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

Abstract

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•) radical formation in unlabeled and 15N-labeled lysine 5,6-aminomutase. In the first place, we show that the CoII–His133β interaction enhances the spin delocalization from CoII to the α-axial ligand, reducing the 5′-dAdo•–CoII recombination probability in situ and allowing nascent 5′-dAdo• to migrate toward the substrate. Next, using [5′-2H]-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•, 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•–product H-exchange triad, which is central in the mechanism of action of B12 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•. 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• and a diamagnetic product. The role of dynamical and reversible spin partitioning as a mechanism for modulating the radical stability is likely common in B12 enzymes. This study leads to a better comprehension about how an enzyme controls the reaction trajectory of 5′-dAdo• 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.