Electron Spin Echo Envelope Modulation Spectroscopy Reveals How Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase Positions the Radical Pair Intermediates and Modulates Their Stabilities for Efficient Catalysis

Electron spin echo envelope modulation (ESEEM) spectroscopy reveals several interactions that serve to weaken the Co-C5' bond, through stabilization of the cleaved state, thereby facilitating the 5'-deoxyadenosyl (5'-dAdo(center dot)) radical formation in unlabeled and N-15-labeled lysine 5,6-aminomutase. In the first place, we show that the Co-II -His133 beta interaction enhances the spin delocalization from Co-II to the alpha-axial ligand, reducing the 5'-dAdo(center dot)-Co-II recombination probability in situ and allowing nascent 5'-dAdo(center dot) to migrate toward the substrate. Next, using [5'-H-2]-deoxyadenosylcobalamin, we show that the C5' methyl group of 5'-dAdoH is at closest contact with the substrate radical. In the case of 5'-dAdo(center dot), this would allow the 5'-methylene radical to be rapidly quenched by H-transfer from the substrate. Finally, we show that the spin density is transferred noncovalently from the substrate radical to the adenine N7 of 5'-dAdoH. In the one-radical substrate-5'-dAdo(center dot)-product H-exchange triad, which is central in the mechanism of action of B-12 enzymes, such transfer would promote the forward H-transfer reaction by dynamically lowering the free energy of the substrate radical, thereby facilitating its formation and quenching of 5'-dAdo(center dot). In the reverse H-transfer reaction following the 1,2-rearrangement, the reversibility of the noncovalent spin transfer allows the product radical to restore full radical character to reform 5'-dAdo(center dot) and a diamagnetic product. The role of dynamical and reversible spin partitioning as a mechanism for modulating the radical stability is likely common in B-12 enzymes. This study leads to a better comprehension about how an enzyme controls the reaction trajectory of 5'-dAdo(center dot) exquisitely with specificity. The ESEEM results support the positioning of the interacting partners in the catalytic form of lysine 5,6-aminomutase obtained by modeling.

Automated summary

The ESEEM spectroscopy uncovers multiple interactions that aid in the formation of the 5'-deoxyadenosyl radical in lysine 5,6-aminomutase, such as the Co-II -His133 beta interaction and the transfer of spin density from the substrate radical to the adenine N7 of 5'-dAdoH. These interactions promote the forward H-transfer reaction and the quenching of 5'-dAdo(center dot), showing how enzymes modulate radical stability.