The vulnerability of radical SAM enzymes to oxidants and soft metals

Radical S-adenosylmethionine enzymes (RSEs) drive diverse biological processes by catalyzing chemically difficult reactions. Each of these enzymes uses a solvent-exposed [4Fe–4S] cluster to coordinate and cleave its SAM co-reactant. This cluster is destroyed during oxic handling, forcing investigators to work with these enzymes under anoxic conditions. Analogous substrate-binding [4Fe–4S] clusters in dehydratases are similarly sensitive to oxygen in vitro; they are also extremely vulnerable to reactive oxygen species (ROS) in vitro and in vivo. These observations suggested that ROS might similarly poison RSEs. This conjecture received apparent support by the observation that when E. coli experiences hydrogen peroxide stress, it induces a cluster-free isozyme of the RSE HemN. In the present study, surprisingly, the purified RSEs viperin and HemN proved quite resistant to peroxide and superoxide in vitro. Furthermore, pathways that require RSEs remained active inside E. coli cells that were acutely stressed by hydrogen peroxide and superoxide. Viperin, but not HemN, was gradually poisoned by molecular oxygen in vitro, forming an apparent [3Fe–4S]⁺ form that was readily reactivated. The modest rate of damage, and the known ability of cells to repair [3Fe–4S]⁺ clusters, suggest why these RSEs remain functional inside fully aerated organisms. In contrast, copper(I) damaged HemN and viperin in vitro as readily as it did fumarase, a known target of copper toxicity inside E. coli. Excess intracellular copper also impaired RSE-dependent biosynthetic processes. These data indicate that RSEs may be targets of copper stress but not of reactive oxygen species.