Details

Autor(en) / Beteiligte

• Yang, Hao
• Ho, Madeline B
• Lundahl, Maike N
• Mosquera, Martín A
• Broderick, William E
• Broderick, Joan B
• Hoffman, Brian M

Titel

ENDOR Spectroscopy Reveals the "Free" 5'-Deoxyadenosyl Radical in a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound 4Fe-4S2+ Cluster

Ist Teil von

• Journal of the American Chemical Society, 2024-02, Vol.146 (6), p.3710

Erscheinungsjahr

2024

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• 1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.