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

ABSTRACT: Members of the radical S-adenosyl-l-methionine (SAM) enzyme superfamily initiate a broad spectrum of radical transformations through reductive cleavage of SAM by a [4Fe-4S]¹⁺ cluster it coordinates to generate the reactive 5'-deoxyadenosyl radical (5'-dAdo^•). However, 5'-dAdo^• is not directly liberated for reaction and instead binds to the unique Fe of the cluster to create the catalytically competent S = 1/2 organometallic intermediate Ω. An alternative mode of reductive SAM cleavage, especially seen photochemically, instead liberates CH₃^•, which forms the analogous S = 1/2 organometallic intermediate with an Fe-CH₃ bond, Ω_M. The presence of a covalent Fe-C bond in both structures was established by the ENDOR observation of ¹³C and ¹H hyperfine couplings to the alkyl groups that show isotropic components indicative of Fe-C bond covalency. The synthetic [Fe₄S₄]³⁺-CH₃ cluster, M-CH₃, is a crystallographically characterized analogue to Ω_M that exhibits the same [Fe₄S₄]³⁺ cluster state as Ω and Ω_M, and thus an analysis of its spectroscopic properties─and comparison with those of Ω and Ω_M─can be grounded in its crystal structure. We report cryogenic (2 K) EPR and ¹³C/^1/2H ENDOR measurements on isotopically labeled M-CH₃. At low temperatures, the complex exhibits EPR spectra from two distinct conformers/subpopulations. ENDOR shows that at 2 K, one contains a static methyl, but in the other, the methyl undergoes rapid tunneling/hopping rotation about the Fe-CH₃ bond. This generates an averaged hyperfine coupling tensor whose analysis requires an extended treatment of rotational averaging. The methyl group ¹³C/^1/2H hyperfine couplings are compared with the corresponding values for Ω and Ω_M.

SUBMITTER: Ho MB

PROVIDER: S-EPMC9529902 | biostudies-literature | 2022 Sep

REPOSITORIES: biostudies-literature

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

Ho Madeline B MB Jodts Richard J RJ Kim Youngsuk Y McSkimming Alex A Suess Daniel L M DLM Hoffman Brian M BM

Journal of the American Chemical Society 20220915 38

Members of the radical S-adenosyl-l-methionine (SAM) enzyme superfamily initiate a broad spectrum of radical transformations through reductive cleavage of SAM by a [4Fe-4S]1+ cluster it coordinates to generate the reactive 5'-deoxyadenosyl radical (5'-dAdo•). However, 5'-dAdo• is not directly liberated for reaction and instead binds to the unique Fe of the cluster to create the catalytically competent S = 1/2 organometallic intermediate Ω. An altern ...[more]

PMID: 36108299

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Project description:A [4Fe-4S](+) cluster reduces a bound S-adenosylmethionine (SAM) molecule, cleaving it into methionine and a 5'-deoxyadenosyl radical (5'-dA(•)). This step initiates the varied chemistry catalyzed by each of the so-called radical SAM enzymes. The strongly oxidizing 5'-dA(•) is quenched by abstracting a H-atom from a target species. In some cases, this species is an exogenous molecule of substrate, for example, L-tyrosine in the [FeFe] hydrogenase maturase, HydG. In other cases, the target is a proteinaceous residue as in all the glycyl radical forming enzymes. The generation of this initial radical species and the subsequent chemistry involving downstream radical intermediates is meticulously controlled by the enzyme so as to prevent unwanted reactions. But the manner in which this control is exerted is unknown. Electron paramagnetic resonance (EPR) spectroscopy has proven to be a valuable tool used to gain insight into these mechanisms. In this Account, we summarize efforts to trap such radical intermediates in radical SAM enzymes and highlight four examples in which EPR spectroscopic results have shed significant light on the corresponding mechanism. For lysine 2,3-aminomutase, nearly each possible intermediate, from an analogue of the initial 5'-dA(•) to the product radical L-β-lysine, has been explored. A paramagnetic intermediate observed in biotin synthase is shown to involve an auxiliary [FeS] cluster whose bridging sulfide is a co-substrate for the final step in the biosynthesis of vitamin B7. In HydG, the L-tyrosine substrate is converted in unprecedented fashion to a 4-oxidobenzyl radical on the way to generating CO and CN(-) ligands for the [FeFe] cluster of hydrogenase. And finally, EPR has confirmed a mechanistic proposal for the antibiotic resistance protein Cfr, which methylates the unactivated sp(2)-hybridized C8-carbon of an adenosine base of 23S ribosomal RNA. These four systems provide just a brief survey of the ever-growing set of radical SAM enzymes. The diverse chemistries catalyzed by these enzymes make them an intriguing target for continuing study, and EPR spectroscopy, in particular, seems ideally placed to contribute to our understanding.

Dataset Information

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

Publications

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

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