Radical SAM Enzyme Spore Photoproduct Lyase: Properties of the Ω Organometallic Intermediate and Identification of Stable Protein Radicals Formed during Substrate-Free Turnover

Abstract

Spore photoproduct lyase is a radical S-adenosyl-l-methionine (SAM) enzyme with the unusual property that addition of SAM to the [4Fe-4S]¹⁺ enzyme absent substrate results in rapid electron transfer to SAM with accompanying homolytic S–C5′ bond cleavage. Herein, we demonstrate that this unusual reaction forms the organometallic intermediate Ω in which the unique Fe atom of the [4Fe-4S] cluster is bound to C5′ of the 5′-deoxyadenosyl radical (5′-dAdo^•). During catalysis, homolytic cleavage of the Fe–C5′ bond liberates 5′-dAdo^• for reaction with substrate, but here, we use Ω formation without substrate to determine the thermal stability of Ω. The reaction of Geobacillus thermodenitrificans SPL (GtSPL) with SAM forms Ω within ∼15 ms after mixing. By monitoring the decay of Ω through rapid freeze–quench trapping at progressively longer times we find an ambient temperature decay time of the Ω Fe–C5′ bond of τ ≈ 5–6 s, likely shortened by enzymatic activation as is the case with the Co–C5′ bond of B₁₂. We have further used hand quenching at times up to 10 min, and thus with multiple SAM turnovers, to probe the fate of the 5′-dAdo^• radical liberated by Ω. In the absence of substrate, Ω undergoes low-probability conversion to a stable protein radical. The WT enzyme with valine at residue 172 accumulates a Val^•; mutation of Val172 to isoleucine or cysteine results in accumulation of an Ile^• or Cys^• radical, respectively. The structures of the radical in WT, V172I, and V172C variants have been established by detailed EPR/DFT analyses.