Spin-Regulated Electron Transfer and Exchange-Enhanced Reactivity in Fe₄S₄-Mediated Redox Reaction of the Dph2 Enzyme During the Biosynthesis of Diphthamide

The [4Fe-4S]-dependent radical S-adenosylmethionine (SAM) proteins is one of large families of redox enzymes that are able to carry a panoply of challenging transformations. Despite the extensive studies of structure–function relationships of radical SAM (RS) enzymes, the electronic state-dependent reactivity of the [4Fe-4S] cluster in these enzymes remains elusive. Using combined MD simulations and QM/MM calculations, we deciphered the electronic state-dependent reactivity of the [4Fe-4S] cluster in Dph2, a key enzyme involved in the biosynthesis of diphthamide. Our calculations show that the reductive cleavage of the S−C_(γ) bond is highly dependent on the electronic structure of [4Fe-4S]. Interestingly, the six electronic states can be classified into a low-energy and a high-energy groups, which are correlated with the net spin of Fe4 atom ligated to SAM. Due to the driving force of Fe4−C_(γ) bonding, the net spin on the Fe4 moiety dictate the shift of the opposite spin electron from the Fe1-Fe2-Fe3 block to SAM. Such spin-regulated electron transfer results in the exchange-enhanced reactivity in the lower-energy group compared with those in the higher-energy group. This reactivity principle provides fundamental mechanistic insights into reactivities of [4Fe-4S] cluster in RS enzymes.