Active-Site Controlled, Jahn-Teller Enabled Regioselectivity in Reductive S-C Bond Cleavage of S-Adenosylmethionine in Radical SAM Enzymes

Abstract

Catalysis by canonical radical S-adenosyl-l-methionine (SAM) enzymes involves electron transfer (ET) from [4Fe-4S]⁺ to SAM, generating an R₃S⁰ radical that undergoes regioselective homolytic reductive cleavage of the S-C5' bond to generate the 5'-dAdo· radical. However, cryogenic photoinduced S-C bond cleavage has regioselectively yielded either 5'-dAdo· or ·CH₃, and indeed, each of the three SAM S-C bonds can be regioselectively cleaved in an RS enzyme. This diversity highlights a longstanding central question: what controls regioselective homolytic S-C bond cleavage upon SAM reduction? We here provide an unexpected answer, founded on our observation that photoinduced S-C bond cleavage in multiple canonical RS enzymes reveals two enzyme classes: in one, photolysis forms 5'-dAdo·, and in another it forms ·CH₃. The identity of the cleaved S-C bond correlates with SAM ribose conformation but not with positioning and orientation of the sulfonium center relative to the [4Fe-4S] cluster. We have recognized the reduced-SAM R₃S⁰ radical is a (²E) state with its antibonding unpaired electron in an orbital doublet, which renders R₃S⁰ Jahn-Teller (JT)-active and therefore subject to vibronically induced distortion. Active-site forces induce a JT distortion that localizes the odd electron in a single priority S-C antibond, which undergoes regioselective cleavage. In photolytic cleavage those forces act through control of the ribose conformation and are transmitted to the sulfur via the S-C5' bond, but during catalysis thermally induced conformational changes that enable ET from a cluster iron generate dominant additional forces that specifically select S-C5' for cleavage. This motion also can explain how 5'-dAdo· subsequently forms the organometallic intermediate Ω.

Figure 1.

Structure of the complex of SAM with a [4Fe–4S]⁺ RS cluster illustrating the three alternative radical products resulting from the reductive cleavage of SAM.

Figure 2.

X-band EPR spectra of 5′dAdo· obtained from the photolysis of reduced RS enzymes PFL-AE, RNR-AE, OspD, and SPL with SAM (left) that collapse to singlets with adenosyl-2,8-D₂-[1′,2′,3′,4′,5′,5″-D₆]-SAM (D₈-SAM) (right). Conditions: microwave frequency, 9.37 GHz; modulation amplitude, 5 G; T = 40 K.

Figure 3.

X-band EPR spectra of ·CH₃ from photolysis of reduced RS enzymes HydG, PoyD, LAM, TmHydE, and CaHydE with SAM and CD₃-SAM. All samples are prepared with H₂O, except that the sample for the bottom spectra is prepared in D₂O solvent. EPR conditions: microwave frequency, 9.37 GHz; modulation amplitude, 5 G; T = 40 K.

Figure 4.

X-band EPR spectra of radical species from the photolysis of reduced PFL-AE and HydG with SAM and SAE, with an EPR spectrum simulation for ·CH₂CH₃ (red): LW = 27 MHz, g = [2.003, 2.003, 2.001], two α-protons from ·CH₂− with A(¹H_a) = [80, 40, 60] MHz, (α, β, γ) = (−60, 0, 0), A(¹H_b) = [80, 40, 60], (α, β, γ) = (60, 0, 0), and three β-protons from the methyl group with a_iso(¹H_a, ¹H_b, ¹H_c) = 110, 20, and 20 MHz. EPR conditions: microwave frequency, 9.37 GHz; modulation amplitude, 5 G; T = 40 K.

Figure 5.

(Upper) Crystallographic structure of SAM bound to the [4Fe–4S] cluster in SPL (left: 4fhf.pdb), which undergoes photochemical liberation of 5′-dAdo·, and in HydE (right: 3iiz.pdb), which undergoes photochemical liberation of ·CH₃. (Lower) Overlay of the two structures. Together, the figures show nearly identical positioning and orientation of the sulfonium center with respect to the unique Fe of the [4Fe–4S] cluster.

Figure 6.

Comparisons of SAM configurations that yield photo-induced cleavage to form 5′-dAdo· or ·CH₃, with orientation adjusted so that in all cases the ribose C2′, C1′, and ring O atoms overlay. (Left) Overlay of PFL-AE (3cb8.pdb) and HydG (4wcx.pdb). (Right) Overlay of (a) SPL (4fhf.pdb), (b) PFL-AE, (c) HydE (3iiz.pdb), (d) LAM (2a5h.pdb), (e) HydG, demonstrating the axial orientation of the C4′–C5′ bond in 5′-dAdo· formers and the equatorial orientation in ·CH₃ formers.

Figure 7.

DFT computed energy levels (left) and MO diagrams (right) for S(CH₃)₃⁺. (Left) Black, carbon–sulfur based orbitals; gray, the highest-lying C–H based bonding orbitals; blue, sulfur lone pair HOMO; red, C–S based antibonding orbitals. (Right) Blue, positive orbital coefficients; red, negative orbital coefficients. The orbitals are visualized with an isodensity of 0.08 au in PyMol. For completeness we note that the components of a degenerate (e) level are not uniquely defined.

Figure 8.

Upper row: (Left) Cartoon of trigonal R₃S⁰ SAM sulfur center; (Others) contour plots of the APES (orange highest energy) for the JT distortion of R₃S⁰ with a singly occupied E-doublet vibronically coupled to an e vibration, within a formal JT model that treats R₃S⁰ as a stable intermediate for progressively more complex treatments (see Figure S8 for corresponding figure for R₃S⁰ as a dissociative state); radial distance ⇔ JT distortion parameter, azimuthal angle, pseudorotation angle of triangle distortion. Lower Row: Cartoons representing the distribution of triangle orientations. (A) Linear JT vibronic coupling term yields rotationally symmetric APES (upper), and pseudorotating distortion with uniform distribution in orientation (lower). (B) Addition of quadratic vibronic coupling terms plus 3-fold potential of S–C bonds yields three energy minima on APES contour plot (upper) corresponding to three equivalent acute isosceles triangles (e.g., Scheme 3) oriented (lower) with the acute vertex (direction of elongation) pointed along the angle φ associated with that minimum: φ = 0, 2π/3, and 4π/3. (C) Inclusion of appropriate active-site distortion forces lowers the energy of a single minimum (upper, arbitrarily chosen here at φ = 0) with elongated priority S–C bond (lower) that ultimately undergoes regioselective homolysis, as illustrated by both contour and perspective plots.

Figure 9.

(Left) LUMO of (CH₃)₃S⁺ (see the Supporting Information) corresponding to the SOMO of the JT-distorted acute-isosceles R₃S⁰ radical (Scheme 3) with a stretched apex priority S–C bond of 2.2 Å, showing strongly antibonding character of the priority (S[3p-like]–C[σ]) orbital at the apex of the triangle of S–C carbons (see the Supporting Information). (Right) Dimensions of the triangle of carbons in the distorted R₃S⁰ radical.

Figure 10.

Crystallographic structures of SAM bound to the [4Fe–4S] clusters in SPL (4fhf.pdb; left) and in HydE (3iiz.pdb; right), as reoriented from Figure 5 to visualize the basis of conformational changes involved in creating an S–Fe contact in the two regioselectivity classes.

Scheme 1.

Radical Displacement Model; See Ref

Scheme 2.

Frey Model for SAM σ-Donation; See Ref

Scheme 3.

Splitting of ²E Orbital Degeneracy with JT Distortion