Mechanism of Reduction of an Aminyl Radical Intermediate in the Radical SAM GTP 3′,8-Cyclase MoaA

The diversity of the reactions catalyzed by radical S-adenosyl-l-methionine (SAM) enzymes is achieved at least in part through the variety of mechanisms to quench their radical intermediates. In the SPASM-twitch family, the largest family of radical SAM enzymes, the radical quenching step is thought to involve an electron transfer to or from an auxiliary 4Fe-4S cluster in or adjacent to the active site. However, experimental demonstration of such functions remains limited. As a representative member of this family, MoaA has one radical SAM cluster ([4Fe-4S]RS) and one auxiliary cluster ([4Fe-4S]AUX), and catalyzes a unique 3′,8-cyclization of GTP into 3′,8-cyclo-7,8-dihydro-GTP (3′,8-cH2GTP) in the molybdenum cofactor (Moco) biosynthesis. Here, we report a mechanistic investigation of the radical quenching step in MoaA, a chemically challenging reduction of 3′,8-cyclo-GTP-N7 aminyl radical. We first determined the reduction potentials of [4Fe-4S]RS and [4Fe-4S]AUX as −510 mV and −455 mV, respectively, using a combination of protein film voltammogram (PFV) and electron paramagnetic resonance (EPR) spectroscopy. Subsequent Q-band EPR characterization of 5′-deoxyadenosine C4′ radical (5′-dA-C4′•) trapped in the active site revealed isotropic exchange interaction (∼260 MHz) between 5′-dA-C4′• and [4Fe-4S]AUX 1+, suggesting that [4Fe-4S]AUX is in the reduced (1+) state during the catalysis. Together with density functional theory (DFT) calculation, we propose that the aminyl radical reduction proceeds through a proton-coupled electron transfer (PCET), where [4Fe-4S]AUX serves as an electron donor and R17 residue acts as a proton donor. These results provide detailed mechanistic insights into the radical quenching step of radical SAM enzyme catalysis.