Broken-Symmetry Density Functional Theory Analysis of the Ω Intermediate in Radical S-Adenosyl-l-methionine Enzymes: Evidence for a Near-Attack Conformer over an Organometallic Species

Abstract

Radical S-adenosyl-L-methionine (SAM) enzymes are found in all domains of life and catalyze a wide range of biochemical reactions. Recently, an organometallic intermediate, Ω, has been experimentally implicated in the 5′-deoxyadenosyl radical generation mechanism of the radical SAM superfamily. In this work, we employ broken-symmetry density functional theory to evaluate several structural models of Ω. The results show that the calculated hyperfine coupling constants (HFCCs) for the proposed organometallic structure of Ω are inconsistent with the experiment. In contrast, a near-attack conformer of SAM bound to the catalytic [4Fe−4S] cluster, in which the distance between the unique iron and SAM sulfur is ∼3 Å, yields HFCCs that are all within 1 MHz of the experimental values. These results clarify the structure of the ubiquitous Ω intermediate and suggest a paradigm shift reversal regarding the mechanism of SAM cleavage by members of the radical SAM superfamily.

Characterization by ENDOR Spectroscopy of the Iron–Alkyl Bond in a Synthetic Counterpart of Organometallic Intermediates in Radical SAM Enzymes

“…Considering the properties of the Fe–alkyl bond itself, methyl group interactions with the “down”-spin on the unique Fe cause both conformers of M-CH 3 to exhibit a small positive and essentially isotropic 13 C coupling constant, indicative of positive (“up”) spin density induced on carbon. The magnitude of the M-CH 3 coupling, a iso ( 13 C) ∼+6 MHz, is quite comparable to that observed for Ω, | a iso ( 13 C)| ∼9 MHz, while both couplings are somewhat smaller than | a iso ( 13 C)| ∼18 MHz for Ω M ; notably, the coupling is more than an order of magnitude smaller than that purported for Ω in a recent DFT study . In contrast, the “down” spin on the unique Fe induces a negative isotropic 1 H hyperfine coupling to the methyl protons of both M-CH 3 conformers, and the magnitude of their a iso ( 1 H) also compares well with the 1 H coupling in Ω, | a iso ( 1 H)| = 7 MHz, as follows .…”

“…The magnitude of the M-CH 3 coupling, a iso ( 13 C) ∼+6 MHz, is quite comparable to that observed for Ω, |a iso ( 13 C)| ∼9 MHz, 12 while both couplings are somewhat smaller than |a iso ( 13 C)| ∼18 MHz for Ω M ; 15 notably, the coupling is more than an order of magnitude smaller than that purported for Ω in a recent DFT study. 37 In contrast, the "down" spin on the unique Fe induces a negative isotropic 1 H hyperfine coupling to the methyl protons of both M-CH 3 conformers, and the magnitude of their a iso ( 1 H) also compares well with the 1 H coupling in Ω, |a iso ( 1 H)| = 7 MHz, as follows. 11 The rotationally averaged methyl hyperfine tensor of M1-CH 3 has an isotropic coupling of a iso ( 1 H) = −8.5 MHz and anisotropic coupling with unique value T1 || av = −4.2 MHz, while the 2D ENDOR pattern of M2-CH 3 (Figure S5) is satisfactorily reproduced by the sum of signals from the three tetrahedrally oriented protons of a static methyl group (Scheme 1) with 1 H tensors that have similar isotropic couplings, a iso ( 1 H) ∼−(8 ↔ 10) MHz, and an average value for the unique component of the anisotropic coupling tensor of, T2 || av = −5 MHz.…”

Mechanism of Radical S-Adenosyl-l-methionine Adenosylation: Radical Intermediates and the Catalytic Competence of the 5′-Deoxyadenosyl Radical

“…Following acceptance of this paper, a computation-only paper appeared in the literature, concluding that Ω is not an organometallic species . We do not agree with the method or conclusions of this work and this will be addressed in future reports.…”

Computational Description of Alkylated Iron–Sulfur Organometallic Clusters

“…This report clearly shows that it is inappropriate to use single-determinant BS-DFT approaches to investigate organometallic iron–sulfur clusters with an Fe–alkyl bond, such as that presented recently in an errant attempt to treat Ω . Perhaps chief among the flaws in that report (which are discussed in detail in the Supporting Information), in calculating alkyl HFCCs, it employed single-determinant BS-DFT computations and used a different projection factor, rather than the required factor, K C = −1/3, that modifies the BS-DFT coupling in 2C-DFT, eq .…”