Selenocysteine Substitution in a Class I Ribonucleotide Reductase

Ribonucleotide reductases (RNRs) employ a complex radical-based mechanism during nucleotide reduction involving multiple active site cysteines that both activate the substrate and reduce it. Using an engineered allo-tRNA, we substituted two active site cysteines with distinct function in the class Ia RNR of Escherichia coli for selenocysteine (U) via amber codon suppression, with efficiency and selectivity enabling biochemical and biophysical studies. Examination of the interactions of the C₄₃₉U α₂ mutant protein with nucleotide substrates and the cognate β₂ subunit demonstrates that the endogenous Y₁₂₂^• of β₂ is reduced under turnover conditions, presumably through radical transfer to form a transient U₄₃₉^• species. This putative U₄₃₉^• species is formed in a kinetically competent fashion but is incapable of initiating nucleotide reduction via 3'-H abstraction. An analogous C₂₂₅U α₂ protein is also capable of radical transfer from Y₁₂₂^•, but the radical-based substrate chemistry partitions between turnover and stalled reduction akin to the reactivity of mechanism-based inhibitors of RNR. The results collectively demonstrate the essential role of cysteine redox chemistry in the class I RNRs and establish a new tool for investigating thiyl radical reactivity in biology.