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

Abstract

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.

Characterization of Protein Contributions to Cobalt−Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase by using Photolysis in the Ternary Complex

“…Ultra-violet/visible absorption, magnetic circular dichroism (MCD) and resonance Raman studies showed that the protein in AdoCbl-dependent methylmalonyl-CoA mutase (MMCM) does not significantly distort AdoCbl in the holo-enzyme, or in holo-enzyme with bound substrate analogs. 19 An absence of Co-C bond length changes by the protein in MMCM was also concluded from infrared studies. 57 Picosecond optical studies suggested an absence of structural perturbations of the Co-C bond in glutamate mutase (GM).…”

“…19 The spectra in Figure 3 for holo-EAL and the ternary complex show that the presence of bound substrate does not induce significant shifts in the α/β band λ max , or significant changes in the wavelength maxima and absorbance values, of other principal UV/visible absorption bands of the bound AdoCbl cofactor. Our results for the ternary complex are obtained under conditions in which the mechanisms that promote the thermal cleavage of the Co-C bond cleavage are poised.…”

Spectroscopic Studies of the Corrinoid/Iron−Sulfur Protein from Moorella thermoacetica

“…54 All spectra were red-shifted by 3000 cm −1 to compensate for the fact that transition energies for Co 3+ -corrinoids and similar systems are consistently overestimated by the B3LYP TD-DFT method. [24][25][26][27]49,53,[55][56][57] …”

Entropic Origin of Cobalt–Carbon Bond Cleavage Catalysis in Adenosylcobalamin-Dependent Ethanolamine Ammonia-Lyase

“…17-20 Spectroscopic studies, including UV-visible absorption, magnetic circular dichroism (MCD), and resonance Raman, have shown that AdoCbl is not significantly distorted in the MCM holoenzyme, or in the presence of substrate analogs, relative to solution. 21 The structure of the cleavage product, cob(II)alamin, is also not significantly influenced by the protein. 22 An absence of significant ground state Co-C bond activation by the enzyme was also concluded from infrared 23 and picosecond optical 24 spectroscopic studies.…”