Co−C Bond Activation in Methylmalonyl-CoA Mutase by Stabilization of the Post-homolysis Product Co²⁺Cobalamin

Abstract

Despite decades of research, the mechanism by which coenzyme B12 (adenosylcobalamin, AdoCbl)-dependent enzymes promote homolytic cleavage of the cofactor's Co-C bond to initiate catalysis has continued to elude researchers. In this work, we utilized magnetic circular dichroism spectroscopy to explore how the electronic structure of the reduced B12 cofactor (i.e., the post-homolysis product Co2+ Cbl) is modulated by the enzyme methylmalonyl-CoA mutase. Our data reveal a fairly uniform stabilization of the Co 3d orbitals relative to the corrin pi/pi*-based molecular orbitals when Co2+ Cbl is bound to the enzyme active site, particularly in the presence of substrate. Contrastingly, our previous studies (Brooks, A. J.; Vlasie, M.; Banerjee, R.; Brunold, T. C. J. Am. Chem. Soc. 2004, 126, 8167-8180.) showed that when AdoCbl is bound to the MMCM active site, no enzymatic perturbation of the Co3+ Cbl electronic structure occurs, even in the presence of substrate (analogues). Collectively, these observations provide direct evidence that enzymatic Co-C bond activation involves stabilization of the post-homolysis product, Co2+ Cbl, rather than destabilization of the Co3+ Cbl "ground" state.

Spectroscopic Studies of the EutT Adenosyltransferase from Salmonella enterica: Mechanism of Four-Coordinate Co(II)Cbl Formation

“…7 The two lowest-energy features in this spectrum have previously been assigned to LF transitions primarily involving electronic excitations from the doubly-occupied Co 3d xz - and 3d yz -based molecular orbitals (MOs) to the singly occupied Co 3d z 2 -based MO. 46 Additional features between 16 000 and 22 000 cm −1 have generally been attributed to metal-to-ligand charge transfer (MLCT) or additional LF transitions, with specific assignments made difficult by the presence of multiple transitions with similar energies in this region.…”

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

“…28,30,38 The low-energy region (<17500 cm -1 ) is dominated by LF transitions involving electronic excitations among the Co 3d-based MOs, while the major features in the high-energy region (>21000 cm -1 ) are due to corrin-centered π→π* transitions. The most intense features observed between these two regions arise from electronic excitations between Co 3d-based and corrin π*-based MOs, corresponding to metal-to-ligand charge transfer (MLCT) transitions (Figure 12).…”

Role of vitamin B12 on methylmalonyl-CoA mutase activity

“…This enzymatic activation involves a decrease in the dissociation enthalpy of the Co-C from 31.4±1.5 to ~14 kcal/mol, thus contributing to a ~17 kcal/mol decrease. Brooks et al (2005) used the magnetic circular dichroism (MCD) to study the spectra of the MCM enzyme with cob(II)alamin at the binding domain to the coenzyme and in the presence and absence of analog substrates. The spectra show differences between the enzyme bound to cob(II)alamin and the enzyme without the cofactor, indicating a conformational change caused by the bond with the cofactor, which could labilize or activate the Co-C bond.…”

“…Comparing these spectra with those obtained for MCM-AdoCbl, some modifications may be observed due to the bond between the enzyme and the cofactor, which increases the peaks of the spectra, because the cofactor adopts a well-defined form at the active site once it stabilizes in one of the various possible isoenergetic orientations that the adenosine molecule may adopt after MCM has bound to the cofactor. Brooks et al (2005) continued using the MCD and ABS techniques to analyze the spectra of cob(II)alamin bound to MCM. Much more pronounced modifications were observed in these spectra, which led them to conclude that distortions of the cofactor in its Co(II) form are a significant source of activation of the Co-C bond.…”