Pyruvate formate-lyase activating enzyme: elucidation of a novel mechanism for glycyl radical formation

Pyruvate formate lyase activating enzyme is a member of a novel superfamily of enzymes that utilize S-adenosylmethionine to initiate radical catalysis. This enzyme has been isolated with several different iron–sulfur clusters, but single turnover monitored by EPR has identified the [4Fe–4S]1+ cluster as the catalytically active cluster; this cluster is believed to be oxidized to the [4Fe–4S]2+ state during turnover. The [4Fe–4S] cluster is coordinated by a three-cysteine motif common to the radical/S-adenosylmethionine superfamily, suggesting the presence of a unique iron in the cluster. The unique iron site has been confirmed by Mössbauer and ENDOR spectroscopy experiments, which also provided the first evidence for direct coordination of S-adenosylmethionine to an iron–sulfur cluster, in this case the unique iron of the [4Fe–4S] cluster. Coordination to the unique iron anchors the S-adenosylmethionine in the active site, and allows for a close association between the sulfonium of S-adenosylmethionine and the cluster as observed by ENDOR spectroscopy. The evidence to date leads to a mechanistic proposal involving inner-sphere electron transfer from the cluster to the sulfonium of S-adenosylmethionine, followed by or concomitant with C–S bond homolysis to produce a 5′-deoxyadenosyl radical; this transient radical abstracts a hydrogen atom from G734 to activate pyruvate formate lyase.