A mechanochemical switch to control radical intermediates

Abstract

B₁₂-dependent enzymes employ radical species with exceptional prowess to catalyze some of the most chemically challenging, thermodynamically unfavorable reactions. However, dealing with highly reactive intermediates is an extremely demanding task, requiring sophisticated control strategies to prevent unwanted side reactions. Using hybrid quantum mechanical/molecular mechanical simulations, we follow the full catalytic cycle of an AdoB₁₂-dependent enzyme and present the details of a mechanism that utilizes a highly effective mechanochemical switch. When the switch is "off", the 5'-deoxyadenosyl radical moiety is stabilized by releasing the internal strain of an enzyme-imposed conformation. Turning the switch "on," the enzyme environment becomes the driving force to impose a distinct conformation of the 5'-deoxyadenosyl radical to avoid deleterious radical transfer. This mechanochemical switch illustrates the elaborate way in which enzymes attain selectivity of extremely chemically challenging reactions.

Figure 1

Adenosylcobalamin cofactor. In (a), the B₁₂ cofactor, doubly coordinated with a histidine residue (DMB-off conformation) and the 5′-deoxyadenosyl moiety (Ado). In (b) and (c), the crystal structures of bound (pdb entry 3REQ) and unbound Ado (pdb entry 4REQ), respectively, demonstrate the change in conformation of the adenine base relative to the B₁₂ corrinoid ring.

Figure 2

Mechanochemical switch. Upon homolysis, illustrated by the stages 1_a to 1_b, Ado· diffuses out of its cavity and undergoes a spontaneous conformational change in which the adenine base rotates by 100° to an orientation perpendicular to the corrinoid ring. The hydrophobic and nonpolar residues lining the cavity around the adenine base allow for this rotation to be diffusion controlled. The panel on the upper right displays the free energy profile for Co···C_5′ bond cleavage. In contrast, during the hydrogen abstraction reaction, illustrated by the stages 2_a to 2_b, the enzyme mediates the conformational change in Ado· from C_3′-endo to C_2′-endo via nearby charged and polar residues which exchange hydrogen bonds with axial O_3′ and equatorial O_3′. The panel on the lower right displays the free energy profile for hydrogen abstraction. The maximum error obtained along the profiles are indicated by the error bars.

Figure 3

Different conformations of Ado· in other B₁₂-dependent enzymes. In (a), the results of a relaxed scan in gas phase along glycosidic bond, the O_4′–C_1′–N₉–C₈ dihedral angle, of an isolated Ado· moiety. The scan uncovers several free energy minimum structures: ϕ of 48, −11, and −110°. All energies (M06/6-31G(d)) are relative to an arbitrarily chosen origin, an orientation of Ado· taken from the QM/MM simulations following AdoCbl bond cleavage (an averaged value of ϕ of 115° ± 8.9). Performing a geometry optimization of Ado· from this bound state gains nearly 5 kcal mol^–1 of energy via the rotation of ϕ from 115° to 48° (indicated by the dashed gray line connecting these two points). In (b), the ϕ values taken from various B₁₂-dependent enzymes, plotted as a function of AdoCbl bond length. The gray points represent the average dihedral value for each cluster. When bound to Co(III), the value of ϕ is 50–80° larger than it is in the unbound state. As previously suggested, the global minimum structure in the gas phase (ϕ = −11°) is stabilized by an intramolecular hydrogen bond between H–O_2′ and N₃, which is not observed in the condensed phase.

Figure 4

Preventing unproductive side reactions. Using QM/MM simulations, the free energy profiles for the native hydrogen (H_1°) abstraction and a deleterious side reaction, the abstraction of the tertiary hydrogen atom (H_3°) on the subsequent carbon atom, are shown at the top. In the native reaction mechanism, shown in green and labeled b, the shift in ribose puckering of the Ado· moiety from C_3′-endo to C_2′-endo occurs spontaneously during the hydrogen abstraction step and is accommodated by MCM via a nearby glutamate residue, E370. In the absence of this conformational change, the Ado· remains in the C_3′-endo conformation, and the abstraction of the “wrong” hydrogen atom (in this case, H_3°) is thermodynamically and kinetically favored over the native reaction (see purple curve, labeled a). In contrast, abstraction of the same hydrogen, H_3°, is no longer favored if Ado· adopts the C_2′-endo conformation (see orange curve labeled c).