytic effect is much smaller, e.g. only 42 kJ/mol for methyl cobalamin. The deformation of the coenzyme is caused mainly by the substrate, a side chain of the coenzyme itself, and a few residues around the adenosine part of the coenzyme.     

Electronic spectroscopy and computational studies of glutathionylco(III)balamin

We have studied glutathionylcobalamin (GS-Cbl) by optical spectroscopy and with density functional theory (DFT) and time-dependent DFT (TD-DFT) electronic structure methods of truncated geometric models. We examined the geometric structure of the models by comparison of DFT calculations with recent high-resolution experimental X-ray structure data ( Hannibal, L. et al. Inorg. Chem. 2010, 49, 9921) for GS-Cbl, and we examined the TD-DFT excitation simulations by comparison of the models with measured optical spectra. The calculations employed the B3LYP hybrid functional and the nonhybrid BP86 functional in both vacuum and water (conductor polarized continuum model (cpcm)) with the 6-311G(d,p) basis set. The optimized geometric structure calculations for six truncated models were made by varying the chemical structure, solvent model, and the two DFT functionals. All showed similar geometry. Charge decomposition analysis (CDA) and extended charge decomposition analysis (ECDA), especially with BP86 shows the similar charge transfer nature of the Co-S bond in GS-Cbl and the Co-C bond in CH(3)Cbl. Mayer and Wiberg bond orders illustrate the similar covalent nature of the two bonds. Finally, absolute optical spectral simulation calculations were compared with the experimental UV-visible extinction spectrum and the electronic circular dichroism (ECD) differential extinction spectrum. The BP86 method shows more spectral features, and the best fit was found for a GS-Cbl model with 5,6-dimethylbenzimidazole at the BP86/6-311G(d,p) level with a water cpcm solvent model. The excited state transitions were investigated with Martin's natural transition orbitals (NTOs). The BP86 calculations also showed π bonding interactions between Co and the axial S of the GS- ligand in the molecular orbitals (MOs) and NTOs.     

Accurate redetermination of the X-ray structure and electronic bonding in adenosylcobalamin

The electronic structure of adenosylcobalamin (B12 coenzyme, AdoCbl) has been calculated by a density functional method, using the orthogonalized linear combination of the atomic orbital method (OLCAO). Since a fixed accurately determined geometry was needed in such calculations, the crystal structure of adenosylcobalamin has been redone and refined to R = 0.065, using synchrotron diffraction data. Comparison with the recently reported electronic structures of cyano- (CNCbl) and methylcobalamin (MeCbl) shows that the net charges and bond orders vary only on the axial donors. The values in the three cobalamins suggest that the Co-C bond in MeCbl has a strength similar to that in AdoCbl, but it is significantly weaker that that in CNCbl. Present results are compared with those previously reported for the analogous corrin derivatives; i.e., simplified cobalamins with the side chains a-f replaced by H atoms. Despite a qualitative agreement, a discrepancy in the calculated HOMO-LUMO gap is found.     

Escherichia coli type I isopentenyl diphosphate isomerase: structural and catalytic roles for divalent metals

Isopentenyl diphosphate isomerase (IDI) catalyzes the essential conversion of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) in the mevalonate entry into the isoprenoid biosynthetic pathway. Two convergently evolved forms of IDI are known. Type I IDI, which is found in Eukarya and many Bacteria, catalyzes the isomerization of IPP and DMAPP by a protonation-deprotonation mechanism. The enzyme requires two divalent metal ions for activity. An X-ray structure of type I IDI from crystals soaked with (N,N-dimethylamino)-1-ethyl diphosphate (NIPP), a potent transition-state analogue for the carbocationic intermediate in the isomerization reaction, shows one of the metals in a His(3)Glu(2) hexacoordinate binding site, while the other forms a bridge between the diphosphate moiety of the substrate and the enzyme (Wouters, J.; et al. J. Biol. Chem. 2003, 278, 11903). Reconstitution of metal-free recombinant Escherichia coli type I IDI with several divalent metals-Mg(2+), Mn(2+), Zn(2+), Co(2+), Ni(2+), and Cd(2+)-generated active enzyme. Freshly purified IDI contained substoichiometric levels of a single metal ion, presumably bound in the hexacoordinate site. When NIPP was added to the disruption and purification buffers of enzyme, the purified protein contained 0.72 equiv of Mg(2+), 0.92 equiv of Zn(2+), and 0.10 equiv of Mn(2+). These results are consistent with a structure in which Mg(2+) facilitates diphosphate binding and Zn(2+) or Mn(2+) occupies the hexacoordinate site.