Paramagnetic Resonance in Mechanistic Studies of Fe-S/Radical Enzymes

Abstract

Paramagnetic resonance studies have provided critical insight into the structural and mechanistic properties of the "radical-SAM" enzymes, a superfamily of enzymes which utilize iron-sulfur clusters and S-adenosylmethionine (SAM or AdoMet) to initiate radical catalysis. Here we report recent studies of pyruvate formate-lyase activating enzyme (PFL-AE), which functions to generate the catalytically essential glycyl radical of pyruvate formate-lyase (PFL). Quantitative EPR studies show that the [4Fe-4S]+ cluster on PFL-AE is the electron source required for generation of the glycyl radical on PFL. Electron-nuclear double resonance (ENDOR) studies show that the co-substrate AdoMet sits close to the [4Fe4S]+ cluster of PFL-AE in the ES complex, with the methyl carbon approximately 4-5 Å, and the methyl hydrogen approximately 3-4 Å, from the closest iron of the cluster. This close association of AdoMet and the Fe-S cluster has significant implications regarding the catalytic mechanism.